Elephant
Bibliographic
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References updated October 2009 by date of publication, most recent first. 

Behr, B., Rath, D., Hildebrandt, T.B., Goeritz, F., Blottner, S., Portas, T.J., Bryant, B.R., Sieg, B., Knieriem, A., de Graaf, S.P., Maxwell, W.M., Hermes, R., 2009. Germany/Australia index of sperm sex sortability in elephants and rhinoceros. Reprod. Domest. Anim 44, 273-277.
Abstract: Flow cytometric sexing of spermatozoa followed by application in artificial insemination or in vitro fertilization provides a unique opportunity to predetermine the sex of offspring and might enhance the conservation management of endangered species in captivity such as the elephant and rhinoceros. To obtain an indication of the sortability of spermatozoa from these species, the relative DNA differences between X and Y chromosome bearing spermatozoa (fresh, frozen thawed, epididymal) from three rhinoceros species [white (Ceratotherium simum), black (Diceros bicornis), Indian (Rhinoceros unicornis)] and both elephant species, the Asian and the African elephant (Elephas maximus, Loxodonta Africana), were determined through separation of spermatozoa into X and Y chromosome bearing populations, using a modified high speed flow cytometer. The head profile areas of spermatozoa from all five species were measured using light microscopy. By multiplying the relative DNA differences and the head profile areas, the sperm sorting indices were calculated to be 47, 48 and 51 for white, black and Indian rhinoceros respectively. The calculated sorting index for the Asian elephant was 66. In the African elephant, we determined the highest sorting index of 76. These results indicate the practicability of flow cytometric sex sorting of spermatozoa from the tested rhinoceros species and both elephant species. The lower sorting indices in rhinos indicate that sex sorting of spermatozoa from the rhinoceros will be more challenging than in elephants

Fulka, J., Jr., Loi, P., Ptak, G., Fulka, H., John, J.S., 2009. Hope for the mammoth? Cloning Stem Cells 11, 1-4.

Garner, M.M., Helmick, K., Ochsenreiter, J., Richman, L.K., Latimer, E., Wise, A.G., Maes, R.K., Kiupel, M., Nordhausen, R.W., Zong, J.C., Hayward, G.S., 2009. Clinico-pathologic features of fatal disease attributed to new variants of endotheliotropic herpesviruses in two Asian elephants (Elephas maximus)
119. Vet. Pathol. 46, 97-104.
Abstract: The first herpesviruses described in association with serious elephant disease were referred to as endotheliotropic herpesviruses (EEHV) because of their ability to infect capillary endothelial cells and cause potentially fatal disease. Two related viruses, EEHV1 and EEHV2, have been described based on genetic composition. This report describes the similarities and differences in clinicopathologic features of 2 cases of fatal endotheliotropic herpesvirus infections in Asian elephants caused by a previously unrecognized virus within the betaherpesvirus subfamily. EEHV3 is markedly divergent from the 2 previously studied fatal probosciviruses, based on polymerase chain reaction sequence analysis of 2 segments of the viral genome. In addition to ascites, widespread visceral edema, petechiae, and capillary damage previously reported, important findings with EEHV3 infection were the presence of grossly visible renal medullary hemorrhage, a tropism for larger veins and arteries in various tissues, relatively high density of renal herpetic inclusions, and involvement of the retinal vessels. These findings indicate a less selective organ tropism, and this may confer a higher degree of virulence for EEHV3

Gobush, K., Kerr, B., Wasser, S., 2009. Genetic relatedness and disrupted social structure in a poached population of African elephants
110. Mol. Ecol. 18, 722-734.
Abstract: We use genetic measures of relatedness and observations of female bonding to examine the demographic signature of historically heavy poaching of a population of free-ranging African elephants. We collected dung samples to obtain DNA and observed behaviour from 102 elephant families over a 25-month period in 2003-2005 in Mikumi National Park, Tanzania. Poaching reduced the population by 75% in the decade prior to the 1989 ivory trade ban; park records indicate that poaching dropped significantly in Mikumi following the ban. Using 10 microsatellite loci, DNA was genotyped in 203 elephants and pair-wise relatedness was calculated among adult females within and between groups. The Mikumi population is characterized by small group size, considerable variation in group relatedness, females with no first-order adult relatives and females that form only weak social bonds. We used gene-drop analysis and a model of a genetically intact pedigree to compare our observed Mikumi group relatedness to a simulated genetically intact unpoached expectation. The majority of groups in Mikumi contain 2 to 3 adults; of these, 45% were classified as genetically disrupted. Bonding, quantified with a pair-wise association index, was significantly correlated with relatedness; however only half of the females formed strong bonds with other females, and relatedness was substantially lower for a given bond strength as compared to an unpoached population. Female African elephants without kin demonstrated considerable behavioural plasticity in this disturbed environment, grouping with other females lacking kin, with established groups, or remaining alone, unable to form any stable adult female-bonds. We interpret these findings as the remaining effect of poaching disturbance in Mikumi, despite a drop in the level of poaching since the commercial trade in ivory was banned 15 years ago

Grus, W.E., Zhang, J., 2009. Origin of the genetic components of the vomeronasal system in the common ancestor of all extant vertebrates. Molecular Biology and Evolution 26, 407-419.
Abstract: Comparative genomics provides a valuable tool for inferring the evolutionary history of physiological systems, particularly when this information is difficult to ascertain by morphological traits. One such example is the vomeronasal system (VNS), a vertebrate nasal chemosensory system that is responsible for detecting intraspecific pheromonal cues as well as environmental odorants. The morphological components of the VNS are found only in tetrapods, but the genetic components of the system have been found in teleost fish, in addition to tetrapods. To determine when the genetic components of the VNS originated, we searched for the VNS-specific genes in the genomes of two early diverging vertebrate lineages: the sea lamprey from jawless fishes and the elephant shark from cartilaginous fishes. Genes encoding vomeronasal type 1 receptors (V1Rs) and Trpc2, two components of the vomeronasal signaling pathway, are present in the sea lamprey genome, and both are expressed in the olfactory organ, revealing that the genetic components of the present-day VNS existed in the common ancestor of all extant vertebrates. Additionally, all three VNS genes, Trpc2, V1Rs, and vomeronasal type 2 receptors (V2Rs), are found in the elephant shark genome. Because V1Rs and V2Rs are related to two families of taste receptors, we also searched the early diverging vertebrate genomes for taste system genes and found them in the shark genome but not in the lamprey. Coupled with known distributions of the genetic components of the vertebrate main olfactory system, our results suggest staggered origins of vertebrate sensory systems. These findings are important for understanding the evolution of vertebrate sensory systems and illustrate the utility of the genome sequences of early diverging vertebrates for uncovering the evolution of vertebrate-specific traits

Hermes, R., Behr, B., Hildebrandt, T.B., Blottner, S., Sieg, B., Frenzel, A., Knieriem, A., Saragusty, J., Rath, D., 2009. Sperm sex-sorting in the Asian elephant (Elephas maximus). Anim Reprod. Sci. 112, 390-396.
Abstract: In captive Asian elephants, there is a strong need for production of female offspring to enhance reproduction, counter premature aging processes in female animals and reduce challenging management situations derived from husbandry of several bulls in one institution. Artificial insemination of flow cytometrically sex-sorted spermatozoa offers the possibility to predetermine the sex of offspring with high accuracy. The aims of this study were to determine a suitable semen extender and basic parameters for flow cytometrical sex-sorting of Asian elephant spermatozoa. In total 18 semen samples were collected by manual rectal stimulation from one bull. Sperm quality parameters and sex sortability of spermatozoa were evaluated after dilution in three semen extenders (MES-HEPES-skim milk, MES-HEPES, TRIS-citric acid) and DNA staining. MES-HEPES-skim milk was the only semen extender found suitable to sex Asian elephant spermatozoa. From 18 ejaculates collected, 12 were successfully sorted with a purity of 94.5+/-0.7% at an average sort rate of 1945.5+/-187.5 spermatozoa per second. Sperm integrity, progressive and total motility were 42.6+/-3.9%, 48.1+/-3.3%, 59.4+/-3.8% after DNA labelling, and 64.8+/-3.2%, 58.0+/-5.0%, 70.8+/-4.4% after sorting, respectively. After liquid storage of sorted spermatozoa for 12h at 4 degrees C, sperm integrity, progressive and total motility were 46.4+/-5.2%, 32.2+/-4.2% and 58.2+/-3.9%, respectively. The obtained results provide a promising base to inseminate Asian elephants with sexed semen

Landolfi, J.A., Schultz, S.A., Mikota, S.K., Terio, K.A., 2009. Development and validation of cytokine quantitative, real time RT-PCR assays for characterization of Asian elephant immune responses
71. Vet. Immunol. Immunopathol. 131, 73-78.
Abstract: Infectious disease is an important factor in Asian elephant health and long-term species survival. In studying disease pathogenesis, it is important to consider not only the pathogen, but also the effectiveness of the host immune response. Currently, there is a paucity of information available on elephant immune function. Measurement of cytokine levels within clinical samples can provide valuable information regarding immune function during health and disease that may elucidate disease susceptibility. To develop tools for assessment of elephant immune function, Asian elephant partial mRNA sequences for interleukin (IL)-2, IL-4, IL-10, IL-12, interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, transforming growth factor (TGF)-beta, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and beta-actin were determined. Sequence information was then utilized to design elephant-specific primers and probes for quantitative, real time, RT-PCR assays for the measurement of cytokine mRNA. Greater than 300bps of Asian elephant mRNA sequence were determined for each cytokine of interest. Consistent and reproducible, real time, RT-PCR assays with efficiencies of greater than 93% were also developed. Assay sensitivities ranged from less than 1 to 5000 DNA copies with the exception of IL-12, which had a sensitivity of 42,200 copies. Employment of molecular techniques utilizing mRNA-based detection systems, such as real time, RT-PCR, facilitate sensitive and specific cytokine detection and measurement in samples from species for which commercial reagents are not available. Future studies utilizing these techniques to compare elephant immune function during health and in the face of infection will be useful for characterizing the contribution of the elephant immune system to disease

Lee, J.C., Hsieh, H.M., Huang, L.H., Kuo, Y.C., Wu, J.H., Chin, S.C., Lee, A.H., Linacre, A., Tsai, L.C., 2009. Ivory identification by DNA profiling of cytochrome b gene. Int. J. Legal Med. 123, 117-121.
Abstract: Ivory can be visually identified in its native form as coming from an elephant species; however, determining from which of the three extant elephant species a section of ivory originates is more problematic. We report on a method that will identify and distinguish the protected and endangered elephant species, Elephas maximus or Loxodonta sp. To identify the species of elephant from ivory products, we developed three groups of nested PCR amplifications within the cytochrome b gene that generate amplification products using highly degraded DNA isolated from confiscated ivory samples dating from 1995. DNA from a total of 382 out of 453 ivory samples were successfully isolated and amplified leading to species identification. All sequences were searched against GenBank and found to match with E. maximus and Loxodonta sp. with at least 99% similarity. The samples that were tested came from eight Asian elephants, 14 African forest elephants (Loxodonta cyclotis), and 360 African savannah elephants (Loxodonta africana). This study demonstrates a high success rate in species identification of ivory by a nested PCR approach within the cytochrome b gene which provides the necessary information for the protection of endangered species conservation

Lei, R., Brenneman, R.A., Schmitt, D.L., Louis, E.E., Jr., 2009. Detection of Cytonuclear Genomic Dissociation in the North American Captive African Elephant Collection. J. Hered.
Abstract: A total of 114 captive elephants (6 Asian; 108 African) from 43 private institutions or North American zoos accredited by the Association of Zoos and Aquariums were sampled and evaluated to investigate genetic status. Because previous analyses of the captive collection indicated potential cytonuclear dissociation between mitochondrial DNA (mtDNA) sequence and microsatellite nuclear DNA genotype data, we investigated this phenomenon within the captive collection with 2 X-linked genes (BGN and PHKA2) and 1 Y-linked gene (AMELY). These data reveal that individuals with forest-derived elephant mtDNA lineages carried only savannah elephant nuclear gene haplotypes. These results are concordant with a previous study of wild populations sampled across Africa, indicating that cytonuclear genomic dissociation was captured in the founders of the North American African elephant collection. These results are important for resolving questions that can potentially impact future management and breeding programs related to the collection

Looringh van Beeck, F.A., Reinink, P., Hermsen, R., Zajonc, D.M., Laven, M.J., Fun, A., Troskie, M., Schoemaker, N.J., Morar, D., Lenstra, J.A., Vervelde, L., Rutten, V.P., van, E.W., Van, R., I, 2009. Functional CD1d and/or NKT cell invariant chain transcript in horse, pig, African elephant and guinea pig, but not in ruminants
109. Mol. Immunol. 46, 1424-1431.
Abstract: CD1d-restricted invariant natural killer T cells (NKT cells) have been well characterized in humans and mice, but it is unknown whether they are present in other species. Here we describe the invariant TCR alpha chain and the full length CD1d transcript of pig and horse. Molecular modeling predicts that porcine (po) invariant TCR alpha chain/poCD1d/alpha-GalCer and equine (eq) invariant TCR alpha chain/eqCD1d/alpha-GalCer form complexes that are highly homologous to the human complex. Since a prerequisite for the presence of NKT cells is the expression of CD1d protein, we performed searches for CD1D genes and CD1d transcripts in multiple species. Previously, cattle and guinea pig have been suggested to lack CD1D genes. The CD1D genes of European taurine cattle (Bos taurus) are known to be pseudogenes because of disrupting mutations in the start codon and in the donor splice site of the first intron. Here we show that the same mutations are found in six other ruminants: African buffalo, sheep, bushbuck, bongo, N'Dama cattle, and roe deer. In contrast, intact CD1d transcripts were found in guinea pig, African elephant, horse, rabbit, and pig. Despite the discovery of a highly homologous NKT/CD1d system in pig and horse, our data suggest that functional CD1D and CD1d-restricted NKT cells are not universally present in mammals

Murata, Y., Yonezawa, T., Kihara, I., Kashiwamura, T., Sugihara, Y., Nikaido, M., Okada, N., Endo, H., Hasegawa, M., 2009. Chronology of the extant African elephant species and case study of the species identification of the small African elephant with the molecular phylogenetic method
70. Gene 441, 176-186.
Abstract: Despite vigorous genetic studies of African elephants (Loxodonta africana and L. cyclotis) during the last decade, their evolutionary history is still obscure. Phylogenetic studies and coalescence time estimation using longer nucleotide sequence data from denser samplings are necessary to better understand the natural history of African elephants. Further, species identification among African elephants is sometimes very difficult using only the external morphological characteristics. This is a serious problem for making an adequate breeding plan in zoological gardens. In this paper, we investigated the continent-wide phylogeographical pattern of the African elephants and estimated the coalescence times among them. From these molecular data and geological evidence, we proposed an evolutionary scenario for the African elephants. We further demonstrated the effectiveness of molecular phylogenetic methods in species identification.

Opazo, J.C., Sloan, A.M., Campbell, K.L., Storz, J.F., 2009. Origin and ascendancy of a chimeric fusion gene: the beta/delta-globin gene of paenungulate mammals
84. Molecular Biology and Evolution 26, 1469-1478.
Abstract: The delta-globin gene (HBD) of eutherian mammals exhibits a propensity for recombinational exchange with the closely linked beta-globin gene (HBB) and has been independently converted by the HBB gene in multiple lineages. Here we report the presence of a chimeric beta/delta fusion gene in the African elephant (Loxodonta africana) that was created by unequal crossing-over between misaligned HBD and HBB paralogs. The recombinant chromosome that harbors the beta/delta fusion gene in elephants is structurally similar to the "anti-Lepore" duplication mutant of humans (the reciprocal exchange product of the hemoglobin Lepore deletion mutant). However, the situation in the African elephant is unique in that the chimeric beta/delta fusion gene supplanted the parental HBB gene and is therefore solely responsible for synthesizing the beta-chain subunits of adult hemoglobin. A phylogenetic survey of beta-like globin genes in afrotherian and xenarthran mammals revealed that the origin of the chimeric beta/delta fusion gene and the concomitant inactivation of the HBB gene predated the radiation of "Paenungulata," a clade of afrotherian mammals that includes three orders: Proboscidea (elephants), Sirenia (dugongs and manatees), and Hyracoidea (hyraxes). The reduced fitness of the human Hb Lepore deletion mutant helps to explain why independently derived beta/delta fusion genes (which occur on an anti-Lepore chromosome) have been fixed in a number of mammalian lineages, whereas the reciprocal delta/beta fusion gene (which occurs on a Lepore chromosome) has yet to be documented in any nonhuman mammal. This illustrates how the evolutionary fates of chimeric fusion genes can be strongly influenced by their recombinational mode of origin

Roca, A.L., Ishida, Y., Nikolaidis, N., Kolokotronis, S.O., Fratpietro, S., Stewardson, K., Hensley, S., Tisdale, M., Boeskorov, G., Greenwood, A.D., 2009. Genetic variation at hair length candidate genes in elephants and the extinct woolly mammoth. BMC. Evol. Biol. 9, 232.
Abstract: BACKGROUND: Like humans, the living elephants are unusual among mammals in being sparsely covered with hair. Relative to extant elephants, the extinct woolly mammoth, Mammuthus primigenius, had a dense hair cover and extremely long hair, which likely were adaptations to its subarctic habitat. The fibroblast growth factor 5 (FGF5) gene affects hair length in a diverse set of mammalian species. Mutations in FGF5 lead to recessive long hair phenotypes in mice, dogs, and cats; and the gene has been implicated in hair length variation in rabbits. Thus, FGF5 represents a leading candidate gene for the phenotypic differences in hair length notable between extant elephants and the woolly mammoth. We therefore sequenced the three exons (except for the 3' UTR) and a portion of the promoter of FGF5 from the living elephantid species (Asian, African savanna and African forest elephants) and, using protocols for ancient DNA, from a woolly mammoth. RESULTS: Between the extant elephants and the mammoth, two single base substitutions were observed in FGF5, neither of which alters the amino acid sequence. Modeling of the protein structure suggests that the elephantid proteins fold similarly to the human FGF5 protein. Bioinformatics analyses and DNA sequencing of another locus that has been implicated in hair cover in humans, type I hair keratin pseudogene (KRTHAP1), also yielded negative results. Interestingly, KRTHAP1 is a pseudogene in elephantids as in humans (although fully functional in non-human primates). CONCLUSION: The data suggest that the coding sequence of the FGF5 gene is not the critical determinant of hair length differences among elephantids. The results are discussed in the context of hairlessness among mammals and in terms of the potential impact of large body size, subarctic conditions, and an aquatic ancestor on hair cover in the Proboscidea

Rodriguez Delgado, C.L., Waters, P.D., Gilbert, C., Robinson, T.J., Graves, J.A., 2009. Physical mapping of the elephant X chromosome: conservation of gene order over 105 million years. Chromosome. Res.
Abstract: All therian mammals (eutherians and marsupials) have an XX female/XY male sex chromosome system or some variant of it. The X and Y evolved from a homologous pair of autosomes over the 166 million years since therian mammals diverged from monotremes. Comparing the sex chromosomes of eutherians and marsupials defined an ancient X conserved region that is shared between species of these mammalian clades. However, the eutherian X (and the Y) was augmented by a recent addition (XAR) that is autosomal in marsupials. XAR is part of the X in primates, rodents, and artiodactyls (which belong to the eutherian clade Boreoeutheria), but it is uncertain whether XAR is part of the X chromosome in more distantly related eutherian mammals. Here we report on the gene content and order on the X of the elephant (Loxodonta africana)-a representative of Afrotheria, a basal endemic clade of African mammals-and compare these findings to those of other documented eutherian species. A total of 17 genes were mapped to the elephant X chromosome. Our results support the hypothesis that the eutherian X and Y chromosomes were augmented by the addition of autosomal material prior to eutherian radiation. Not only does the elephant X bear the same suite of genes as other eutherian X chromosomes, but gene order appears to have been maintained across 105 million years of evolution, perhaps reflecting strong constraints posed by the eutherian X inactivation system

Saragusty, J., Hildebrandt, T.B., Behr, B., Knieriem, A., Kruse, J., Hermes, R., 2009. Successful cryopreservation of Asian elephant (Elephas maximus) spermatozoa. Anim Reprod. Sci. 115, 255-266.
Abstract: Reproduction in captive elephants is low and infant mortality is high, collectively leading to possible population extinction. Artificial insemination was developed a decade ago; however, it relies on fresh-chilled semen from just a handful of bulls with inconsistent sperm quality. Artificial insemination with frozen-thawed sperm has never been described, probably, in part, due to low semen quality after cryopreservation. The present study was designed with the aim of finding a reliable semen freezing protocol. Screening tests included freezing semen with varying concentrations of ethylene glycol, propylene glycol, trehalose, dimethyl sulfoxide and glycerol as cryoprotectants and assessing cushioned centrifugation, rapid chilling to suprazero temperatures, freezing extender osmolarity, egg yolk concentration, post-thaw dilution with cryoprotectant-free BC solution and the addition of 10% (v/v) of autologous seminal plasma. The resulting optimal freezing protocol uses cushioned centrifugation, two-step dilution with isothermal 285 m Osm/kg Berliner Cryomedium (BC) with final glycerol concentration of 7% and 16% egg yolk, and freezing in large volume by the directional freezing technique. After thawing, samples are diluted 1:1 with BC solution. Using this protocol, post-thaw evaluations results were: motility upon thawing: 57.2+/-5.4%, motility following 30 min incubation at 37 degrees C: 58.5+/-6.0% and following 3h incubation: 21.7+/-7.6%, intact acrosome: 57.1+/-5.2%, normal morphology: 52.0+/-5.8% and viability: 67.3+/-6.1%. With this protocol, good quality semen can be accumulated for future use in artificial inseminations when and where needed

Saragusty, J., Hermes, R., Goritz, F., Schmitt, D.L., Hildebrandt, T.B., 2009. Skewed birth sex ratio and premature mortality in elephants. Anim Reprod. Sci. 115, 247-254.
Abstract: Sex allocation theories predict equal offspring number of both sexes unless differential investment is required or some competition exists. Left undisturbed, elephants reproduce well and in approximately even numbers in the wild. We report an excess of males are born and substantial juvenile mortality occurs, perinatally, in captivity. Studbook data on captive births (CB, n=487) and premature deaths (PD, <5 years of age; n=164) in Asian and African elephants in Europe and North America were compared with data on Myanmar timber (Asian) elephants (CB, n=3070; PD, n=738). Growth in CB was found in three of the captive populations. A significant excess of male births occurred in European Asian elephants (ratio: 0.61, P=0.044) and in births following artificial insemination (0.83, P=0.003), and a numerical inclination in North American African elephants (0.6). While juvenile mortality in European African and Myanmar populations was 21-23%, it was almost double (40-45%) in all other captive populations. In zoo populations, 68-91% of PD were within 1 month of birth with stillbirth and infanticide being major causes. In Myanmar, 62% of juvenile deaths were at >6 months with maternal insufficient milk production, natural hazards and accidents being the main causes. European Asian and Myanmar elephants PD was biased towards males (0.71, P=0.024 and 0.56, P<0.001, respectively). The skewed birth sex ratio and high juvenile mortality hinder efforts to help captive populations become self-sustaining. Efforts should be invested to identify the mechanism behind these trends and seek solutions for them.

Schwarz, C., Debruyne, R., Kuch, M., McNally, E., Schwarcz, H., Aubrey, A.D., Bada, J., Poinar, H., 2009. New insights from old bones: DNA preservation and degradation in permafrost preserved mammoth remains
89. Nucleic Acids Res. 37, 3215-3229.
Abstract: Despite being plagued by heavily degraded DNA in palaeontological remains, most studies addressing the state of DNA degradation have been limited to types of damage which do not pose a hindrance to Taq polymerase during PCR. Application of serial qPCR to the two fractions obtained during extraction (demineralization and protein digest) from six permafrost mammoth bones and one partially degraded modern elephant bone has enabled further insight into the changes which endogenous DNA is subjected to during diagenesis. We show here that both fractions exhibit individual qualities in terms of the prevailing type of DNA (i.e. mitochondrial versus nuclear DNA) as well as the extent of damage, and in addition observed a highly variable ratio of mitochondrial to nuclear DNA among the six mammoth samples. While there is evidence suggesting that mitochondrial DNA is better preserved than nuclear DNA in ancient permafrost samples, we find the initial DNA concentration in the bone tissue to be as relevant for the total accessible mitochondrial DNA as the extent of DNA degradation post-mortem. We also evaluate the general applicability of indirect measures of preservation such as amino-acid racemization, bone crystallinity index and thermal age to these exceptionally well-preserved samples

Schwarz, C., Debruyne, R., Kuch, M., McNally, E., Schwarcz, H., Aubrey, A.D., Bada, J., Poinar, H., 2009. New insights from old bones: DNA preservation and degradation in permafrost preserved mammoth remains. Nucleic Acids Res March 24.

Sherwood, C.C., Stimpson, C.D., Butti, C., Bonar, C.J., Newton, A.L., Allman, J.M., Hof, P.R., 2009. Neocortical neuron types in Xenarthra and Afrotheria: implications for brain evolution in mammals. Brain Struct. Funct. 213, 301-328.
Abstract: Interpreting the evolution of neuronal types in the cerebral cortex of mammals requires information from a diversity of species. However, there is currently a paucity of data from the Xenarthra and Afrotheria, two major phylogenetic groups that diverged close to the base of the eutherian mammal adaptive radiation. In this study, we used immunohistochemistry to examine the distribution and morphology of neocortical neurons stained for nonphosphorylated neurofilament protein, calbindin, calretinin, parvalbumin, and neuropeptide Y in three xenarthran species-the giant anteater (Myrmecophaga tridactyla), the lesser anteater (Tamandua tetradactyla), and the two-toed sloth (Choloepus didactylus)-and two afrotherian species-the rock hyrax (Procavia capensis) and the black and rufous giant elephant shrew (Rhynchocyon petersi). We also studied the distribution and morphology of astrocytes using glial fibrillary acidic protein as a marker. In all of these species, nonphosphorylated neurofilament protein-immunoreactive neurons predominated in layer V. These neurons exhibited diverse morphologies with regional variation. Specifically, high proportions of atypical neurofilament-enriched neuron classes were observed, including extraverted neurons, inverted pyramidal neurons, fusiform neurons, and other multipolar types. In addition, many projection neurons in layers II-III were found to contain calbindin. Among interneurons, parvalbumin- and calbindin-expressing cells were generally denser compared to calretinin-immunoreactive cells. We traced the evolution of certain cortical architectural traits using phylogenetic analysis. Based on our reconstruction of character evolution, we found that the living xenarthrans and afrotherians show many similarities to the stem eutherian mammal, whereas other eutherian lineages display a greater number of derived traits

Thongtip, N., Mahasawangkul, S., Thitaram, C., Pongsopavijitr, P., Kornkaewrat, K., Pinyopummin, A., Angkawanish, T., Jansittiwate, S., Rungsri, R., Boonprasert, K., Wongkalasin, W., Homkong, P., Dejchaisri, S., Wajjwalku, W., Saikhun, K., 2009. Successful artificial insemination in the Asian elephant (Elephas maximus) using chilled and frozen-thawed semen. Reprod. Biol. Endocrinol. 7, 75.
Abstract: BACKGROUND: Artificial insemination (AI) using frozen-thawed semen is well established and routinely used for breeding in various mammalian species. However, there is no report of the birth of elephant calves following AI with frozen-thawed semen. The objective of the present study was to investigate the fertilizing ability of chilled and frozen-thawed semen in the Asian elephant following artificial insemination (AI). METHODS: Semen samples were collected by from 8 bulls (age range, 12-to 42-years) by manual stimulation. Semen with high quality were either cooled to 4 degrees C or frozen in liquid nitrogen (-196 degrees C) before being used for AI. Blood samples collected from ten elephant females (age range, 12-to 52-years) were assessed for estrus cycle and elephants with normal cycling were used for AI. Artificial insemination series were conducted during 2003 to 2008; 55 and 2 AI trials were conducted using frozen-thawed and chilled semen, respectively. Pregnancy was detected using transrectal ultrasonography and serum progestagen measurement. RESULTS: One female (Khod) inseminated with chilled semen became pregnant and gave birth in 2007. The gestation length was 663 days and the sex of the elephant calf was male. One female (Sao) inseminated with frozen-thawed semen showed signs of pregnancy by increasing progestagen levels and a fetus was observed for 5 months by transrectal ultrasonography. CONCLUSION: This is the first report showing pregnancy following AI with frozen-thawed semen in the Asian elephant. Successful AI in the Asian elephant using either chilled or frozen-thawed semen is a stepping stone towards applying this technology for genetic improvement of the elephant population.

Vidya, T.N., Sukumar, R., Melnick, D.J., 2009. Range-wide mtDNA phylogeography yields insights into the origins of Asian elephants. Proc. Biol. Sci. 276, 893-902.
Abstract: Recent phylogeographic studies of the endangered Asian elephant (Elephas maximus) reveal two highly divergent mitochondrial DNA (mtDNA) lineages, an elucidation of which is central to understanding the species's evolution. Previous explanations for the divergent clades include introgression of mtDNA haplotypes between ancestral species, allopatric divergence of the clades between Sri Lanka or the Sunda region and the mainland, historical trade of elephants, and retention of divergent lineages due to large population sizes. However, these studies lacked data from India and Myanmar, which host approximately 70 per cent of all extant Asian elephants. In this paper, we analyse mtDNA sequence data from 534 Asian elephants across the species's range to explain the current distribution of the two divergent clades. Based on phylogenetic reconstructions, estimates of times of origin of clades, probable ancestral areas of origin inferred from dispersal-vicariance analyses and the available fossil record, we believe both clades originated from Elephas hysudricus. This probably occurred allopatrically in different glacial refugia, the alpha clade in the Myanmar region and the beta clade possibly in southern India-Sri Lanka, 1.6-2.1Myr ago. Results from nested clade and dispersal-vicariance analyses indicate a subsequent isolation and independent diversification of the beta clade in both Sri Lanka and the Sunda region, followed by northward expansion of the clade. We also find more recent population expansions in both clades based on mismatch distributions. We therefore suggest a contraction-expansion scenario during severe climatic oscillations of the Quaternary, with range expansions from different refugia during warmer interglacials leading to the varying geographical overlaps of the two mtDNA clades. We also demonstrate that trade in Asian elephants has not substantially altered the species's mtDNA population genetic structure

Wallis, M., 2009. Prolactin in the Afrotheria: characterization of genes encoding prolactin in elephant (Loxodonta africana), hyrax (Procavia capensis) and tenrec (Echinops telfairi). J. Endocrinol. 200, 233-240.
Abstract: Pituitary prolactin shows an episodic pattern of molecular evolution, with occasional short bursts of rapid change imposed on a generally rather slow evolutionary rate. In mammals, episodes of rapid change occurred in the evolution of primates, cetartiodactyls, rodents and the elephant. The bursts of rapid evolution in cetartiodactyls and rodents were followed by duplications of the prolactin gene that gave rise to large families of prolactin-related proteins including placental lactogens, while in primates the burst was followed by corresponding duplications of the related GH gene. The position in elephant is less clear. Extensive data relating to the genomic sequences of elephant and two additional members of the group Afrotheria are now available, and have been used here to characterize the prolactin genes in these species and explore whether additional prolactin-related genes are present. The results confirm the rapid evolution of elephant (Loxodonta africana) prolactin - the sequence of elephant prolactin is substantially different from that predicted for the ancestral placental mammal. Hyrax (Procavia capensis) prolactin is even more divergent but tenrec (Echinops telfairi) prolactin is strongly conserved. No evidence was obtained from searches of public databases for additional genes encoding prolactin-like proteins in any of these species. Detailed analysis of evolutionary rates, and other factors, indicates that the episode of rapid change in hyrax, and probably elephant, was adaptive, though the nature of the associated biological change(s) is not clear

Wasser, S.K., Clark, B., Laurie, C., 2009. The ivory trail
42. Scientific American 301, 68-74, 76.

Wittemyer, G., Okello, J.B., Rasmussen, H.B., Arctander, P., Nyakaana, S., Douglas-Hamilton, I., Siegismund, H.R., 2009. Where sociality and relatedness diverge: the genetic basis for hierarchical social organization in African elephants. Proc Royal Soc Biol 276, 3513-3521.
Abstract: Hierarchical properties characterize elephant fission-fusion social organization whereby stable groups of individuals coalesce into higher order groups or split in a predictable manner. This hierarchical complexity is rare among animals and, as such, an examination of the factors driving its emergence offers unique insight into the evolution of social behaviour. Investigation of the genetic basis for such social affiliation demonstrates that while the majority of core social groups (second-tier affiliates) are significantly related, this is not exclusively the case. As such, direct benefits received through membership of these groups appear to be salient to their formation and maintenance. Further analysis revealed that the majority of groups in the two higher social echelons (third and fourth tiers) are typically not significantly related. The majority of third-tier members are matrilocal, carrying the same mtDNA control region haplotype, while matrilocality among fourth-tier groups was slightly less than expected at random. Comparison of results to those from a less disturbed population suggests that human depredation, leading to social disruption, altered the genetic underpinning of social relations in the study population. These results suggest that inclusive fitness benefits may crystallize elephant hierarchical social structuring along genetic lines when populations are undisturbed. However, indirect benefits are not critical to the formation and maintenance of second-, third- or fourth-tier level bonds, indicating the importance of direct benefits in the emergence of complex, hierarchical social relations among elephants. Future directions and conservation implications are discussed

Wong, K., 2009. Decoding the mammoth
108. Scientific American 300, 26-27.

Archie, E.A., Maldonado, J.E., Hollister-Smith, J.A., Poole, J.H., Moss, C.J., Fleischer, R.C., Alberts, S.C., 2008. Fine-scale population genetic structure in a fission-fusion society. Mol. Ecol. 17, 2666-2679.
Abstract: Nonrandom patterns of mating and dispersal create fine-scale genetic structure in natural populations - especially of social mammals - with important evolutionary and conservation genetic consequences. Such structure is well-characterized for typical mammalian societies; that is, societies where social group composition is stable, dispersal is male-biased, and males form permanent breeding associations in just one or a few social groups over the course of their lives. However, genetic structure is not well understood for social mammals that differ from this pattern, including elephants. In elephant societies, social groups fission and fuse, and males never form permanent breeding associations with female groups. Here, we combine 33 years of behavioural observations with genetic information for 545 African elephants (Loxodonta africana), to investigate how mating and dispersal behaviours structure genetic variation between social groups and across age classes. We found that, like most social mammals, female matrilocality in elephants creates co-ancestry within core social groups and significant genetic differentiation between groups (Phi(ST) = 0.058). However, unlike typical social mammals, male elephants do not bias reproduction towards a limited subset of social groups, and instead breed randomly across the population. As a result, reproductively dominant males mediate gene flow between core groups, which creates cohorts of similar-aged paternal relatives across the population. Because poaching tends to eliminate the oldest elephants from populations, illegal hunting and poaching are likely to erode fine-scale genetic structure. We discuss our results and their evolutionary and conservation genetic implications in the context of other social mammals

Baker, C.S., 2008. A truer measure of the market: the molecular ecology of fisheries and wildlife trade. Mol. Ecol. 17, 3985-3998.
Abstract: Wildlife and fisheries markets are end-points in the supply chain of both legitimate and illegitimate or unregulated trade in species and natural products. Molecular ecology provides powerful tools for surveillance and estimation of this trade. Here, I review the application of these tools to market surveys and species in trade, including species identification and molecular taxonomy, population assignment and 'mixed-stock' analysis, genetic tracking and capture-recapture by individual identification. I consider the analogy of markets to natural populations and also the unique features that require novel analytical approaches and sampling design. In the most developed of these applications, the molecular ecology of market surveys and confiscated trade shipments has provided independent estimates of illegal, unregulated or unreported exploitation for sharks, elephants and whales. Although each study has taken advantage of information from trade records or official government reports concerning the ostensible levels of exploitation, it is telling that the truer measure of exploitation seems to arise from the market end-point of the supply chain

Bechert, U., Southern, S., Chase, M. Minimally invasive molecular health analysis in elephants. Proc American Associaton of Zoo Veterinarians and Assoc of Reptile and Amphibian Veterinarians.  88. 2008. 11-10-2008.
Ref Type: Conference Proceeding
Abstract: This paper describes the application of a new assay platform called Stress Response Profiling (SRP) to the analysis of health status in elephants. SRP assays use a large biomarker panel as an indicator of chronically perturbed physiologic homeostasis ("chronic stress"),1,2 which is a known predictor of increased morbidity, infertility and mortality rates.3-8 SRP assays have a broad-based sensitivity to diverse types of stressors in multiple species of vertebrates.2 A minimally invasive SRP assay is based on skin microsamples obtained using routine biopsy procedures.9 The skin SRP assay was applied to captive African elephants with clinically diagnosed gastrointestinal infections and to healthy wild elephants.10 The elephant health status was classified using a reference database of SR biomarker profiles corresponding to eight species of normal and stressed animals. The biomarker profiles were converted into pathway profiles indicating that the molecular mechanism of the elephant gastrointestinal infections preferentially involved responses to misfolded proteins and DNA lesions. To rapidly and economically screen samples from 70 free-ranging African elephants sampled in Northern Botswana, we used a multiplexed SRP assay called multi-SRP.1,2 Statistical analysis of the multi-SRP scores showed correlations with population density, movements, and human-elephant conflict reports. In
summary, this paper documents that SRP and multi-SRP assays are suitable for the elephant skin and relevant to both symptomatic diseases and asymptomatic effects of environmental and anthropogenic stressors. We anticipate that the SRP technology might have a wide range of potential applications in veterinary medicine and ecosystem conservation.
LITERATURE CITED
1. Southern, S.O., A.C. Allen, and N. Kellar. 2002. Molecular signature of physiological stress in dolphins based on protein expression profiling of skin. Administrative Report LJ-02-27, National Marine Fisheries Service, SW Fisheries Science Center, La Jolla, California.
2. Southern, S.O., and G.W. Lilienthal. 2008. New technology for early detection of health threats. Proc. SPIE 69450F.
3. Camougrand, N., and M. Rigoulet. 2001. Aging and oxidative stress: studies of some genes involved both in aging and in response to oxidative stress. Respir. Physiol. 128:393-40.
4. Epel, E.S., J. Lin, F.H. Wilhelm, O.M. Wolkowitz, R. Cawthon, N.E. Adler, C. Dolbier, W.B. Mendes, and E.H. Blackburn. 2006. Cell aging in relation to stress arousal and cardiovascular disease risk factors. Psychoneuroendocrinology. 31:277-87.
5. Feder, M.E., and G.E. Hofmann. 1999. Heat-shock proteins, molecular chaperones, and the stress response: evolutionary and ecological physiology. Ann. Rev. Physiol. 61:243-82.
6. Kapahi, P., M.E. Boulton, and T.B.L. Kirkwood. 1999. Positive correlation between mammalian life span and cellular resistance to stress. Free Radical Biol. Med. 26:495-500.
7. Selye, H.A. 1936. Syndrome produced by diverse nocuous agents. Nature 138:32.
8. Wilson, J.F., and E.J. Kopitzke 2002. Stress and infertility Curr. Womens Health Rep. 2: 194

Gilbert, C., Pace, J.K., Waters, P.D., 2008. Target site analysis of RTE1_LA and its AfroSINE partner in the elephant genome. Gene 425 , 1-8.
Abstract: SINEs retrotranspose using their partner LINE's enzymatic machinery. It has recently been proposed that AfroSINEs ending with GGTTT 3' tandem repeats were mobilized by RTE elements ending with CAA 3' tandem repeats in the Afrotherian genome. Using sequences from the elephant genome, we show that AfroSINEs derive from RTE ending with GGTTT-like 3' tandem repeats, a subgroup of RTE1_LA that only reached low copy number, and confirm that they were most likely mobilized by RTE ending with CAA(n) tandem repeats (RTE1_LA-CAA(n)). This partnership is supported by sequence similarity between two regions of the elements, overlap in the timing of their activity, common features of their target site consensus that are not shared by other members of the RTE family, and their high copy number. Detailed analyses of pre-insertion loci reveal that like many other apurinic/apyrimidinic endonuclease encoding elements, RTE1_LA-CAA(n) shows loose target site specificity. In addition, the RTE1_LA-CAA(n) target site consensus shares several structural and primary sequence features with that of LINE1, suggesting that these two elements share close functional similarity in the target primed reverse transcription (TPRT) reaction. Interestingly, although globally similar, the target site consensus of AfroSINE(Anc) and RTE1_LA-CAA(n) differ in several aspects. These differences, not observed among all SINE/LINE pairs so far examined, are most likely due to the fact that AfroSINEs and RTE1_LA-CAA(n) are terminated by a different tandem repeat motif. We propose that these differences reflect constraints imposed by base pairing interactions between the mRNA 3' terminal tandem repeats and the target DNA at the onset of TPRT. So in addition to the endonuclease nicking preference, the mRNA of these elements appears to play an important role in integration site choice through a passive, post-nicking, selective process

Gilbert, M.T., Drautz, D.I., Lesk, A.M., Ho, S.Y., Qi, J., Ratan, A., Hsu, C.H., Sher, A., Dalen, L., Gotherstrom, A., Tomsho, L.P., Rendulic, S., Packard, M., Campos, P.F., Kuznetsova, T.V., Shidlovskiy, F., Tikhonov, A., Willerslev, E., Iacumin, P., Buigues, B., Ericson, P.G., Germonpre, M., Kosintsev, P., Nikolaev, V., Nowak-Kemp, M., Knight, J.R., Irzyk, G.P., Perbost, C.S., Fredrikson, K.M., Harkins, T.T., Sheridan, S., Miller, W., Schuster, S.C., 2008. Intraspecific phylogenetic analysis of Siberian woolly mammoths using complete mitochondrial genomes. Proc. Natl. Acad. Sci. U. S. A 105, 8327-8332.
Abstract: We report five new complete mitochondrial DNA (mtDNA) genomes of Siberian woolly mammoth (Mammuthus primigenius), sequenced with up to 73-fold coverage from DNA extracted from hair shaft material. Three of the sequences present the first complete mtDNA genomes of mammoth clade II. Analysis of these and 13 recently published mtDNA genomes demonstrates the existence of two apparently sympatric mtDNA clades that exhibit high interclade divergence. The analytical power afforded by the analysis of the complete mtDNA genomes reveals a surprisingly ancient coalescence age of the two clades, approximately 1-2 million years, depending on the calibration technique. Furthermore, statistical analysis of the temporal distribution of the (14)C ages of these and previously identified members of the two mammoth clades suggests that clade II went extinct before clade I. Modeling of protein structures failed to indicate any important functional difference between genomes belonging to the two clades, suggesting that the loss of clade II more likely is due to genetic drift than a selective sweep

Gobush, K.S., Mutayoba, B.M., Wasser, S.K., 2008. Long-term impacts of poaching on relatedness, stress physiology, and reproductive output of adult female african elephants. Conserv. Biol. 22, 1590-1599.
Abstract: Widespread poaching prior to the 1989 ivory ban greatly altered the demographic structure of matrilineal African elephant (Loxodonta africana) family groups in many populations by decreasing the number of old, adult females. We assessed the long-term impacts of poaching by investigating genetic, physiological, and reproductive correlates of a disturbed social structure resulting from heavy poaching of an African elephant population in Mikumi National Park, Tanzania, prior to 1989. We examined fecal glucocorticoid levels and reproductive output among 218 adult female elephants from 109 groups differing in size, age structure, and average genetic relatedness over 25 months from 2003 to 2005. The distribution in group size has changed little since 1989, but the number of families with tusked old matriarchs has increased by 14.2%. Females from groups that lacked an old matriarch, first-order adult relatives, and strong social bonds had significantly higher fecal glucocorticoid values than those from groups with these features (all females R(2)= 0.31; females in multiadult groups R(2)= 0.46). Females that frequented isolated areas with historically high poaching risk had higher fecal glucocorticoid values than those in low poaching risk areas. Females with weak bonds and low group relatedness had significantly lower reproductive output (R(2)[U]=0.21). Females from disrupted groups, defined as having observed average group relatedness 1 SD below the expected mean for a simulated unpoached family, had significantly lower reproductive output than females from intact groups, despite many being in their reproductive prime. These results suggest that long-term negative impacts from poaching of old, related matriarchs have persisted among adult female elephants 1.5 decades after the 1989 ivory ban was implemented

Hofreiter, M., 2008. DNA sequencing: Mammoth genomics. Nature 456, 330-331.

Huang, S., 2008. Ancient fossil specimens of extinct species are genetically more distant to an outgroup than extant sister species are. Riv. Biol. 101, 93-108.
Abstract: There exists a remarkable correlation between genetic distance as measured by protein or DNA dissimilarity and time of species divergence as inferred from fossil records. This observation has provoked the molecular clock hypothesis. However, data inconsistent with the hypothesis have steadily accumulated in recent years from studies of extant organisms. Here the published DNA and protein sequences from ancient fossil specimens were examined to see if they would support the molecular clock hypothesis. The hypothesis predicts that ancient specimens cannot be genetically more distant to an outgroup than extant sister species are. Also, two distinct ancient specimens cannot be genetically more distant than their extant sister species are. The findings here do not conform to these predictions. Neanderthals are more distant to chimpanzees and gorillas than modern humans are. Dinosaurs are more distant to frogs than extant birds are. Mastodons are more distant to opossums than other placental mammals are. The genetic distance between dinosaurs and mastodons is greater than that between extant birds and mammals. Therefore, while the molecular clock hypothesis is consistent with some data from extant organisms, it has yet to find support from ancient fossils. Far more damaging to the hypothesis than data from extant organisms, which merely question the constancy of mutation rate, the study of ancient fossil organisms here challenges for the first time the fundamental premise of modern evolution theory that genetic distances had always increased with time in the past history of life on Earth

Jackson, T., van Aarde, R., 2008. CSI:Africa. Africa Geographic 16, 35-39.
Abstract: In 1989, prompted by the wholesale slaughter of elephants for their tusks, CITES (the Convention on International Trade in Endangered Species) implemented a worldwide ban on the trade in ivory. Nearly 20 years later, elephant populations in some parts of Africa have stabilised, some are even increasing, and yet, seizures of ivory destined for the black market continue. While imposing tougher punishments on poachers, middlemen and dealers is an obvious measure, first prize would be preventing the elephants from being killed in the first place. To do this effectively, however, you need to know which populations are at risk. So, how can we find out where all the ivory is coming from? The answers, Tim Jackson finds, range from the seemingly low-tech collection of elephant dung to the very latest developments in DNA analysis.

Konnai, S., Mekata, H., Odbileg, R., Simuunza, M., Chembensof, M., Witola, W.H., Tembo, M.E., Chitambo, H., Inoue, N., Onuma, M., Ohashi, K., 2008. Detection of Trypanosoma brucei in field-captured tsetse flies and identification of host species fed on by the infected flies. Vector. Borne. Zoonotic. Dis. 8, 565-573.
Abstract: The prevalence of trypanosome infections in tsetse flies in the Chiawa area of Lower Zambezi in Zambia, with endemic trypanosomosis, was determined by a polymerase chain reaction (PCR) method that allowed the detection of trypanosome DNA and determination of the type of animal host fed on by the tsetse fly Glossina pallidipes, using tsetse-derived DNA extracts as templates. Ninety G. pallidipes (82 females and 8 males; 18.3%) of the 492 flies captured by baited biconical traps tested positive for the presence of Trypanosoma brucei species genomic DNA. Of the 90 T. brucei-positive flies, 47 (52.2%) also tested positive for vertebrate mitochondrial DNA. Sequence analysis of the vertebrate mitochondrial DNA amplicons established that they originated from 8 different vertebrate species, namely, human (Homo sapiens), African elephant (Loxodonta cyclotis), African buffalo (Syncerus caffer), waterbuck (Kobus ellipsiprymnus), roan antelope (Hippotragus equinus), greater kudu (Tragelaphus strepsiceros), warthog (Phacochoerus africanus), and goat (Capra hircus). Furthermore, to investigate the prevalence of trypanosome infections in domestic goats in the same area where trypanosomes had been detected in tsetse files, a total of 86 goats were randomly selected from 6 different herds. Among the selected goats, 36 (41.9%) were found to be positive for T. brucei species. This combined detection method would be an ideal approach not only for mass screening for infection prevalence in tsetse populations, but also for the prediction of natural reservoirs in areas endemic for trypanosomosis

Kurth, A., Wibbelt, G., Gerber, H.P., Petschaelis, A., Pauli, G., Nitsche, A., 2008. Rat-to-elephant-to-human transmission of cowpox virus. Emerg. Infect. Dis. 14, 670-671.

Liu, A.G., Seiffert, E.R., Simons, E.L., 2008. Stable isotope evidence for an amphibious phase in early proboscidean evolution. Proc. Natl. Acad. Sci. U. S. A 105, 5786-5791.
Abstract: The order Proboscidea includes extant elephants and their extinct relatives and is closely related to the aquatic sirenians (manatees and dugongs) and terrestrial hyracoids (hyraxes). Some analyses of embryological, morphological, and paleontological data suggest that proboscideans and sirenians shared an aquatic or semiaquatic common ancestor, but independent tests of this hypothesis have proven elusive. Here we test the hypothesis of an aquatic ancestry for advanced proboscideans by measuring delta(18)O in tooth enamel of two late Eocene proboscidean genera, Barytherium and Moeritherium, which are sister taxa of Oligocene-to-Recent proboscideans. The combination of low delta(18)O values and low delta(18)O standard deviations in Barytherium and Moeritherium matches the isotopic pattern seen in aquatic and semiaquatic mammals, and differs from that of terrestrial mammals. delta(13)C values of these early proboscideans suggest that both genera are likely to have consumed freshwater plants, although a component of C(3) terrestrial vegetation cannot be ruled out. The simplest explanation for the combined evidence from isotopes, dental functional morphology, and depositional environments is that Barytherium and Moeritherium were at least semiaquatic and lived in freshwater swamp or riverine environments, where they grazed on freshwater vegetation. These results lend new support to the hypothesis that Oligocene-to-Recent proboscideans are derived from amphibious ancestors

Lotfy, W.M., Brant, S.V., DeJong, R.J., Le, T.H., Demiaszkiewicz, A., Rajapakse, R.P., Perera, V.B., Laursen, J.R., Loker, E.S., 2008. Evolutionary origins, diversification, and biogeography of liver flukes (Digenea, Fasciolidae). American Journal of Tropical Medicine and Hygiene 79, 248-255.
Abstract: Fasciolid flukes are among the largest and best known digenetic trematodes and have considerable historical and veterinary significance. Fasciola hepatica is commonly implicated in causing disease in humans. The origins, patterns of diversification, and biogeography of fasciolids are all poorly known. We have undertaken a molecular phylogenetic study using 28S, internal transcribed spacer 1 and 2 (ITS-1 and ITS-2) of nuclear ribosomal DNA, and mitochondrial nicotinamide dehydrogenase subunit 1 (nad1) that included seven of the nine recognized species in the family. The fasciolids examined comprise a monophyletic group with the most basal species recovered from African elephants. We hypothesize fasciolids migrated from Africa to Eurasia, with secondary colonization of Africa. Fasciolids have been conservative in maintaining relatively large adult body size, but anatomical features of their digestive and reproductive systems are available. These flukes have been opportunistic, with respect to switching to new snail (planorbid to lymnaeid) and mammalian hosts and from intestinal to hepatic habitats within mammals

Lynch, V.J., Tanzer, A., Wang, Y., Leung, F.C., Gellersen, B., Emera, D., Wagner, G.P., 2008. Adaptive changes in the transcription factor HoxA-11 are essential for the evolution of pregnancy in mammals. Proc. Natl. Acad. Sci. U. S. A 105, 14928-14933.
Abstract: Evolutionary change in gene regulation can result from changes in cis-regulatory elements, leading to differences in the temporal and spatial expression of genes or in the coding region of transcription factors leading to novel functions or both. Although there is a growing body of evidence supporting the importance of cis-regulatory evolution, examples of protein-mediated evolution of novel developmental pathways have not been demonstrated. Here, we investigate the evolution of prolactin (PRL) expression in endometrial cells, which is essential for placentation/pregnancy in eutherian mammals and is a direct regulatory target of the transcription factor HoxA-11. Here, we show that (i) endometrial PRL expression is a derived feature of placental mammals, (ii) the PRL regulatory gene HoxA-11 experienced a period of strong positive selection in the stem-lineage of eutherian mammals, and (iii) only HoxA-11 proteins from placental mammals, including the reconstructed ancestral eutherian gene, are able to up-regulate PRL from the promoter used in endometrial cells. In contrast, HoxA-11 from the reconstructed therian ancestor, opossum, platypus, and chicken are unable to up-regulate PRL expression. These results demonstrate that the evolution of novel gene expression domains is not only mediated by the evolution of cis-regulatory elements but can also require evolutionary changes of transcription factor proteins themselves

Mekata, H., Konnai, S., Simuunza, M., Chembensofu, M., Kano, R., Witola, W.H., Tembo, M.E., Chitambo, H., Inoue, N., Onuma, M., Ohashi, K., 2008. Prevalence and source of trypanosome infections in field-captured vector flies (Glossina pallidipes) in southeastern Zambia. J. Vet. Med. Sci. 70, 923-928.
Abstract: The prevalence of trypanosome infections in tsetse flies, Glossina pallidipes, collected from Chiawa and Chakwenga in Zambia with endemic trypanosomosis was assessed by polymerase chain reaction (PCR). Out of the 550 G. pallidipes, 58 (10.5%) flies were found to harbor trypanosome DNA. Infection rates of tsetse with Trypanosoma vivax universal, Trypanosoma congolense savannah, T. congolense forest and T. congolense kilifi were 4.2% (23/550), 4.7% (26/550), 1.1% (6/550) and 1.6% (9/550), respectively. To determine the mammalian hosts of T. congolense and T. vivax infections from the tsetse flies, mammalian mitochondrion DNA of blood meal in these flies were analyzed by PCR and subsequent gene sequence analysis of the amplicons. Sequence analysis showed the presence of cytochrome b gene (cyt b) of 7 different mammalian species such as human, elephant, buffalo, goat, warthog, greater kudu and cattle. Goats which were main livestock in these areas were further examined to know the extent of its contribution in spreading the infection. We examined the prevalence of trypanosome infections in the domestic goat population in 6 settlements in Chiawa alone. Of the 86 goats sampled, 4 (4.6%), 5 (5.8%), 4 (4.6%) and 4 (4.6%) were positive for T. vivax universal, T. congolense savannah, forest and kilifi, respectively. These findings showed that the host-source of trypanosome infections in vector fly give a vital information about spread of infection. The result of this study will certainly contribute in elucidating more the epidemiology of trypanosomosis

Miller, W., Drautz, D.I., Ratan, A., Pusey, B., Qi, J., Lesk, A.M., Tomsho, L.P., Packard, M.D., Zhao, F., Sher, A., Tikhonov, A., Raney, B., Patterson, N., Lindblad-Toh, K., Lander, E.S., Knight, J.R., Irzyk, G.P., Fredrikson, K.M., Harkins, T.T., Sheridan, S., Pringle, T., Schuster, S.C., 2008. Sequencing the nuclear genome of the extinct woolly mammoth. Nature 456, 387-390.
Abstract: In 1994, two independent groups extracted DNA from several Pleistocene epoch mammoths and noted differences among individual specimens. Subsequently, DNA sequences have been published for a number of extinct species. However, such ancient DNA is often fragmented and damaged, and studies to date have typically focused on short mitochondrial sequences, never yielding more than a fraction of a per cent of any nuclear genome. Here we describe 4.17 billion bases (Gb) of sequence from several mammoth specimens, 3.3 billion (80%) of which are from the woolly mammoth (Mammuthus primigenius) genome and thus comprise an extensive set of genome-wide sequence from an extinct species. Our data support earlier reports that elephantid genomes exceed 4 Gb. The estimated divergence rate between mammoth and African elephant is half of that between human and chimpanzee. The observed number of nucleotide differences between two particular mammoths was approximately one-eighth of that between one of them and the African elephant, corresponding to a separation between the mammoths of 1.5-2.0 Myr. The estimated probability that orthologous elephant and mammoth amino acids differ is 0.002, corresponding to about one residue per protein. Differences were discovered between mammoth and African elephant in amino-acid positions that are otherwise invariant over several billion years of combined mammalian evolution. This study shows that nuclear genome sequencing of extinct species can reveal population differences not evident from the fossil record, and perhaps even discover genetic factors that affect extinction

Nicholls, H., 2008. Darwin 200: Let's make a mammoth. Nature 456, 310-314.

Nishihara, H., Okada, N., 2008. Retroposons: genetic footprints on the evolutionary paths of life. Methods Mol. Biol. 422, 201-225.
Abstract: Retroposons such as short interspersed elements (SINEs) and long interspersed elements are abundant transposable elements in eukaryote genomes. Recent large-scale comparative genome analyses have revealed that retroposons are a major component of genomes, wherein they provide structural diversity between species and uniqueness to each species. SINEs have been used as powerful markers in phylogenetic analyses of various species. This approach, which has been termed the SINE insertion method, infers phylogenetic relationships based on the presence/absence of SINEs among lineages. However, the method is not yet used extensively among biologists, especially molecular phylogenetists, because it is based on an understanding of the molecular mechanisms of retroposition, which may be unfamiliar to many researchers. Moreover, the method may require a large amount of bench work to characterize a new SINE family and to screen genomic libraries of the species of interest. In this chapter, we present the basic theory and detailed technical steps involved in a SINE insertion analysis. Furthermore, we explain the isolation and characterization of a new SINE family from the genome of a species of interest using as an example a known SINE family in mammals

Okello, J.B., Wittemyer, G., Rasmussen, H.B., Arctander, P., Nyakaana, S., Douglas-Hamilton, I., Siegismund, H.R., 2008. Effective population size dynamics reveal impacts of historic climatic events and recent anthropogenic pressure in African elephants. Mol. Ecol. 17, 3788-3799.
Abstract: Two hundred years of elephant hunting for ivory, peaking in 1970-1980s, caused local extirpations and massive population declines across Africa. The resulting genetic impacts on surviving populations have not been studied, despite the importance of understanding the evolutionary repercussions of such human-mediated events on this keystone species. Using Bayesian coalescent-based genetic methods to evaluate time-specific changes in effective population size, we analysed genetic variation in 20 highly polymorphic microsatellite loci from 400 elephants inhabiting the greater Samburu-Laikipia region of northern Kenya. This area experienced a decline of between 80% and 90% in the last few decades when ivory harvesting was rampant. The most significant change in effective population size, however, occurred approximately 2500 years ago during a mid-Holocene period of climatic drying in tropical Africa. Contrary to expectations, detailed analyses of four contemporary age-based cohorts showed that the peak poaching epidemic in the 1970s caused detectable temporary genetic impacts, with genetic diversity rebounding as juveniles surviving the poaching era became reproductively mature. This study demonstrates the importance of climatic history in shaping the distribution and genetic history of a keystone species and highlights the utility of coalescent-based demographic approaches in unravelling ancestral demographic events despite a lack of ancient samples. Unique insights into the genetic signature of mid-Holocene climatic change in Africa and effects of recent poaching pressure on elephants are discussed

Okello, J.B., Masembe, C., Rasmussen, H.B., Wittemyer, G., Omondi, P., Kahindi, O., Muwanika, V.B., Arctander, P., Douglas-Hamilton, I., Nyakaana, S., Siegismund, H.R., 2008. Population genetic structure of savannah elephants in Kenya: conservation and management implications. J. Hered. 99, 443-452.
Abstract: We investigated population genetic structure and regional differentiation among African savannah elephants in Kenya using mitochondrial and microsatellite markers. We observed mitochondrial DNA (mtDNA) nucleotide diversity of 1.68% and microsatellite variation in terms of average number of alleles, expected and observed heterozygosities in the total study population of 10.20, 0.75, and 0.69, respectively. Hierarchical analysis of molecular variance of mtDNA variation revealed significant differentiation among the 3 geographical regions studied (F(CT) = 0.264; P < 0.05) and a relatively lower differentiation among populations within regions (F(SC) = 0.218; P < 0.0001). Microsatellite variation significantly differentiated among populations within regions (F(SC) = 0.019; P < 0.0001) but not at the regional levels (F(CT) = 0.000; P > 0.500). We attribute the high differentiation at the mitochondrial genome to the matrilineal social structure of elephant populations, female natal philopatry, and probably ancient vicariance. Lack of significant regional differentiation at the nuclear loci vis-a-vis strong differences at mtDNA loci between regions is likely the effect of subsequent homogenization through male-mediated gene flow. Our results depicting 3 broad regional mtDNA groups and the observed population genetic differentiation as well as connectivity patterns should be incorporated in the planning of future management activities such as translocations

Organ, C.L., Schweitzer, M.H., Zheng, W., Freimark, L.M., Cantley, L.C., Asara, J.M., 2008. Molecular phylogenetics of mastodon and Tyrannosaurus rex. Science 320, 499.
Abstract: We report a molecular phylogeny for a nonavian dinosaur, extending our knowledge of trait evolution within nonavian dinosaurs into the macromolecular level of biological organization. Fragments of collagen alpha1(I) and alpha2(I) proteins extracted from fossil bones of Tyrannosaurus rex and Mammut americanum (mastodon) were analyzed with a variety of phylogenetic methods. Despite missing sequence data, the mastodon groups with elephant and the T. rex groups with birds, consistent with predictions based on genetic and morphological data for mastodon and on morphological data for T. rex. Our findings suggest that molecular data from long-extinct organisms may have the potential for resolving relationships at critical areas in the vertebrate evolutionary tree that have, so far, been phylogenetically intractable

Pacheco, J.M., Traulsen, A., Antal, T., Dingli, D., 2008. Cyclic neutropenia in mammals. Am. J. Hematol. 83, 920-921.
Abstract: Cyclic neutropenia (CN) has been well documented in humans and the gray collie. A recent model of the architecture and dynamics of hematopoiesis has been used to provide insights into the mechanism of cycling of this disorder. It provides a link between the cycling period and the cells where the mutated ELA2 is expressed. Assuming that the biologic defect in CN is the same in dogs, and the observation that the structure of hematopoiesis is invariant across mammals, we use allometric scaling techniques to correctly predict the period of cycling in the gray collie and extend it to other mammals from mice to elephants. This work provides additional support for the relevance of animal models to understand disease but cautions that disease dynamics in model animals are different and this has to be taken into consideration when planning experiments

Page, J.E., Murphy, W.J., 2008. Construction of radiation hybrid panels. Methods Mol. Biol. 422, 51-64.
Abstract: Whole-genome radiation hybrid (RH) mapping has proven to be a powerful tool for mapping genes and comparing genome architecture. We describe a protocol for constructing RH panels by rescuing irradiated fibroblast donor cells of any mammalian species by polyethylene glycol fusion to a thymidine kinase-deficient hamster cell line. Characterization and expansion of a panel of 90-100 cell lines can be used to map virtually any PCR-based marker that can be distinguished from the recipient hamster genome. The described procedure has been used successfully to create RH panels from diverse mammalian species such as macaques, elephants, alpacas, and armadillos, and may be applicable to nonmammalian vertebrates as well

Perelygin, A.A., Zharkikh, A.A., Astakhova, N.M., Lear, T.L., Brinton, M.A., 2008. Concerted evolution of vertebrate CCR2 and CCR5 genes and the origin of a recombinant equine CCR5/2 gene. J. Hered. 99, 500-511.
Abstract: Chemokine receptors (CCRs) play an essential role in the initiation of an innate immune host response. Several of these receptors have been shown to modulate the outcome of viral infections. The recent availability of complete genome sequences from a number of species provides a unique opportunity to analyze the evolution of the CCR genes. A phylogenetic analysis revealed that the CCR2 gene evolved in concert with the paralogous CCR5 gene, but not with another paralogous gene, CCR3, in the opossum, platypus, rabbit, guinea pig, cat, and rodent lineages. In addition, evidence of concerted evolution of the CCR2 and CCR5 genes was observed in chicken and lizard genomes. A unique CCR5/2 gene that originated by unequal crossing over between the CCR2 and CCR5 genes was detected in the domestic horse. The CCR2, CCR5, and CCR5/2 genes were mapped to ECA16q21 using fluorescent in situ hybridization (FISH). Single-nucleotide polymorphisms identified in the equine CCR5 gene and characterized within 5 horse breeds provide haplotype markers for future case/control studies investigating the genetic bases of horse susceptibility to infectious diseases

Thongtip, N., Saikhun, J., Mahasawangkul, S., Kornkaewrat, K., Pongsopavijitr, P., Songsasen, N., Pinyopummin, A., 2008. Potential factors affecting semen quality in the Asian elephant (Elephas maximus). Reprod. Biol. Endocrinol. 6, 9.
Abstract: BACKGROUND: One of the major obstacles in using artificial insemination to manage genetics of elephant population in captivity is the large variations in semen quality among ejaculates within the same and among individuals. The objectives of this study were to determine the influences of (1) age (2) seasonality (3) and circulating testosterone (SrTest), triiodothyronine (SrT3) and tetraiodothyronine (SrT4), as well as seminal (4) testosterone (SpTest), zinc (SpZn) and protein (SpTP) on semen quality in the Asian elephant METHODS: Analyses, including motility, viability and morphology were performed in semen samples collected twice monthly from 13 elephant bulls (age range, 10-to 72-years) by manual stimulation between July 2004 and June 2005. Serum samples obtained monthly were assessed for SrTest, SrT3, SrT4, and seminal plasma samples were evaluated for, SpTest, SpZn and SpTP. RESULTS: The highest semen quality was observed at age 23 to 43 years. Percentages of progressive motility and viable sperm were lowest at age 51 to 70 years (P < 0.05); on the other hand, sperm concentration was lowest at age 10 to 19 years (P < 0.05). Percentage of sperm with normal morphology was highest at age 23 to 43 years. The levels of SrT3, SrTest, SpTest and SpZn were lowest at age 51 to 70 years, whereas SrT4 was lowest at age 23 to 43 years. Seasonality significantly affected semen characteristics in which percentage of viable sperm and cell concentration were highest during rainy season and lowest during summer months (P < 0.05). However, percentage of sperm with normal morphology was highest in summer and lowest in rainy season (P < 0.05). Seasonality significantly influenced SrTest with elevated concentrations observed in rainy season and winter (P < 0.05). CONCLUSION: This study indicates that age and seasonality had influence on semen characteristics in the Asian elephant. The knowledge obtained in this study will improve our understanding of the reproductive biology of this species

von, A., I, Nimzyk, R., Klemke, M., Bullerdiek, J., 2008. A microRNA encoded in a highly conserved part of the mammalian HMGA2 gene. Cancer Genet. Cytogenet. 187, 43-44.
Abstract: The high mobility group protein HMGA2 plays an important role as a chromatin component of stem cells and as a protein causally related to the development of a variety of benign tumors (e.g., uterine leiomyomas, lipomas, and pleomorphic adenomas of the salivary glands). Herein, the existence of a highly conserved region within intron 3 of HMGA2 encoding a microRNA is described. The co-expression with HMGA2 suggests that as an intronic microRNA, this microRNA may cooperate with HMGA2 in its physiological and/or aberrant functions

Wallis, M., 2008. Mammalian genome projects reveal new growth hormone (GH) sequences. Characterization of the GH-encoding genes of armadillo (Dasypus novemcinctus), hedgehog (Erinaceus europaeus), bat (Myotis lucifugus), hyrax (Procavia capensis), shrew (Sorex araneus), ground squirrel (Spermophilus tridecemlineatus), elephant (Loxodonta africana), cat (Felis catus) and opossum (Monodelphis domestica). Gen. Comp Endocrinol. 155, 271-279.
Abstract: Mammalian growth hormone (GH) sequences have been shown previously to display episodic evolution: the sequence is generally strongly conserved but on at least two occasions during mammalian evolution (on lineages leading to higher primates and ruminants) bursts of rapid evolution occurred. However, the number of mammalian orders studied previously has been relatively limited, and the availability of sequence data via mammalian genome projects provides the potential for extending the range of GH gene sequences examined. Complete or nearly complete GH gene sequences for six mammalian species for which no data were previously available have been extracted from the genome databases-Dasypus novemcinctus (nine-banded armadillo), Erinaceus europaeus (western European hedgehog), Myotis lucifugus (little brown bat), Procavia capensis (cape rock hyrax), Sorex araneus (European shrew), Spermophilus tridecemlineatus (13-lined ground squirrel). In addition incomplete data for several other species have been extended. Examination of the data in detail and comparison with previously available sequences has allowed assessment of the reliability of deduced sequences. Several of the new sequences differ substantially from the consensus sequence previously determined for eutherian GHs, indicating greater variability than previously recognised, and confirming the episodic pattern of evolution. The episodic pattern is not seen for signal sequences, 5' upstream sequence or synonymous substitutions-it is specific to the mature protein sequence, suggesting that it relates to the hormonal function. The substitutions accumulated during the course of GH evolution have occurred mainly on the side of the hormone facing away from the receptor, in a non-random fashion, and it is suggested that this may reflect interaction of the receptor-bound hormone with other proteins or small ligands

Wasser, S.K., Joseph, C.W., Drori, O., Stephen, K.E., Mailand, C., Mutayoba, B., Stephens, M., 2008. Combating the illegal trade in African elephant ivory with DNA forensics. Conserv. Biol. 22, 1065-1071.
Abstract: International wildlife crime is burgeoning in this climate of global trade. We contend that the most effective way to contain this illegal trade is to determine where the wildlife is being removed. This allows authorities to direct law enforcement to poaching hot spots, potentially stops trade before the wildlife is actually killed, prevents countries from denying their poaching problems at home, and thwarts trade before it enters into an increasingly complex web of international criminal activity. Forensic tools have been limited in their ability to determine product origin because the information they can provide typically begins only at the point of shipment. DNA assignment analyses can determine product origin, but its use has been limited by the inability to assign samples to locations where reference samples do not exist. We applied new DNA assignment methods that can determine the geographic origin(s) of wildlife products from anywhere within its range. We used these methods to examine the geographic origin(s) of 2 strings of seizures involving large volumes of elephant ivory, 1 string seized in Singapore and Malawi and the other in Hong Kong and Cameroon. These ivory traffickers may comprise 2 of the largest poaching rings in Africa. In both cases all ivory seized in the string had common origins, which indicates that crime syndicates are targeting specific populations for intense exploitation. This result contradicts the dominant belief that dealers are using a decentralized plan of procuring ivory stocks as they became available across Africa. Large quantities of ivory were then moved, in multiple shipments, through an intermediate country prior to shipment to Asia, as a risk-reduction strategy that distances the dealer from the poaching locale. These smuggling strategies could not have been detected by forensic information, which typically begins only at the shipping source

Asara, J.M., Schweitzer, M.H., Freimark, L.M., Phillips, M., Cantley, L.C., 2007. Protein sequences from mastodon and Tyrannosaurus rex revealed by mass spectrometry. Science 316, 280-285.
Abstract: Fossilized bones from extinct taxa harbor the potential for obtaining protein or DNA sequences that could reveal evolutionary links to extant species. We used mass spectrometry to obtain protein sequences from bones of a 160,000- to 600,000-year-old extinct mastodon (Mammut americanum) and a 68-million-year-old dinosaur (Tyrannosaurus rex). The presence of T. rex sequences indicates that their peptide bonds were remarkably stable. Mass spectrometry can thus be used to determine unique sequences from ancient organisms from peptide fragmentation patterns, a valuable tool to study the evolution and adaptation of ancient taxa from which genomic sequences are unlikely to be obtained. Division of Signal Transduction, Beth Israel Deaconess Medical Center, Boston, MA 02115, USA. jasara@bidmc.harvard.edu

Barnes, I., Shapiro, B., Lister, A., Kuznetsova, T., Sher, A., Guthrie, D., Thomas, M.G., 2007. Genetic structure and extinction of the woolly mammoth, Mammuthus primigenius. Curr. Biol. 17, 1072-1075.
Abstract: The interval since circa 50 Ka has been a period of significant species extinctions among the large mammal fauna. However, the relative roles of an increasing human presence and a synchronous series of complex environmental changes in these extinctions have yet to be fully resolved. Recent analyses of fossil material from Beringia have clarified our understanding of the spatiotemporal pattern of Late Pleistocene extinctions, identifying periods of population turnover well before the last glacial maximum (LGM: circa 21 Ka) or subsequent human expansion. To examine the role of pre-LGM population changes in shaping the genetic structure of an extinct species, we analyzed the mitochondrial DNA of woolly mammoths in western Beringia and across its range. We identify genetic signatures of a range expansion of mammoths, from eastern to western Beringia, after the last interglacial (circa 125 Ka), and then an extended period during which demographic inference indicates no population-size increase. The most marked change in diversity at this time is the loss of one of two major mitochondrial lineages

Binladen, J., Gilbert, M.T., Willerslev, E., 2007. 800,000 year old mammoth DNA, modern elephant DNA or PCR artefact? Biol. Lett. 3, 55-56.
Abstract: Poulakakis and colleagues (Poulakakis et al. 2006: Biol. Lett. 2, 451-454), report the recovery of 'authentic' mammoth DNA from an 800,000-year-old fragment of bone excavated on the island of Crete. In light of results from other ancient DNA studies that indicate how DNA survival is unlikely in samples, which are recovered from warm environments and are relatively old (e.g. more than 100,000 years), these findings come as a great surprise. Here, we show that problems exist with the methodological approaches used in the study. First, the nested PCR technique as reported is nonsensical--one of the second round 'nested' primers falls outside the amplicon of the first round PCR. More worryingly, the binding region of one of the first round primers (Elcytb320R) falls within the short 43 base pair reported mammoth sequence, specifically covering two of the three reportedly diagnostic Elephas polymorphisms. Finally, we demonstrate using a simple BLAST search in GenBank that the claimed 'uniquely derived character state' for mammoths is in fact also found within modern elephants

Gilbert, M.T., Binladen, J., Miller, W., Wiuf, C., Willerslev, E., Poinar, H., Carlson, J.E., Leebens-Mack, J.H., Schuster, S.C., 2007. Recharacterization of ancient DNA miscoding lesions: insights in the era of sequencing-by-synthesis
422. Nucleic Acids Res. 35, 1-10.
Abstract: Although ancient DNA (aDNA) miscoding lesions have been studied since the earliest days of the field, their nature remains a source of debate. A variety of conflicting hypotheses exist about which miscoding lesions constitute true aDNA damage as opposed to PCR polymerase amplification error. Furthermore, considerable disagreement and speculation exists on which specific damage events underlie observed miscoding lesions. The root of the problem is that it has previously been difficult to assemble sufficient data to test the hypotheses, and near-impossible to accurately determine the specific strand of origin of observed damage events. With the advent of emulsion-based clonal amplification (emPCR) and the sequencing-by-synthesis technology this has changed. In this paper we demonstrate how data produced on the Roche GS20 genome sequencer can determine miscoding lesion strands of origin, and subsequently be interpreted to enable characterization of the aDNA damage behind the observed phenotypes. Through comparative analyses on 390,965 bp of modern chloroplast and 131,474 bp of ancient woolly mammoth GS20 sequence data we conclusively demonstrate that in this sample at least, a permafrost preserved specimen, Type 2 (cytosine-->thymine/guanine-->adenine) miscoding lesions represent the overwhelming majority of damage-derived miscoding lesions. Additionally, we show that an as yet unidentified guanine-->adenine analogue modification, not the conventionally argued cytosine-->uracil deamination, underpins a significant proportion of Type 2 damage. How widespread these implications are for aDNA will become apparent as future studies analyse data recovered from a wider range of substrates

Hollister-Smith, J.A., Poole, J.H., Archie, E.A., Vance, E.A., Georgiadis, N.J., Moss, C.J., Alberts, S.C., 2007. Age, musth and paternity success in wild maleAfrican elephants, Loxodonta africana. Animal Behaviour 74, 287-296.
Abstract: Male African elephants experience intense intrasexual selection in gaining access to oestrous females, who represent a very scarce and highly mobile resource. An unusual combination of behavioural and physiologica ltraits in males probably reflects this intense selection pressure. Males show prolonged growth, growing throughout much or perhaps all of their long life span (ca. 60-65 years), and they show musth,a physiological and behavioural condition exclusive to elephants, which is manifested by bouts of elevated testosterone and aggression and heightened sexual activity. Most observed matings are by males over 35years of age and in musth, suggesting that age and musth are both important factors contributing to male reproductive success. Here we report the results of a genetic paternity analysis of a well-studied population of wild African elephants. Patterns of paternity for 119 calves born over a 22-year period showed significant effects of both age and musth on paternity success. Among males in musth, paternity success increased significantly with age until the very oldest age classes, when it modestly declined. When not inmusth, males experienced relatively constant, low levels of paternity success at all ages. Thus, despite the importance of both musth and age in determining male paternity success, adult males both in and out ofmusth, and of all ages, produced calves. In general, however, older males had markedly elevated paternitysuccess compared with younger males, suggesting the possibility of sexual selection for longevity in this species.

Hubbard, T.J., Aken, B.L., Beal, K., Ballester, B., Caccamo, M., Chen, Y., Clarke, L., Coates, G., Cunningham, F., Cutts, T., Down, T., Dyer, S.C., Fitzgerald, S., Fernandez-Banet, J., Graf, S., Haider, S., Hammond, M., Herrero, J., Holland, R., Howe, K., Howe, K., Johnson, N., Kahari, A., Keefe, D., Kokocinski, F., Kulesha, E., Lawson, D., Longden, I., Melsopp, C., Megy, K., Meidl, P., Ouverdin, B., Parker, A., Prlic, A., Rice, S., Rios, D., Schuster, M., Sealy, I., Severin, J., Slater, G., Smedley, D., Spudich, G., Trevanion, S., Vilella, A., Vogel, J., White, S., Wood, M., Cox, T., Curwen, V., Durbin, R., Fernandez-Suarez, X.M., Flicek, P., Kasprzyk, A., Proctor, G., Searle, S., Smith, J., Ureta-Vidal, A., Birney, E., 2007. Ensembl 2007. Nucleic Acids Res. 35, D610-D617.
Abstract: The Ensembl (http://www.ensembl.org/) project provides a comprehensive and integrated source of annotation of chordate genome sequences. Over the past year the number of genomes available from Ensembl has increased from 15 to 33, with the addition of sites for the mammalian genomes of elephant, rabbit, armadillo, tenrec, platypus, pig, cat, bush baby, common shrew, microbat and european hedgehog; the fish genomes of stickleback and medaka and the second example of the genomes of the sea squirt (Ciona savignyi) and the mosquito (Aedes aegypti). Some of the major features added during the year include the first complete gene sets for genomes with low-sequence coverage, the introduction of new strain variation data and the introduction of new orthology/paralog annotations based on gene trees

Kellogg, M.E., Burkett, S., Dennis, T.R., Stone, G., Gray, B.A., McGuire, P.M., Zori, R.T., Stanyon, R., 2007. Chromosome painting in the manatee supports Afrotheria and Paenungulata. BMC. Evol. Biol. 7, 6.
Abstract: BACKGROUND: Sirenia (manatees, dugongs and Stellar's sea cow) have no evolutionary relationship with other marine mammals, despite similarities in adaptations and body shape. Recent phylogenomic results place Sirenia in Afrotheria and with elephants and rock hyraxes in Paenungulata. Sirenia and Hyracoidea are the two afrotherian orders as yet unstudied by comparative molecular cytogenetics. Here we report on the chromosome painting of the Florida manatee. RESULTS: The human autosomal and X chromosome paints delimited a total of 44 homologous segments in the manatee genome. The synteny of nine of the 22 human autosomal chromosomes (4, 5, 6, 9, 11, 14, 17, 18 and 20) and the X chromosome were found intact in the manatee. The syntenies of other human chromosomes were disrupted in the manatee genome into two to five segments. The hybridization pattern revealed that 20 (15 unique) associations of human chromosome segments are found in the manatee genome: 1/15, 1/19, 2/3 (twice), 3/7 (twice), 3/13, 3/21, 5/21, 7/16, 8/22, 10/12 (twice), 11/20, 12/22 (three times), 14/15, 16/19 and 18/19. CONCLUSION: There are five derived chromosome traits that strongly link elephants with manatees in Tethytheria and give implicit support to Paenungulata: the associations 2/3, 3/13, 8/22, 18/19 and the loss of the ancestral eutherian 4/8 association. It would be useful to test these conclusions with chromosome painting in hyraxes. The manatee chromosome painting data confirm that the associations 1/19 and 5/21 phylogenetically link afrotherian species and show that Afrotheria is a natural clade. The association 10/12/22 is also ubiquitous in Afrotheria (clade I), present in Laurasiatheria (clade IV), only partially present in Xenarthra (10/12, clade II) and absent in Euarchontoglires (clade III). If Afrotheria is basal to eutherians, this association could be part of the ancestral eutherian karyotype. If afrotherians are not at the root of the eutherian tree, then the 10/12/22 association could be one of a suite of derived associations linking afrotherian taxa

Kirkpatrick, J.F., 2007. Measuring the effects of wildlife contraception: the argument for comparing apples with oranges. Reprod. Fertil. Dev. 19, 548-552.
Abstract: There are few wildlife populations existing today that can be supported without some form of management. Wildlife fertility control, as one option, has moved from the research stage to actual application with a number of species, including wild horses, urban deer, captive exotic species and even African elephants, but this approach remains controversial in many quarters. Strident debate has arisen over the possible effects of contraception on behaviour, genetics, stress and even management economics, among other parameters. Part of the debate arises from the fact that critics often fail to recognise that some form of alternative management will be applied, and a second problem arises when critics fail to identify and demand the same concern for the consequences of the alternative management approaches. Thus, any rational debate on the merits or possible effects of contraceptive management of wildlife must also recognise all alternative management approaches and apply the same concern and questions to these alternative approaches--including 'no management'--as are currently being applied to fertility control. Only then will the stewards of wildlife be in a position to make wise and informed decisions about management options

Kullberg, M., Hallström, B., Arnason, U., Janke, A., 2007. Expressed sequence tags as a tool for phylogenetic analysis of placental mammal evolution. PLoS ONE E publication Aug 22;2(1):e775.
Abstract: BACKGROUND: We investigate the usefulness of expressed sequence tags, ESTs, for establishing divergences within the tree of placental mammals. This is done on the example of the established relationships among primates (human), lagomorphs (rabbit), rodents (rat and mouse), artiodactyls (cow), carnivorans (dog) and proboscideans (elephant). METHODOLOGY/PRINCIPAL FINDINGS: We have produced 2000 ESTs (1.2 mega bases) from a marsupial mouse and characterized the data for their use in phylogenetic analysis. The sequences were used to identify putative orthologous sequences from whole genome projects. Although most ESTs stem from single sequence reads, the frequency of potential sequencing errors was found to be lower than allelic variation. Most of the sequences represented slowly evolving housekeeping-type genes, with an average amino acid distance of 6.6% between human and mouse. Positive Darwinian selection was identified at only a few single sites. Phylogenetic analyses of the EST data yielded trees that were consistent with those established from whole genome projects. CONCLUSIONS: The general quality of EST sequences and the general absence of positive selection in these sequences make ESTs an attractive tool for phylogenetic analysis. The EST approach allows, at reasonable costs, a fast extension of data sampling from species outside the genome projects.

Lynn, D.J., Bradley, D.G., 2007. Discovery of alpha-defensins in basal mammals. Dev. Comp Immunol. 31, 963-967.
Abstract: Alpha-defensins are essential molecules of the innate immune system that have broad spectrum antimicrobial activity against a range of bacteria and viruses. To date, alpha-defensins have only been identified in the Euarchontoglires branch of the mammals. This has led to speculation that alpha-defensins may be specific to this group, a somewhat surprising finding, given their importance in the immune system. The mammalian genome project provided us with the opportunity to search for alpha-defensins in previously unexamined mammalian superorders. Using hidden Markov model (HMM) profile searching, we report the discovery of alpha-defensins in the African savanna elephant, the lesser hedgehog tenrec, and the nine-banded armadillo genomes representing two of the most basal mammalian superorders, Afrotheria and Xenarthra. Furthermore, we identify an alpha-defensin-like gene in the gray short-tailed opossum, suggesting that alpha-defensins may have evolved much earlier than previously thought, before the divergence of placental mammals and marsupials approximately 130 mya

Mailand, C., Wasser, S.K., 2007. Isolation of DNA from small amounts of elephant ivory. Nat Protoc 2, 2228-2232.
Abstract: This protocol describes a method for the extraction of DNA from elephant ivory. These techniques are being used to assign geographic origin to poached ivory by comparing the ivory genotype to a geographic-based gene frequency map, developed separately. The method has three components: ivory pulverization, decalcification and DNA extraction. Pulverization occurs in a freezer mill while the sample is deep frozen in liquid nitrogen, preventing degradation of DNA during the process. Decalcification involves repeated agitation of the sample in 0.5 M ethylenediaminetetraacetic acid over a 4-d period. Extraction follows a modified Qiagen protocol for the extraction of DNA from animal tissue. This method can be used on all forms of ivory. However, DNA recovery is highest when the outermost layer of the tusk, the cementum, is used. When applied to extract DNA from 11 samples, in duplicate, the entire protocol can be completed in 6 d, although much of this time consists of pause points that do not require effort. The protocol provides 0.8 +/- 0.11 ng microl(-1) (mean +/- s.e., n = 48) of DNA per sample.

Matsumoto, K., Parola, P., Rolain, J.M., Jeffery, K., Raoult, D., 2007. Detection of "Rickettsia sp. strain Uilenbergi" and "Rickettsia sp. strain Davousti" in Amblyomma tholloni ticks from elephants in Africa. BMC Microbiol 7, 74 [Epub ahead of print].
Abstract: ABSTRACT: BACKGROUND: To date, 6 tick-borne rickettsiae pathogenic for humans are known to occur in Africa and 4 of them were first identified in ticks before being recognized as human pathogens. RESULTS: We examined 33 and 5 Amblyomma tholloni ticks from African elephants in the Central African Republic and Gabon, respectively, by PCR amplification and sequencing of a part of gltA and ompA genes of the genus Rickettsia. The partial sequences of gltA and ompA genes detected in tick in Gabon had 99.1% similarity with those of R. heilongjiangensis and 97.1% with those of Rickettsia sp. HL-93 strain, respectively. The partial gltA and ompA gene sequences detected in tick in the Central African Republic were 98.9% and 95.1% similar to those of Rickettsia sp. DnS14 strain and R. massiliae, respectively. Phylogenetic analysis showed Rickettsia sp. detected in Gabon clusters with R. japonica and R. heilongjiangensis in a phylogenetic tree based on the partial gltA and ompA genes. The genotype of the Rickettsia sp. detected in the Central African Republic is close to those of R. massiliae group in the phylogenetic tree based on partial gltA gene sequences, and distantly related to other rickettsiae in the tree based on partial ompA gene. CONCLUSIONS: The degrees of similarity of partial gltA and ompA genes with recognized species indicate the rickettsiae detected in this study may be new species although we could only study the partial sequences of 2 genes regarding the amount of DNA that was available. We propose the Rickettsia sp. detected in Gabon be provisionally named "Rickettsia sp. stain Davousti" and Rickettsia sp. detected in the Central African Republic be named "Rickettsia sp. strain Uilenbergi".

Murphy, W.J., Pringle, T.H., Crider, T.A., Springer, M.S., Miller, W., 2007. Using genomic data to unravel the root of the placental mammal phylogeny. Genome Res. 17, 413-421.
Abstract: The phylogeny of placental mammals is a critical framework for choosing future genome sequencing targets and for resolving the ancestral mammalian genome at the nucleotide level. Despite considerable recent progress defining superordinal relationships, several branches remain poorly resolved, including the root of the placental tree. Here we analyzed the genome sequence assemblies of human, armadillo, elephant, and opossum to identify informative coding indels that would serve as rare genomic changes to infer early events in placental mammal phylogeny. We also expanded our species sampling by including sequence data from >30 ongoing genome projects, followed by PCR and sequencing validation of each indel in additional taxa. Our data provide support for a sister-group relationship between Afrotheria and Xenarthra (the Atlantogenata hypothesis), which is in turn the sister-taxon to Boreoeutheria. We failed to recover any indels in support of a basal position for Xenarthra (Epitheria), which is suggested by morphology and a recent retroposon analysis, or a hypothesis with Afrotheria basal (Exafricoplacentalia), which is favored by phylogenetic analysis of large nuclear gene data sets. In addition, we identified two retroposon insertions that also support Atlantogenata and none for the alternative hypotheses. A revised molecular timescale based on these phylogenetic inferences suggests Afrotheria and Xenarthra diverged from other placental mammals approximately 103 (95-114) million years ago. We discuss the impacts of this topology on earlier phylogenetic reconstructions and repeat-based inferences of phylogeny

Orlando, L., Pages, M., Calvignac, S., Hughes, S., Hanni, C., 2007. Does the 43 bp sequence from an 800,000 year old cretan dwarf elephantid really rewrite the textbook on mammoths? Biol. Lett. 3, 57-59.
Abstract: Pigmy elephants inhabited the islands from the Mediterranean region during the Pleistocene period but became extinct in the course of the Holocene. Despite striking distinctive anatomical characteristics related to insularity, some similarities with the lineage of extant Asian elephants have suggested that pigmy elephants could be most probably seen as members of the genus Elephas. Poulakakis et al (2006) have recently challenged this view by recovering a short mtDNA sequence from an 800 000 year old fossil of the Cretan pigmy elephant (Elephas creticus). According to the authors of this study, a deep taxonomic revision of Cretan dwarf elephants would be needed, as the sequence exhibits clear affinities with woolly mammoth haplotypes. However, we point here many aspects that seriously weaken the strength of the ancient DNA evidence reported

Orlando, L., Hanni, C., Douady, C.J., 2007. Mammoth and Elephant Phylogenetic Relationships: Mammut Americanum, the Missing Outgroup. Evol. Bioinform. Online. 3, 45-51.
Abstract: At the morphological level, the woolly mammoth has most often been considered as the sister-species of Asian elephants, but at the DNA level, different studies have found support for proximity with African elephants. Recent reports have increased the available sequence data and apparently solved the discrepancy, finding mammoths to be most closely related to Asian elephants. However, we demonstrate here that the three competing topologies have similar likelihood, bayesian and parsimony supports. The analysis further suggests the inadequacy of using Sirenia or Hyracoidea as outgroups. We therefore argue that orthologous sequences from the extinct American mastodon will be required to definitively solve this long-standing question

Orlando, L., Pages, M., Calvignac, S., Hughes, S., Hanni, C., 2007. Does the 43 bp sequence from an 800,000 year old Cretan dwarf elephantid really rewrite the textbook on mammoths? Biology Letters 3, 57-59.
Abstract: Pigmy elephants inhabited the islands from the Mediterranean region during the Pleistocene period but became extinct in the course of the Holocene. Despite striking distinctive anatomical characteristics related to insularity, some similarities with the lineage of extant Asian elephants have suggested that pigmy elephants could be most probably seen as members of the genus Elephas. Poulakakis et al. (2006) have recently challenged this view by recovering a short mtDNA sequence from an 800 000 year old fossil of the Cretan pigmy elephant (Elephas creticus). According to the authors of this study, a deep taxonomic revision of Cretan dwarf elephants would be needed, as the sequence exhibits clear affinities with woolly mammoth haplotypes. However, we point here many aspects that seriously weaken the strength of the ancient DNA evidence reported.

Pan, D., 2007. Hippo signaling in organ size control. Genes Dev. 21, 886-897.
Abstract: The control of organ (or organism) size is a fundamental aspect of life that has long captured human imagination. What makes an elephant grow a million times larger than a mouse? How do our two hands develop independently of each other yet reach very similar size? How does a liver precisely regenerate its original mass when two-thirds of it is removed? The recent discovery of a novel signaling network in Drosophila, known as the Hippo (Hpo) pathway, might provide an important entry point to these fascinating questions. The Hpo pathway consists of several negative growth regulators acting in a kinase cascade that ultimately phosphorylates and inactivates Yorkie (Yki), a transcriptional coactivator that positively regulates cell growth, survival, and proliferation. Components of the Hpo pathway are highly conserved throughout evolution, suggesting that this pathway may function as a global regulator of tissue homeostasis in all metazoan animals. Here, I provide a historical review of this potent growth-regulatory pathway and highlight outstanding questions that will likely be the focus of future investigation

Pardini, A.T., O'Brien, P.C., Fu, B., Bonde, R.K., Elder, F.F., Ferguson-Smith, M.A., Yang, F., Robinson, T.J., 2007. Chromosome painting among Proboscidea, Hyracoidea and Sirenia: support for Paenungulata (Afrotheria, Mammalia) but not Tethytheria. Proc. Biol. Sci. 274, 1333-1340.
Abstract: Despite marked improvements in the interpretation of systematic relationships within Eutheria, particular nodes, including Paenungulata (Hyracoidea, Sirenia and Proboscidea), remain ambiguous. The combination of a rapid radiation, a deep divergence and an extensive morphological diversification has resulted in a limited phylogenetic signal confounding resolution within this clade both at the morphological and nucleotide levels. Cross-species chromosome painting was used to delineate regions of homology between Loxodonta africana (2n=56), Procavia capensis (2n=54), Trichechus manatus latirostris (2n=48) and an outgroup taxon, the aardvark (Orycteropus afer, 2n=20). Changes specific to each lineage were identified and although the presence of a minimum of 11 synapomorphies confirmed the monophyly of Paenungulata, no change characterizing intrapaenungulate relationships was evident. The reconstruction of an ancestral paenungulate karyotype and the estimation of rates of chromosomal evolution indicate a reduced rate of genomic repatterning following the paenungulate radiation. In comparison to data available for other mammalian taxa, the paenungulate rate of chromosomal evolution is slow to moderate. As a consequence, the absence of a chromosomal character uniting two paenungulates (at the level of resolution characterized in this study) may be due to a reduced rate of chromosomal change relative to the length of time separating successive divergence events

Redi, C.A., Garagna, S., Zuccotti, M., Capanna, E., 2007. Genome size: a novel genomic signature in support of Afrotheria. Journal of Molecular Evolution 64, 484-487.
Abstract: Molecular phylogenetic analyses suggest an emerging phylogeny for the extant Placentalia (eutherian) that radically departs from morphologically based constructions of the past. Placental mammals are partitioned into four supraordinal clades: Afrotheria, Xenarthra, Laurasiatheria, and Euarchontoglires. Afrotheria form an endemic African clade that includes elephant shrews, golden moles, tenrecs, aardvarks, hyraxes, elephants, dugongs, and manatees. Datamining databases of genome size (GS) shows that till today just one afrotherian GS has been evaluated, that of the aardvark Orycteropus afer. We show that the GSs of six selected representatives across the Afrotheria supraordinal group are among the highest for the extant Placentalia, providing a novel genomic signature of this enigmatic group. The mean GS value of Afrotheria, 5.3 +/- 0.7 pg, is the highest reported for the extant Placentalia. This should assist in planning new genome sequencing initiatives

Roca, A.L., Georgiadis, N., O'Brien, S.J., 2007. Cyto-nuclear genomic dissociation and the African elephant species question. Quat. Int. 169-170, 4-16.
Abstract: Studies of skull morphology and of nuclear DNA have strongly concluded that African elephants comprise two species. Nonetheless, Debruyne (2005) has suggested a single-species model for Loxodonta based on the polyphyly of a single genetic locus, mitochondrial DNA (mtDNA). Discordant patterns between mitochondrial and nuclear DNA markers were subsequently reported in some African savanna elephant populations, further supporting a two-species model, and prompting us to re-examine here the geographic distribution of different elephant morphotypes and their relationship to nuclear and mtDNA phylogeographic patterns. We used exact tests to compare the distribution of forest elephant-typical and savanna elephant-typical characteristics across eight published datasets containing morphological, mtDNA or nuclear DNA data for African elephants. Among the elephants examined by Debruyne (2005), we found that patterns of forest vs. savanna characteristics were significantly different (p < 10(-5)) between mtDNA and morphology, suggesting the presence of cyto-nuclear genomic dissociation. We show that the eight African elephant continent-wide datasets compared, including that of Debruyne (2005), together support a two-species model with cyto-nuclear genomic dissociation rather than a one-species model, and together indicate that Africa harbors two species of elephant

Rohland, N., Malaspinas, A.S., Pollack, J.L., Slatkin, M., Matheus, P., 2007. Proboscidean mitogenomics: Chronology and mode of elephant evolution using mastodon as outgroup. PLoS Biol 5,  e207. doi:10.1371/journal.pbio.0050207.
Abstract: We have sequenced the complete mitochondrial genome of the extinct American mastodon (Mammut americanum) from an Alaskan fossil that is between 50,000 and 130,000 y old, extending the age range of genomic analyses by almost a complete glacial cycle. The sequence we obtained is substantially different from previously reported partial mastodon mitochondrial DNA sequences. By comparing those partial sequences to other proboscidean sequences, we conclude that we have obtained the first sequence of mastodon DNA ever reported. Using the sequence of the mastodon, which diverged 24-28 million years ago (mya) from the Elephantidae lineage, as an outgroup, we infer that the ancestors of African elephants diverged from the lineage leading to mammoths and Asian elephants approximately 7.6 mya and that mammoths and Asian elephants diverged approximately 6.7 mya. We also conclude
that the nuclear genomes of the African savannah and forest elephants diverged approximately 4.0 mya, supporting the view that these two groups represent different species. Finally, we found the mitochondrial mutation rate of proboscideans to be roughly half of the rate in primates during at least the last 24 million years.

Sreekumar, E., Janki, M.B., Arathy, D.S., Hariharan, R., Premraj, C.A., Rasool, T.J., 2007. Molecular characterization and expression of interferon-gamma of Asian elephant (Elephas maximus). Vet. Immunol. Immunopathol. 118, 75-83.
Abstract: Tuberculosis (TB) caused by Mycobacterial organisms has emerged as one of the major diseases in captive elephants. In vitro Interferon-gamma (IFN-gamma) assay is being used as an ancillary test for early detection of TB in domestic and captive wild animals. In the present study, basic sequence information and immunological cross-reactivity of this major cytokine of Asian elephants were explored. At predicted amino acid level, IFN-gamma of Asian elephant showed maximum identity to that of horse (73%). Other IFN-gamma amino acid sequences that showed high level identity were that of giant panda (72%), dog (71%), nine-banded armadillo (69%), cattle (63%) and human (62%). IFN-gamma promoter sequences of Asian elephant, human, cattle and mouse showed high level conservation of the putative transcription factor binding sites, TATA box and transcriptional start site. The functionally important human IFN-gamma promoter elements, such as AP-2IRE-BE, YY1-gammaIFN-BED, ATFCS and AP-1gammaINF binding sites, were absolutely conserved in the corresponding elephant sequence. There was only a single nucleotide variation in the other two important elements, NFAT-gammaINF and IFN-gammaPE, indicating the highly conserved regulation of IFN-gamma expression across different species. Phylogenetic analysis based on IFN-gamma protein sequences revealed a closer relation of Asian elephants and nine-banded armadillo. This shows a closer evolution of these members of Afrotheria and Xenarthra, respectively; and supports the previous reports based on mitochondrial DNA studies. In Western blot analysis, IFN-gamma of Asian elephant expressed in Escherichia coli was detected using an anti-bovine IFN-gamma monoclonal antibody, indicating immunological cross-reactivity

Tabuce, R., Marivaux, L., Adaci, M., Bensalah, M., Hartenberger, J.L., Mahboubi, M., Mebrouk, F., Tafforeau, P., Jaeger, J.J., 2007. Early Tertiary mammals from North Africa reinforce the molecular Afrotheria clade. Proc. Biol. Sci. 274, 1159-1166.
Abstract: The phylogenetic pattern and timing of the radiation of mammals, especially the geographical origins of major crown clades, are areas of controversy among molecular biologists, morphologists and palaeontologists. Molecular phylogeneticists have identified an Afrotheria clade, which includes several taxa as different as tenrecs (Tenrecidae), golden moles (Chrysochloridae), elephant-shrews (Macroscelididae), aardvarks (Tubulidentata) and paenungulates (elephants, sea cows and hyracoids). Molecular data also suggest a Cretaceous African origin for Afrotheria within Placentalia followed by a long period of endemic evolution on the Afro-Arabian continent after the mid-Cretaceous Gondwanan breakup (approx. 105-25 Myr ago). However, there was no morphological support for such a natural grouping so far. Here, we report new dental and postcranial evidence of Eocene stem hyrax and macroscelidid from North Africa that, for the first time, provides a congruent phylogenetic view with the molecular Afrotheria clade. These new fossils imply, however, substantial changes regarding the historical biogeography of afrotheres. Their long period of isolation in Africa, as assumed by molecular inferences, is now to be reconsidered inasmuch as Eocene paenungulates and elephant-shrews are here found to be related to some Early Tertiary Euramerican 'hyopsodontid condylarths' (archaic hoofed mammals). As a result, stem members of afrotherian clades are not strictly African but also include some Early Paleogene Holarctic mammals

Wasser, S.K., Mailand, C., Booth, R., Mutayoba, B., Kisamo, E., Clark, B., Stephens, M., 2007. Using DNA to track the origin of the largest ivory seizure since the 1989 trade ban. Proc. Natl. Acad. Sci. U. S. A 104 , 4228-4233.
Abstract: The illegal ivory trade recently intensified to the highest levels ever reported. Policing this trafficking has been hampered by the inability to reliably determine geographic origin of contraband ivory. Ivory can be smuggled across multiple international borders and along numerous trade routes, making poaching hotspots and potential trade routes difficult to identify. This fluidity also makes it difficult to refute a country's denial of poaching problems. We extend an innovative DNA assignment method to determine the geographic origin(s) of large elephant ivory seizures. A Voronoi tessellation method is used that utilizes genetic similarities across tusks to simultaneously infer the origin of multiple samples that could have one or more common origin(s). We show that this joint analysis performs better than sample-by-sample methods in assigning sample clusters of known origin. The joint method is then used to infer the geographic origin of the largest ivory seizure since the 1989 ivory trade ban. Wildlife authorities initially suspected that this ivory came from multiple locations across forest and savanna Africa. However, we show that the ivory was entirely from savanna elephants, most probably originating from a narrow east-to-west band of southern Africa, centered on Zambia. These findings enabled law enforcement to focus their investigation to a smaller area and fewer trade routes and led to changes within the Zambian government to improve antipoaching efforts. Such outcomes demonstrate the potential of genetic analyses to help combat the expanding wildlife trade by identifying origin(s) of large seizures of contraband ivory. Broader applications to wildlife trade are discussed

Weiss, B., Faus, H., Haendler, B., 2007. Phylogenetic conservation of the androgen receptor AR45 variant form in placental mammals. Gene 399, 105-111.
Abstract: A cDNA coding for a tissue-specific AR45 variant form of the androgen receptor (AR) has recently been identified in humans, with highest expression levels found in heart. The deduced protein comprises the DNA-binding domain, hinge region and ligand-binding domain of the AR, but not the N-terminal domain which is replaced by a unique, short, seven amino-acid-long stretch. This sequence is encoded by the mutually exclusive exon 1B, located between exons 1 and 2 of the human AR gene. As transcript variants of the steroid receptor family have been shown to have important implications for hormone function, we set out to analyse the genomes of different organisms for potential AR45 expression. We found exon 1B to be conserved in the syntenic chromosomal region of non-human primates such as the chimpanzee Pan troglodytes, the orang-utan Pongo pygmaeus, the macaque Macaca mulatta and the marmoset Callithrix jacchus, and of the elephant Loxondonta africana, the pig Sus scrofa and the dog Canis familiaris. Quantification of AR45 transcript levels in heart, skeletal muscle and lung of Macaca fascicularis showed the heart to be the main organ of expression. A complete AR45 cDNA was furthermore isolated from the heart of this species. Comparative analysis of the identified AR45 exon 1B regions and of the deduced amino acids revealed a high conservation among species. The four N-terminal residues were identical in all eight species, whereas a few changes were seen in the other three residues in the marmoset, elephant and pig. In contrast, we observed more divergence in the mouse Mus musculus and rat Rattus norvegicus syntenic regions. Here a stop codon was found downstream of the potential start codon in the putatively deduced protein sequence and it can be inferred that no protein corresponding to AR45 exists in these two species. The existence of AR45 in different placental mammals with the exception of mouse and rat suggests a disappearance in rodents late in evolution, before the separation of the mouse and rat lineages, about 16 million years ago. In view of the potential function of AR45 as a regulator of AR function, and considering the multiple roles of androgens in normal physiology and in several diseases, these findings have important implications with regard to subtle differences in the action of the male sexual hormone in various organisms

Wellehan, J.F., Johnson, A.J., Childress, A.L., Harr, K.E., Isaza, R., 2007. Six novel gammaherpesviruses of Afrotheria provide insight into the early divergence of the Gammaherpesvirinae. Vet Microbiol 2007 Aug 19; [Epub ahead of print].
Abstract: The Afrotheria represent an early branching of placental mammals. Only two herpesviruses from Afrotheria have been previously identified, and the genus Proboscivirus in the subfamily Betaherpesvirinae has been proposed for them. Six novel gammaherpesviruses were identified in four species in the superorder Afrotheria by detection and analysis of their DNA polymerase genes. Elephantid herpesvirus 3 (ElHV3) and Elephantid herpesvirus 4 (ElHV4) were identified from conjunctival swabs from Asian elephants (Elephas maximus). ElHV3 was also found
in a vaginal swab from one elephant with vaginitis. Elephantid herpesvirus 5 (ElHV5) was identified from vaginal swabs of two Asian elephants with vaginal plaques. Elephantid herpesvirus 6 was discovered in a conjunctival swab from an African elephant (Loxodonta africana). Procavid herpesvirus 1 (PrHV1) was found in spleen and conjunctival swabs of rock hyrax (Procavia capensis). Trichechid herpesvirus 1 (TrHV1) was identified from skin and buffy coats of Florida manatees (Trichechus manatus latirostris). ElHV3 and ElHV4 form a distinct cluster, and ElHV5, ElHV6, TrHV1, and PrHV1 form a second cluster. These viruses may have codiverged with their host species. Phylogenetic analysis of these novel herpesviruses suggests that two separate groups of gammaherpesviruses may have codiverged with the Afrotheria.

Archie, E.A., Moss, C.J., Alberts, S.C., 2006. The ties that bind: genetic relatedness predicts the fission and fusion of social groups in wild African elephants
490. Proc. Biol. Sci. 273, 513-522.
Abstract: Many social animals live in stable groups. In contrast, African savannah elephants (Loxodonta africana) live in unusually fluid, fission-fusion societies. That is, 'core' social groups are composed of predictable sets of individuals; however, over the course of hours or days, these groups may temporarily divide and reunite, or they may fuse with other social groups to form much larger social units. Here, we test the hypothesis that genetic relatedness predicts patterns of group fission and fusion among wild, female African elephants. Our study of a single Kenyan population spans 236 individuals in 45 core social groups, genotyped at 11 microsatellite and one mitochondrial DNA (mtDNA) locus. We found that genetic relatedness predicted group fission; adult females remained with their first order maternal relatives when core groups fissioned temporarily. Relatedness also predicted temporary fusion between social groups; core groups were more likely to fuse with each other when the oldest females in each group were genetic relatives. Groups that shared mtDNA haplotypes were also significantly more likely to fuse than groups that did not share mtDNA. Our results suggest that associations between core social groups persist for decades after the original maternal kin have died. We discuss these results in the context of kin selection and its possible role in the evolution of elephant sociality

Ball, R.L., Dumonceaux, G., Olsen, J.H., Burton, M.S., Lyashchenko, K. Comparison of trunk wash results matched to multiantigen print immunoassay (MAPIA) in a group of captive Asian elephants (Elephas maximus). 2006 Proceedings American Association of Zoo Veterinarians.  303-304. 2006.
Ref Type: Conference Proceeding
Abstract: Introduction: Between 1994 and June 2005, there were 34 confirmed cases of tuberculosis in elephants in the U.S. population. Thirty-one Asian (Elephas maximus) and three African (Loxodonta africana) elephants were affected. Mycobacterium tuberculosis was the etiologic agent in 33 cases and M. bovis in one case. Cases of tuberculosis caused by an unusual nontuberculous mycobacteria, M. szulgai have recently occurred as well.  Currently, TB in elephants remains a diagnostic dilemma. The sensitivity of trunk wash culture, the currently recommended test for diagnosis, is unknown. False negatives have been documented (trunk wash negative elephants that were subsequently found to be culture positive at necropsy).  Other non-culture techniques for TB diagnosis include ELISA, and PCR. A novel technology, MultiAntigen Print ImmunoAssay (MAPIA) and lateral-flow technology (Rapid Test)  has been evaluated and used to diagnose tuberculosis in captive elephants with encouraging results.  One concern with this serologic testing is the possibility of Mycobacterium other than tuberculosis (MOTT) cross-reacting with the antigen used in the Rapid Test or the MAPIA and leading to a false positive.  With numerous MOTT routinely cultured from trunk washes, this is a valid concern. Methods and Materials: A retrospective analysis was done at Busch Gardens Tampa Bay and Chembio, Inc. that matched trunk wash results to serum samples.  All serum was collected within 7 days of the trunk wash and analyzed with the Rapid Test and MAPIA. Four Asian elephants with a total of 18 samples met this criteria and had serum submitted for testing. Results and Discussion: Table 1 lists the results and the organisms cultured. While the sampling is limited in this pilot project, it appears that MOTT does not evoke a response when assayed with the Rapid Test or MAPIA. The recent cases of M. szulgai do demonstrate the potential usefulness for this test when a disease develops from MOTT.  The usefulness of this new technology, taken in conjunction with other clinical data including trunk washes when indicated, is a valuable tool in the healthcare of captive elephants.

LITERATURE CITED
1 Lacasse, C., K.C. Gamble, K. Terio, L.L. Farina, D.A. Travis, and M.Miller. 2005. Mycobacterium szulgai osteroarthritis and pneumonia in an African elephant (Loxdonta africana). Proc. Am. Assoc. Zoo Vet. Ann. Meet. Pp. 170-172.
2 Larsen, R.S., M.D. Salman, S.K. Mikota, R. Isaza, R.J. Montali, and J. Triantis.  2000.  Evaluation of a multiple-antigen enzyme-linked immunosorbent assay for detection of Mycobacterium tuberculosis infection in captive elephants.  J. Zoo Wildl. Med. 31:291-302.
3 Lyashchenko, K., et al.  2000. A multiantigen print immunoassay for the serological diagnosis of infectious diseases.  J. Immunol. Methods  242:91-100
4 Lyashchenko, K., M. Miller, and  W.R. Waters. 2005. Application of multiple antigen print immunoassay and rapid lateral flow technology for tuberculosis testing of elephants. Proc. Am. Assoc. Zoo Vet. Ann. Meet.  Pp. 64-65

Bojesen, A.M., Olsen, K.E., Bertelsen, M.F., 2006. Fatal enterocolitis in Asian elephants (Elephas maximus) caused by Clostridium difficile. Vet Microbiol Epub ahead of print.
Abstract: Two cases of fatal enteritis caused by Clostridium difficile in captive Asian elephants are reported from an outbreak affecting five females in the same zoo. Post mortem examination including histopathology demonstrated fibrinonecrotic enterocolitis. C. difficile was isolated by selective cultivation from two dead and a third severely affected elephant. Four isolates were obtained and found positive for toxin A and B by PCR. All isolates were positive in a toxigenic culture assay and toxin was demonstrated in the intestinal content from one of the fatal cases and in a surviving but severely affected elephant. PCR ribotyping demonstrated that the C. difficile isolates shared an identical profile, which was different from an epidemiologically unrelated strain, indicating that the outbreak was caused by the same C. difficile clone. It is speculated that the feeding of large quantities of broccoli, a rich source of sulforaphane, which has been shown to inhibit the growth of many intestinal microorganisms may have triggered a subsequent overgrowth by C. difficile. This is the first report of C. difficile as the main cause of fatal enterocolitis in elephants. The findings emphasize the need to regard this organism as potentially dangerous for elephants and caution is recommended concerning antibiotic treatment and feeding with diets containing antimicrobials, which may trigger an expansion of a C. difficile population in the gut.

Bojesen, A.M., Olsen, K.E., Bertelsen, M.F., 2006. Fatal enterocolitis in Asian elephants (Elephas maximus) caused by Clostridium difficile
456. Vet. Microbiol. 116, 329-335.
Abstract: Two cases of fatal enteritis caused by Clostridium difficile in captive Asian elephants are reported from an outbreak affecting five females in the same zoo. Post mortem examination including histopathology demonstrated fibrinonecrotic enterocolitis. C. difficile was isolated by selective cultivation from two dead and a third severely affected elephant. Four isolates were obtained and found positive for toxin A and B by PCR. All isolates were positive in a toxigenic culture assay and toxin was demonstrated in the intestinal content from one of the fatal cases and in a surviving but severely affected elephant. PCR ribotyping demonstrated that the C. difficile isolates shared an identical profile, which was different from an epidemiologically unrelated strain, indicating that the outbreak was caused by the same C. difficile clone. It is speculated that the feeding of large quantities of broccoli, a rich source of sulforaphane, which has been shown to inhibit the growth of many intestinal microorganisms may have triggered a subsequent overgrowth by C. difficile. This is the first report of C. difficile as the main cause of fatal enterocolitis in elephants. The findings emphasize the need to regard this organism as potentially dangerous for elephants and caution is recommended concerning antibiotic treatment and feeding with diets containing antimicrobials, which may trigger an expansion of a C. difficile population in the gut

Capelli, C., MacPhee, R.D., Roca, A.L., Brisighelli, F., Georgiadis, N., O'Brien, S.J., Greenwood, A.D., 2006. A nuclear DNA phylogeny of the woolly mammoth (Mammuthus primigenius)
468. Mol. Phylogenet. Evol. 40, 620-627.

Cooper, A., 2006. The year of the mammoth. PLoS Biology 4, 1-3.
Abstract: Mammoth mitochondrial (mt) genomes are apparently on a similar schedule to London buses-you wait for ages and then suddenly three come along at once. Within the past six weeks, three studies [1-3] have independently determined all, or most, of the mammoth mt genome sequence, some 16,800 base pairs (bp). Encouragingly, the partial sequence was a byproduct of a study that generated some 13 million bp of mammoth genomic DNA using a new, massively parallel sequencing approach. The very divergent methods used in these three studies also neatly represent the past, present, and future of ancient DNA (aDNA) research. aDNA methods provide an opportunity to characterise the genetic composition of species and populations in the past, and to actually observe evolutionary change through real time. Such a record has great potential to reveal the processes that have generated the diversity and distribution of taxa in our modern environment, and to examine phenomena such as speciation, domestication, morphological evolution, and the impacts of major environmental changes. aDNA data also provide an important opportunity to test our ability to accurately reconstruct evolutionary history via the fossil record or via extrapolation from the genetic data of modern species. Unfortunately, the potential of aDNA remains largely untapped because research has been severely limited by the technical diffi culties of retrieving and studying the trace amounts of  highly fragmented DNA that survive in ancient specimens.

Eckhart, L., Uthman, A., Sipos, W., Tschachler, E., 2006. Genome sequence comparison reveals independent inactivation of the caspase-15 gene in different evolutionary lineages of mammals
428. Molecular Biology and Evolution 23, 2081-2089.
Abstract: We have recently demonstrated that placental mammalian species such as pig and dog express a novel proapoptotic protease, caspase-15, whereas mouse and humans lack this enzyme. Here we investigated the evolutionary fate of the caspase-15 gene in different mammalian lineages by analyzing whole-genome shotgun sequences of 30 mammalian species for the presence of caspase-15 orthologs. Caspase-15 gene sequences were found in representatives of all major mammalian clades except for the superorders Afrotheria (tenrec, rock hyrax, and elephant) and Euarchontoglires (rodents, rabbit, tree shrew, and primates), which either lacked any caspase-15-like sequences or contained mutated remnants of the caspase-15 gene. Polymerase chain reaction screenings confirmed the results of the database searches and showed that the caspase-15 gene is expressed not only in various placental mammals but also in the marsupial, Monodelphis domestica. The observed species distribution implies that caspase-15 has originated in an early ancestor of modern mammals and has been conserved, over more than 180 Myr, in marsupials and many placental mammals, whereas it was independently lost in 2 phylogenetically distant clades of placental mammals, that is, Afrotheria and Euarchontoglires. Our data suggest that the inactivation of the caspase-15 gene was not counteracted by, and may even have been driven by, evolutionary constraints in these clades, and therefore, caution against the uncritical use of gene absence for the inference of phylogenetic relationships

Ehlers, B., Dural, G., Marschall, M., Schregel, V., Goltz, M., Hentschke, J., 2006. Endotheliotropic elephant herpesvirus, the first betaherpesvirus with a thymidine kinase gene
411. Journal of General Virology 87, 2781-2789.
Abstract: Endotheliotropic elephant herpesvirus (elephantid herpesvirus 1; ElHV-1) is apathogenic for African elephants (Loxodonta africana), but causes fatal haemorrhagic disease in Asian elephants (Elephas maximus). This is thought to occur through transmission from African elephants in places where both species are housed, such as zoological gardens. The virus has caused considerable losses in North American and European zoological gardens and thus severely impedes breeding of the endangered Asian elephant. Previously, the ultrastructural and genetic characterization of ElHV-1 from a male Asian elephant that died from the disease at the Berlin zoological gardens in 1998 have been reported. Here, a partial characterization of the ElHV-1 genome is presented. A 60 kbp locus, spanning 34 open reading frames, was analysed. Most of the detected genes were found to be conserved among the herpesviruses and showed an overall arrangement most similar to that of betaherpesviruses, in particular Human herpesvirus 6 and Human herpesvirus 7. Most importantly, in addition to a protein kinase gene that is homologous to the human cytomegalovirus UL97 gene, a thymidine kinase (TK) gene was found, which is generally missing in betaherpesvirus genomes. Thus, ElHV-1 is the only known betaherpesvirus to encode a TK gene. This peculiarity might contribute to the fulminant pathogenicity of ElHV-1, but also provide a crucial enzymic activity for developing an efficient antiviral therapy with currently available nucleoside analogues

Gee, H., 2006. Evolution: memories of mammoths
505. Nature 439, 673.

Gupta, S.K., Thangaraj, K., Singh, L., 2006. A simple and inexpensive molecular method for sexing and identification of the forensic samples of elephant origin
430. J. Forensic Sci. 51, 805-807.
Abstract: The population of the Asian elephant is being dramatically reduced due to poaching of the ivory from the male. As poaching occurs in remote forests, it often takes weeks or longer for it to be discovered and it is therefore often very difficult to determine the sex of the decomposed body. Data suggest that in the recent past, over 2000 male elephants have been poached in South India. We have developed a technique based on molecular markers to determine that the carcass is an elephant and that it is a male. Using DNA sequence information from Genbank, we have developed two primer pairs: one for the mitochondrial DNA (mtDNA) and the other for the sex-determining region of Y chromosome (SRY) gene of the Indian elephant. After PCR amplification of known elephant DNA, we found that the mtDNA was common in both males and females, whereas the SRY-specific amplicon was observed only in the male

Hildebrandt, T.B., Hermes, R., Ratanakorn, P., Rietschel, W., Fickel, J., RetNat, D., Frey, R., Reid, C., Goeritz, F. Ultrasonographic assessment and ultrasound-guided biopsy of the retropharyngeal lymph nodes in elephants.  2006 Proceedings American Association of Zoo Veterinarians.  117-118. 2006.
Ref Type: Conference Proceeding
Abstract: So far there are no valid diagnostic tools available for identifying latent carriers of endotheliotropic elephant herpes virus (EEHV).  For this reason, the lateral retropharyngeal lymph node complex (LARELYNOC) of elephants, identified during postmortem studies as target organ for EEHV and suitable for transcutaneous biopsy, was grossly described. Transcutaneous ultrasound (3.5 MHz) was applied behind the ear region to identify the LARELYNOC containing up to four single lymph nodes on each side. The lymph node tissue is situated 20-50 mm below the skin surface. An ultrasonographic assessment of the LARELYNOC and two biopsies were performed on 39 healthy Asian elephants (Elephas maximus). Samples were tested for EEHV via PCR. Whole blood samples were also collected and tested for active EEHV infection. Lymph nodes were ultrasonographically classified as active (calculated mean volume=17.4 ± 6.9 cm3, P>0.001), inactive (calculated mean volume=3.1 ± 0.6 cm3, P<0.001), or chronic active (calculated mean volume=10.6 ± 1.0 cm3, P<0.05). Histology confirmed not only the presence of lymph tissue but also the ultrasonographically diagnosed reactivity status of the lymph node biospies.  Although all samples including whole blood were found to be negative for the EEHV DNA particles, the successful development of this procedure in elephants could prove beneficial for the screening of not only latent EEHV infections but might also be a less dangerous alternative method for the diagnosis of zoonotic infections such as tuberculosis.

Hofreiter, M., Lister, A., 2006. Mammoths
459. Curr. Biol. 16, R347-R348.

Kitakado, T., Kitada, S., Kishino, H., Skaug, H.J., 2006. An integrated-likelihood method for estimating genetic differentiation between populations
453. Genetics 173, 2073-2082.
Abstract: The aim of this article is to develop an integrated-likelihood (IL) approach to estimate the genetic differentiation between populations. The conventional maximum-likelihood (ML) and pseudolikelihood (PL) methods that use sample counts of alleles may cause severe underestimations of FST, which means overestimations of theta=4Nm, when the number of sampling localities is small. To reduce such bias in the estimation of genetic differentiation, we propose an IL method in which the mean allele frequencies over populations are regarded as nuisance parameters and are eliminated by integration. To maximize the IL function, we have developed two algorithms, a Monte Carlo EM algorithm and a Laplace approximation. Our simulation studies show that the method proposed here outperforms the conventional ML and PL methods in terms of unbiasedness and precision. The IL method was applied to real data for Pacific herring and African elephants

Krause, J., Dear, P.H., Pollack, J.L., Slatkin, M., Spriggs, H., Barnes, I., Lister, A.M., Ebersberger, I., Paabo, S., Hofreiter, M., 2006. Multiplex amplification of the mammoth mitochondrial genome and the evolution of Elephantidae
533. Nature 439, 724-727.
Abstract: In studying the genomes of extinct species, two principal limitations are typically the small quantities of endogenous ancient DNA and its degraded condition, even though products of up to 1,600 base pairs (bp) have been amplified in rare cases. Using small overlapping polymerase chain reaction products, longer stretches of sequences or even whole mitochondrial genomes can be reconstructed, but this approach is limited by the number of amplifications that can be performed from rare samples. Thus, even from well-studied Pleistocene species such as mammoths, ground sloths and cave bears, no DNA sequences of more than about 1,000 bp have been reconstructed. Here we report the complete mitochondrial genome sequence of the Pleistocene woolly mammoth Mammuthus primigenius. We used about 200 mg of bone and a new approach that allows the simultaneous retrieval of multiple sequences from small amounts of degraded DNA. Our phylogenetic analyses show that the mammoth was more closely related to the Asian than to the African elephant. However, the divergence of mammoth, African and Asian elephants occurred over a short time, corresponding to only about 7% of the total length of the phylogenetic tree for the three evolutionary lineages

Perelygin, A.A., Lear, T.L., Zharkikh, A.A., Brinton, M.A., 2006. Comparative analysis of vertebrate EIF2AK2 (PKR) genes and assignment of the equine gene to ECA15q24-q25 and the bovine gene to BTA11q12-q15
416. Genet. Sel Evol. 38, 551-563.
Abstract: The structures of the canine, rabbit, bovine and equine EIF2AK2 genes were determined. Each of these genes has a 5' non-coding exon as well as 15 coding exons. All of the canine, bovine and equine EIF2AK2 introns have consensus donor and acceptor splice sites. In the equine EIF2AK2 gene, a unique single nucleotide polymorphism that encoded a Tyr329Cys substitution was detected. Regulatory elements predicted in the promoter region were conserved in ungulates, primates, rodents, Afrotheria (elephant) and Insectifora (shrew). Western clawed frog and fugu EIF2AK2 gene sequences were detected in the USCS Genome Browser and compared to those of other vertebrate EIF2AK2 genes. A comparison of EIF2AK2 protein domains in vertebrates indicates that the kinase catalytic domains were evolutionarily more conserved than the nucleic acid-binding motifs. Nucleotide substitution rates were uniform among the vertebrate sequences with the exception of the zebrafish and goldfish EIF2AK2 genes, which showed substitution rates about 20% higher than those of other vertebrates. FISH was used to physically assign the horse and cattle genes to chromosome locations, ECA15q24-q25 and BTA11q12-15, respectively. Comparative mapping data confirmed conservation of synteny between ungulates, humans and rodents

Poinar, H.N., Schwarz, C., Qi, J., Shapiro, B., MacPhee, R.D., Buigues, B., Tikhonov, A., Huson, D.H., Tomsho, L.P., Auch, A., Rampp, M., Miller, W., Schuster, S.C., 2006. Metagenomics to paleogenomics: large-scale sequencing of mammoth DNA
529. Science 311, 392-394.
Abstract: We sequenced 28 million base pairs of DNA in a metagenomics approach, using a woolly mammoth (Mammuthus primigenius) sample from Siberia. As a result of exceptional sample preservation and the use of a recently developed emulsion polymerase chain reaction and pyrosequencing technique, 13 million base pairs (45.4%) of the sequencing reads were identified as mammoth DNA. Sequence identity between our data and African elephant (Loxodonta africana) was 98.55%, consistent with a paleontologically based divergence date of 5 to 6 million years. The sample includes a surprisingly small diversity of environmental DNAs. The high percentage of endogenous DNA recoverable from this single mammoth would allow for completion of its genome, unleashing the field of paleogenomics

Poulakakis, N., Parmakelis, A., Lymberakis, P., Mylonas, M., Zouros, E., Reese, D.S., Glaberman, S., Caccone, A., 2006. Ancient DNA forces reconsideration of evolutionary history of Mediterranean pygmy elephantids. Biol. Lett. 2, 451-454.
Abstract: During the Pleistocene pygmy elephantids, some only a quarter of their ancestors' size, were present on Mediterranean islands until about 10,000 years ago (y.a.). Using a new methodology for ancient DNA (aDNA) studies, the whole genomic multiple displacement amplification method, we were able to retrieve cytochrome b (cytb) DNA fragments from 4200 to 800,000 y.a. specimens from island and mainland samples, including pygmy and normal-sized forms. The short DNA sequence (43 bp) retrieved from the 800,000 y.a. sample is one of the oldest DNA fragment ever retrieved. Duplication of the experiments in two laboratories, the occurrence of three diagnostic sites and the results of the phylogenetic analyses strongly support its authenticity. Our results challenge the prevailing view that pygmy elephantids of the eastern Mediterranean originated exclusively from Elephas, suggesting independent histories of dwarfism and the presence of both pygmy mammoths and elephant-like taxa on these islands. Based on our molecular data, the origin of the Tilos and Cyprus elephantids from a lineage within the genus Elephas is confirmed, while the DNA sequence from the Cretan sample falls clearly within the mammoth clade. Thus, the name Mammuthus creticus rather than Elephas creticus, seems to be justified for this form. Our findings also suggest a need to re-evaluate the evolutionary history of the Sicilian/Maltese species, traditionally included in the genus Elephas

Reid, C.E., Hildebrandt, T.B., Marx, N., Hunt, M., Thy, N., Reynes, J.M., Schaftenaar, W., Fickel, J., 2006. Endotheliotropic elephant herpes virus (EEHV) infection. The first PCR-confirmed fatal case in Asia
436. Vet. Q. 28, 61-64.
Abstract: Since 1995, 4 suspected cases of Endotheliotropic Elephant Herpes Virus (EEHV) infection, i.e. based on clinical presentation, have occurred in Asia without resulting in epidemic outbreaks as expected. In order to confirm the presence of EEHV on the continent of Asia, viral DNA particles from liver samples of a wild-caught 3-year-old elephant found dead at a Cambodian elephant sanctuary and clinically diagnosed with EEHV, were PCR processed using known EEHV strain primers. The presence of EEHV viral nucleic acids was confirmed and the nucleic acids had a 99% sequence similarity to the U.S.A strain (gene bank locus: AF117265) and 97% sequence similarity to the European strain (gene bank locus: AF354746) assigning this case to the EEHV-1 cluster. More than the confirmation of EEHV on the continent of Asia, is the phylogenic relationship to the USA and European strains with no corresponding contact or transport of USA or European elephants to Asia. Thus, this brings many of the traditional theories into question. Although almost forgotten, this disease is still ramped in captive elephant populations worldwide and continues to devastate particularly the neonatal and weaning-age population. Special attention and continued research are needed specifically in the area of basic virology and epidemiology

Rogaev, E.I., Moliaka, Y.K., Malyarchuk, B.A., Kondrashov, F.A., Derenko, M.V., Chumakov, I., Grigorenko, A.P., 2006. Complete mitochondrial genome and phylogeny of Pleistocene mammoth Mammuthus primigenius
507. PLoS. Biol. 4, e73.
Abstract: Phylogenetic relationships between the extinct woolly mammoth (Mammuthus primigenius), and the Asian (Elephas maximus) and African savanna (Loxodonta africana) elephants remain unresolved. Here, we report the sequence of the complete mitochondrial genome (16,842 base pairs) of a woolly mammoth extracted from permafrost-preserved remains from the Pleistocene epoch--the oldest mitochondrial genome sequence determined to date. We demonstrate that well-preserved mitochondrial genome fragments, as long as approximately 1,600-1700 base pairs, can be retrieved from pre-Holocene remains of an extinct species. Phylogenetic reconstruction of the Elephantinae clade suggests that M. primigenius and E. maximus are sister species that diverged soon after their common ancestor split from the L. africana lineage. Low nucleotide diversity found between independently determined mitochondrial genomic sequences of woolly mammoths separated geographically and in time suggests that north-eastern Siberia was occupied by a relatively homogeneous population of M. primigenius throughout the late Pleistocene

Rompler, H., Rohland, N., Lalueza-Fox, C., Willerslev, E., Kuznetsova, T., Rabeder, G., Bertranpetit, J., Schoneberg, T., Hofreiter, M., 2006. Nuclear gene indicates coat-color polymorphism in mammoths
439. Science 313, 62.
Abstract: By amplifying the melanocortin type 1 receptor from the woolly mammoth, we can report the complete nucleotide sequence of a nuclear-encoded gene from an extinct species. We found two alleles and show that one allele produces a functional protein whereas the other one encodes a protein with strongly reduced activity. This finding suggests that mammoths may have been polymorphic in coat color, with both dark- and light-haired individuals co-occurring

Sa-Ardrit, M., Saikhun, J., Thongtip, N., Damyang, M., Mahasawangkul, S., Angkawanish, T., Jansittiwate, S., Faisaikarm, T., Kitiyanant, Y., Pavasuthipaisit, K., Pinyopummin, A., 2006. Ultrastructural alterations of frozen-thawed Asian elephant (Elephas maximus) spermatozoa
491. Int. J. Androl 29, 346-352.
Abstract: Intact plasma and acrosome membranes and functional mitochondria following cryopreservation are important attributes for the fertilizing ability of spermatozoa. In the present study, functional and ultrastructural changes of Asian elephant spermatozoa after cryopreservation either in TEST + glycerol or HEPT + dimethyl sulphoxide (DMSO) were evaluated by fluorescent techniques and electron microscopy. Sperm frozen in TEST + glycerol had higher proportion of sperm with intact plasma (49.1 +/- 9.2% vs. 30.9 +/- 3.9%) and acrosomal (53.7 +/- 4.9% vs. 35.8 +/- 6.1%) membranes, as well as active mitochondria (57.0 +/- 7.2% vs. 42.0 +/- 5.0%) than those cryopreserved in HEPT + DMSO. The results obtained from electron microscopy were similar to those obtained by fluorescence microscopy. The percentage of normal spermatozoa was higher when spermatozoa were frozen in TEST + glycerol than those frozen in HEPT + DMSO (31.8 +/- 5.6 vs. 28.5 +/- 6.4). The ultrastructural alterations revealed by transmission electron microscopy could be classified as (i) distension of plasma membrane, while the acrosome was swollen; (ii) disruption or loss of plasma membrane, while acrosome was swollen with distended outer acrosomal membrane; (iii) disruption or loss of plasma and outer acrosomal membrane with leakage of acrosome content; (iv) extensive vesiculation of plasma and outer acrosomal membrane and leakage of acrosome content; (v) a complete loss of both plasma membrane and outer acrosomal membrane; and (vi) swelling of mitochondria. These findings suggest that the freezing and thawing procedure caused structural damage to elephant spermatozoa, especially in the plasma membrane, acrosome and mitochondria. Fluorescence and electron microscopic evaluations are potentially a powerful tool in the analysis of elephant spermatozoa after freezing and thawing

Shoshani, J., Walter, R.C., Abraha, M., Berhe, S., Tassy, P., Sanders, W.J., Marchant, G.H., Libsekal, Y., Ghirmai, T., Zinner, D., 2006. A proboscidean from the late Oligocene of Eritrea, a "missing link" between early Elephantiformes and Elephantimorpha, and biogeographic implications
384. Proc. Natl. Acad. Sci. U. S. A 103, 17296-17301.
Abstract: We report on a late Oligocene proboscidean species from Eritrea, dated to 26.8 +/- 1.5 Mya. This "missing link" between early elephantiformes and Elephantimorpha is the oldest known nongomphothere proboscidean to probably display horizontal tooth displacement, typical of elephants [Elephantimorpha consists of Mammutida (mastodons) and Elephantida, and Elephantida includes gomphotheres, stegodons, and elephants]. Together with the newly discovered late Oligocene gomphotheres from Chilga, Ethiopia, the Eritrean taxon points to the importance of East Africa as a major area for the knowledge of the early evolution of Elephantimorpha before the faunal exchange between Eurasia and Africa

Thitaram, C., Thongtip, N., Somgird, C., Colenbrander, B., Van Boxtel, D.C.J., Lenstra, J.A. Molecular tool for genetic management and parentage test to control poaching in Asian elephants. Proceedings International Elephant Conservation & Research Symposium.  205-209. 2006.
Ref Type: Conference Proceeding

Vidya, T.N.C., Sukumar, R. Social organization of the Asian elephant (Elephas maximus) in southern India as inferred from microsatellite DNA. Proceedings International Elephant Conservation & Research Symposium.  214-216. 2006. 2006.
Ref Type: Conference Proceeding

Zuba, J.R., Oosterhuis, J.E., Pessier, A.P. The toenail "abscess" in elephants: treatment options including cryotherapy and pathologic similarities with equine proliferative pododermatitis (canker).  2006 Proceedings American Association of Zoo Veterinarians.  187-190. 2006.
Ref Type: Conference Proceeding
Abstract: Foot problems potentially represent the single most important clinical disease of captive elephants.  Predisposing factors include obesity, lack of exercise, nail or sole overgrowth, improper foot care, poor hygiene, inappropriate enclosure surfaces, poor conformation, malnutrition and secondary skeletal disorders such as degenerative joint disease.  Furthermore, factors such as elephant management philosophy, disposition of elephants, facilities and competency of staff in caring for elephant feet will contribute significantly to the foot health of captive animals.  It is important to note that these conditions are rarely reported in free-ranging elephants. The elephant toenail abscess is characterized grossly by proliferative outgrowth of "crab meat-like" tissue that may acutely rupture through the surface of the nail wall and/or adjacent cuticle or sole. True abscess formation with localized collections of suppurative material is not a consistent clinical feature.  In most cases, the inciting cause of these lesions are typically not found and are likely due to one or more of the predisposing factors listed above.  Once established, these frustrating lesions require extensive, intensive and prolonged medical attention.  If not cared for properly, these wounds may progress to phalangeal osteomyelitis and the need for surgical intervention.  Sole abscesses are equally frustrating and difficult to manage with proposed etiologies similar to toenail lesions. There are no reports in the literature describing the pathology of the classic proliferative abscess tissue of the elephant nail abscess.  Although variously interpreted as fibrous or granulation tissue, the authors are unaware of previous histologic descriptions of this tissue.  Biopsy samples of toenail abscess tissue from two Asian elephants (Elephas maximus) at the San Diego Wild Animal Park (SDWAP) consisted of stratified squamous epithelium arranged in columns resembling horn tubules.  The predominant histologic finding was marked, near diffuse, hydropic degeneration of keratinocytes.  There were multifocal areas of suppurative inflammation with admixed bacterial colonies.  Inflammatory foci comprised only a small portion of the lesion and were interpreted as the external surfaces of the biopsy with likely secondary bacterial colonization. Because descriptions of the normal histology of the elephant toenail could not be located, a grossly normal toenail from a different Asian elephant was obtained to compare histologic features with those of the toenail abscesses.  Sections demonstrated formation of the toenail in a manner similar to that of the hoof of the horse and cattle with tubular, intertubular and laminar horn.  Primary and secondary epidermal laminae were identified.  Proliferative lesions of horn-producing epithelium associated with ballooning degeneration and inadequate keratinization of keratinocytes, have been described in horses as equine "canker" and coronary band dystrophy.  Equine canker is most commonly observed in the hind feet of draft horses and begins in the frog sometimes with extension to the sole and hoof wall.  Grossly, lesions are characterized by soft white papillary to "cauliflower-like" tissue associated with a foul odor. Similar to what is noted in elephant foot problems, predisposing factors for the development of equine canker include poor hygiene or wet environmental conditions. There is a lack of gross and histologic description of the normal nail and sole tissue of the elephant and further investigations are warranted.  A review of the anatomy and histology of the normal equine hoof may provide a basic understanding of the elephant nail until more specific and detailed elephant information is available.  From our investigation, the authors offer that a more accurate description of the elephant toenail abscess would be proliferative pododermatitis, the term synonymous with equine canker.  A more colloquial term such as "elephant canker" may be appropriate, as well. Canker in the horse is an uncommon but difficult to treat disease of the hoof.  Historically, treatment options for elephant toenail abscesses include corrective trimming, superficial debridement and application of topical disinfectants or antibiotics. Others have constructed innovative sandals to treat and protect the affected sole or nail with success. The use of regional intravenous perfusion of the affected limb with antibiotics has also been successful. Since the elephant nail abscess now appears to be histologically and clinically comparable to equine canker, this novel characterization of an old disease may offer unique insight for treatment.  In the least, it has provided our practice with a new list of treatment options and experienced equine clinicians for consultation who have been managing patients with a similar disease for many years. One of the Asian elephants at the SDWAP has had chronic toenail abscesses for over 2 yr. Radiographs of the affected digits, as reported by others to assess degree of involvement, have fortunately been negative for evidence of osteomyelitis.  Several bacterial and fungal cultures of deep tissue biopsies and swabs of affected lesions have resulted in a mixture of organisms with no consistent single etiologic agent.  Biopsies were found negative for presence of viral DNA (elephant papillomavirus and herpesvirus) by PCR.  Typical elephant foot care at the SDWAP includes trimming and debriding with hoof knives, foot soaks and topical antibiotics.  Although difficult, attempts are made in keeping the affected foot clean and dry.  Following recommendations for the treatment of equine canker, we recently implemented the routine use of cryotherapy in all elephants with proliferative pododermatitis with improved success in the control and recession of exuberant nail lesions. The proliferative tissue of the nail is first cleaned then disinfected, debrided, trimmed with hoof knives and allowed to dry. Modified brass branding tools with contact surfaces of variable size (2-5 cm diameter) and shape (round or ovoid) are placed into liquid nitrogen (-196 C) for several minutes and then placed directly on the cankerous tissue for 30-60 sec.  This process is then repeated 4-5 min later, following a complete thaw of tissue.  Within 24 hr, the cryoburned tissue becomes macerated and necrotic and is readily removed with gentle scrubbing.  Cryotherapy offers the advantage of destroying tissue to a deeper level than trimming alone and provides hemostasis, as well.  Because of decreased sensation at the cryotherapy treatment site, a memorable painful event is avoided and the elephant patient is more routinely accepting of this technique. With the use of hoof knives, we typically remove 2-3 mm of proliferative tissue before the patient refuses further treatment, presumably due to discomfort.  With cryotherapy, we are able to remove an additional 3-5 mm of tissue by cell freezing and necrosis.  The result is quicker resolution of cankerous lesions without the need for aggressive, and potentially painful, interventions. In conclusion, it appears that elephant nail abscesses can best be described as proliferative pododermatitis, or canker, as is seen in other species.  Further gross and microscopic descriptions of normal and pathologic nail or sole lesions are necessary.  Routine cryotherapy has shown promise in the treatment of these chronic, frustrating and potentially devastating lesions of our captive elephants.

Bertelsen, M.F., Bojesen, M., Olsen, K.E.P. Fatal enterocolitis in two Asian elephants (Elephas maximus) caused by Clostridium difficile. 2005 Proceedings AAZV, AAWV, AZA Nutrition Advisory Group.  66-67. 2005.
Ref Type: Conference Proceeding
Abstract: Altered behavior, anorexia and listlessness were observed in four of five adult captive female Asian elephants (Elephas maximus). Two animals recovered, while two died after 2 days. The dead elephants were subjected to post mortem examination including histopathology, demonstrating fibrinonecrotic enteritis and colitis. Clostridium difficile was isolated from both dead elephants and from the feces of the two surviving affected animals, and identified by selective cultivation and PCR identification. All isolates had the tcdA and tcdB toxin genes and were positive in a toxigenic culture assay. C. difficile toxin from the intestinal content of one of the fatal cases was demonstrated using cell-culture based cytotoxin assays. Clostridium perfringens type A and Clostridium septicum were also isolated from both dead animals. Although C. perfringens has been associated with ulcerative enteritis in an elephant,¹ in this case these isolates likely are incidental, as C. perfringens enterotoxin was not demonstrated, and as C. septicum is well known for producing rapid post mortem overgrowth.  Amplified fragment length polymorphism typing, showed that the C. difficile isolates recovered from the outbreak, all had the same fingerprint profile, indicating that all four elephants were affected by the same bacterial clone. These findings appear to be the first to demonstrate that C. difficile may cause enterocolitis in elephants. The results emphasize the need to regard this organism as potentially dangerous for elephants. Although there was no prior exposure to antibiotic agents in this case, caution is recommended when treating elephants with antibiotics, as this may trigger C. difficile induced enterocolitis in other species, most notably humans and horses.²
LITERATURE CITED
1 Bacciarini, L.N., O. Pagan, J. Frey, and A. Grone. 2001. Clostridium perfringens beta2-toxin in an African elephant (Loxodonta africana) with ulcerative enteritis. Vet. Rec. 149: 618-20.
2 Songer, J.G. 1996. Clostridial enteric diseases of domestic animals. Clin. Microbiol. Rev. 9: 216-234.

Debruyne, R., 2005. A case study of apparent conflict between molecular phylogenies: the interrelationships of African elephants. Cladistics 21, 31-50.
Abstract: Recent molecular phylogenies of the African elephants suggest that there is an evolutionary structure within Loxodonta africana. Some nuclear results (Roca et al., 2001) support the separation of the forest African elephant subspecies L. a. cyclotis as a species distinct from the savannah elephant L. a. africana, on the basis of the recognition of both forming highly divergent (reciprocally monophyletic) clades. Conversely, a mitochondrial survey (Eggert et al., 2002), while admitting a geographic partitioning of the genetic structure within African elephants, suggests retaining the status quo. They recognize three diagnosible entities (western, central and south-eastern Africa) with non-overlapping ranges within L. africana sensu lato. In order to address these con.icting views (historical fragmentation and speciation or isolation by distance, respectively), we have sequenced two datasets of 1961 bp (for 50 elephants) and about 3700 bp, respectively (for 20 elephants) of the mitochondrial DNA for both forms of elephants (cyclotis and africana). They span the cytochrome b gene, the control region and several RNAs. When compared with former mtDNA data, they provide the most comprehensive view of the African elephant phylogeny (78 mtDNA haplotypes, of which 44 are new) and provide the .rst insight into populations from the Democratic Republic of Congo. The genetic diversity of mtDNA was appraised and the stability of alternative phylogenetic trees was investigated. Our results are inconsistent with both those prior studies. They revealed two highly divergent molecular clades referred to as F and S, that do not conform to the morphological delineations of cyclotis and africana. A non-negligible proportion of specimens of L. a. africana display haplotypes prevailing in forest elephant populations (clade F). The geographic distribution of clades and areas of their co-occurrence support the hypothesis of incomplete isolation between forest and savannah African elephant populations, followed by recurrent interbreeding between the two forms. We state that the conclusions of prior studies resulted from insufficient character and / or geographic sampling. We conclude that there is no satisfying argument which can recognize two or more species of African elephants. We briefly comment on the meaning of such an attitude in a conservation viewpoint.

Gibbons, A., 2005. Ancient DNA. New methods yield mammoth samples
527. Science 310, 1889.

Glickman, S.E., Short, R.V., Renfree, M.B., 2005. Sexual differentiation in three unconventional mammals: spotted hyenas, elephants and tammar wallabies
566. Horm. Behav. 48, 403-417.
Abstract: The present review explores sexual differentiation in three non-conventional species: the spotted hyena, the elephant and the tammar wallaby, selected because of the natural challenges they present for contemporary understanding of sexual differentiation. According to the prevailing view of mammalian sexual differentiation, originally proposed by Alfred Jost, secretion of androgen and anti-Mullerian hormone (AMH) by the fetal testes during critical stages of development accounts for the full range of sexually dimorphic urogenital traits observed at birth. Jost's concept was subsequently expanded to encompass sexual differentiation of the brain and behavior. Although the central focus of this review involves urogenital development, we assume that the novel mechanisms described in this article have potentially significant implications for sexual differentiation of brain and behavior, a transposition with precedent in the history of this field. Contrary to the "specific" requirements of Jost's formulation, female spotted hyenas and elephants initially develop male-type external genitalia prior to gonadal differentiation. In addition, the administration of anti-androgens to pregnant female spotted hyenas does not prevent the formation of a scrotum, pseudoscrotum, penis or penile clitoris in the offspring of treated females, although it is not yet clear whether the creation of masculine genitalia involves other steroids or whether there is a genetic mechanism bypassing a hormonal mediator. Wallabies, where sexual differentiation occurs in the pouch after birth, provide the most conclusive evidence for direct genetic control of sexual dimorphism, with the scrotum developing only in males and the pouch and mammary glands only in females, before differentiation of the gonads. The development of the pouch and mammary gland in females and the scrotum in males is controlled by genes on the X chromosome. In keeping with the "expanded" version of Jost's formulation, secretion of androgens by the fetal testes provides the best current account of a broad array of sex differences in reproductive morphology and endocrinology of the spotted hyena, and androgens are essential for development of the prostate and penis of the wallaby. But the essential circulating androgen in the male wallaby is 5alpha androstanediol, locally converted in target tissues to DHT, while in the pregnant female hyena, androstenedione, secreted by the maternal ovary, is converted by the placenta to testosterone (and estradiol) and transferred to the developing fetus. Testicular testosterone certainly seems to be responsible for the behavioral phenomenon of musth in male elephants. Both spotted hyenas and elephants display matrilineal social organization, and, in both species, female genital morphology requires feminine cooperation for successful copulation. We conclude that not all aspects of sexual differentiation have been delegated to testicular hormones in these mammals. In addition, we suggest that research on urogenital development in these non-traditional species directs attention to processes that may well be operating during the sexual differentiation of morphology and behavior in more common laboratory mammals, albeit in less dramatic fashion

Hildebrandt, T.B., Hermes, R., Ratanakorn, P., Rietschel, W., Fickel, J., Frey, R., Wibbelt, G., Reid, C., Goritz, F., 2005. Ultrasonographic assessment and ultrasound-guided biopsy of the retropharyngeal lymph nodes in Asian elephants (Elephas maximus)
552. Veterinary Record 157, 544-548.
Abstract: Endotheliotropic herpesvirus causes a fatal disease in young Asian elephants, but there are no methods for identifying latent carriers of the virus. During the postmortem study of one female African elephant and three male and two female Asian elephants, a lymph node located bilaterally caudoventral to the parotid gland, approximately 1.5 to 5 cm below the skin, was identified as suitable for transcutaneous ultrasound-guided biopsy. An ultrasonographic assessment and two biopsies were performed on 39 Asian elephants, and these lymph nodes were classified ultrasonographically as active, inactive or chronically active. The calculated mean (se) volume of 10 active lymph nodes was 17.4 (6.9) cm(3), and that of three chronically active lymph nodes was 10.6 (1.0) cm(3), whereas the mean volume of 17 inactive lymph nodes was 3.1 (0.6) cm(3). The presence of lymph node tissue in samples obtained by ultrasound-guided biopsy from three animals that were maintained under conditions that allowed for additional sampling was confirmed histologically. The dna extracted from the lymphoid tissue and the whole blood of all the elephants was negative for endotheliotropic herpesvirus by PCR.

Lacasse, C., Gamble, K.C., Terio, K., Farina, L.L., Travis, D.A., Miller, M. Mycobacterium szulgai osteoarthritis and pneumonia in an African elephant (Loxodonta Africana). 2005 Proceedings AAZV, AAWV, AZA Nutrition Advisory Group.  170-172. 2005.
Ref Type: Conference Proceeding
Abstract: Tuberculosis, particularly Mycobacterium bovis and M. tuberculosis, is an important health issue in zoological collections.  Zoos are a particular public health concern because of the close contact between tuberculosis-susceptible animals and humans, specifically animal handlers and visitors.¹⁶ Evidence of M. tuberculosis transmission between humans and elephants, confirmed by DNA fingerprinting, has been reported.¹³ Between 1994 and 2001, M. tuberculosis was isolated from trunk washes of captive elephants from 11 herds in the United States.¹⁷  To date, most reported cases of tuberculosis have occurred in captive Asian elephants (Elephas maximus).¹⁴ In 1997, the National Tuberculosis Working Group for Zoo and Wildlife Species partnered with the USDA to formulate the "Guidelines for the Control of Tuberculosis in Elephants." ¹⁵ This document outlines criteria for the testing, surveillance, and treatment of tuberculosis in elephants. The guidelines recommend annual monitoring of elephants by mycobacterial culture of three direct trunk washes collected over 1 wk.  Isolation of Mycobacterium avium and non-tuberculous mycobacteria from elephant trunk wash samples is common, but these organisms have not been associated with clinical disease.^14,18 This case report details clinical disease with fatal complications of an atypical mycobacterial infection in an African elephant (Loxodonta africana). In September 2003, an African elephant presented with acute, severe lameness of the left rear limb with subsequent swelling of the stifle.  Diagnostic procedures included aspiration cytology of the swelling, radiographs, and thermographic imaging.  The exact location of the injury could not be detected, but a lesion to the stifle or coxofemoral articulation was suspected.  After 13 mo of treatment, including pulse therapy with a variety of nonsteroidal anti-inflammatory drugs (NSAIDs), weekly to biweekly injections of polysulfated glycosaminoglycan, and intensive foot care efforts to treat secondary pedal lesions of both rearlimbs, the animal died acutely.  Gross necropsy revealed granulomatous osteomyelitis with necrosis/loss of the femoral head and acetabulum and pulmonary granulomas.  Both of these lesions contained acid-fast bacteria on cytology. While awaiting confirmatory culture results, quarantine procedures were established for the elephant facility and a program was established to screen all zoo personnel in close contact with the elephant or who participated in the necropsy.  All personnel were tested by the Chicago Department of Public Health without documented conversion. Mycobacterium szulgai was ultimately cultured from both coxofemoral and pulmonary lesions. Mycobacterium szulgai is an uncommon nontuberculous mycobacterium that is usually isolated from pathologic lesions in humans.²¹ This bacterial species was first identified in 1972.¹¹ The lungs are the main locality for pathologic manifestation in humans and several cases have been in patients with acquired immunodeficiency syndrome.^9,20,21 Infection due to M. szulgai most frequently produces thin-walled cavities in lungs resembling tuberculosis.⁴ Other documented sites of infection include the skin, bone, and tendon sheath (causing a carpal tunnel syndrome).^{2,9,10,12,19,20}  Intra-operative contamination from ice water has led to M. szulgai keratitis after laser-assisted ophthalmic surgeries^.6 A case of disseminated disease in a previously healthy young human has been reported.⁵  No evidence of human-to-human transmission of this organism has been documented and human cases are believed to originate from environmental sources.¹²  The natural habitat of the organism is unknown, but previous reports suggest an association of the bacteria with water of swimming pools and fish tanks.^1,21 The organism has been cultured from a snail and tropical fish.^1,3 No standard recommendation for the treatment of M. szulgai infection currently exists.  In general, triple antibiotic therapies used in standard mycobacterial treatments are reported with a low rate of relapses and sterilization of sputum cultures within a mean of 3 mo.³ Pulmonary lesions in this elephant were chronic; it was not possible to determine when initial infection occurred. Infection could have occurred in captivity or in the wild prior to captivity. Three trunk washes over the past year had been negative for mycobacterial culture. Osteomyelitis in the hip may have developed secondary to hematogenous spread from the lungs with the acute lameness resulting from a pathologic fracture associated with this infection. Alternatively, though considered less likely, a traumatic fracture of the hip could have occurred, with bacterial inoculation and secondary osteomyelitis as a result of increased blood flow to the site. The source of infection for this elephant remains unknown.  Prevalence of this organism in the natural habitat or captive environment of the elephants has not been previously documented.
LITERATURE CITED
1 Abalain-Colloc, M.L., D. Guillerm, M. Salaun, S. Gouriou, V. Vincent, and B. Picard.  2003.  Mycobacterium szulgai isolated from a patient, a tropical fish, and aquarium water.  Eur. J. Clin. Microbiol. Infect. Dis.  22: 768-769.
2.Cross, G.M., M. Guill, and J.K. Aton.  1985.  Cutaneous Mycobacterium szulgai infection. Arch. Dermatol. 121: 247-249.
3. Davidson, P.T. 1976. Mycobacterium szulgai: a new pathogen causing infection of the lung.  Chest 69: 799- 801.
4. Dylewski, J.S., H.M. Zackon, A.H. Latour, and G.R. Berry.  1987.  Mycobacterium szulgai: an unusual pathogen.  Rev. Infect. Dis.  9: 578-580.
5. Gur, H., S. Porat, H. Haas, Y. Naparstek, and M. Eliakim.  1984.  Disseminated mycobacterial disease caused by Mycobacterium szulgai. Arch. Intern. Med. 144: 1861-1863.
6.Holmes, G.P., G. Bond, R.C. Fader, and S.F. Fulcher.  2002. A cluster of cases of Mycobacterium szulgai keratitis that occurred after laser-assisted in situ keratomileusis.  Clin. Infect. Dis. 34: 1039-1046.
7.Horusitzky, A., X. Puechal, D. Dumont, T. Begue, M. Robineau, and M. Boissier.  2000.  Carpal tunnel syndrome caused by Mycobacterium szulgai. J. Rheumatol 27: 1299-1302.
8.Hurr, H., and T. Sorg.  1998.  Mycobacterium szulgai osteomyelitis.  J. Infect.  37: 191-192.
9.Luque, A.E., D. Kaminski, R. Reichman, and D. Hardy. 1998.  Mycobacterium szulgai osteomyelitis in an AIDS patient. Scand. J. Infect. Dis. 30: 88-91.
10.Maloney, J.M., C.R. Gregg, D.S. Stephens, F.A. Manian, and D. Rimland.  1987.  Infections caused by Mycobacterium szulgai in humans.  Rev. Infect. Dis.  9: 1120-1126.
11.Marks, J., P.A. Jenkins, and M. Tsukamura.  1972.  Mycobacterium szulgai: a new pathogen.  Tubercle 53: 210.
12.Merlet, C., S. Aberrane, F. Chilot, and J. Laroche.  2000.  Carpal tunnel syndrome complicating hand flexor tenosynovitis due to Mycobacterium szulgai. Joint Bone Spine 67: 247-248.
13.Michalak, K., C. Austin, S. Diesel, J.M. Bacon, P. Zimmerman, and J. N. Maslow.  1998. Mycobacterium tuberculosis infection as a zoonotic disease: transmission between humans and elephants. Emerg. Infect. Dis. 4: 283-287.
14.Mikota, S.K., R.S. Larsen, and R.J. Montali.  2000.  Tuberculosis in elephants in North America.  Zoo Biol. 19: 393-403.
15.National Tuberculosis Working Group for Zoo and Wildlife Species. 2000. Guidelines for the control of tuberculosis in elephants.  USDA Animal and Plant Health Inspection Services.
16.Oh, P., R. Granich, J. Scott, B. Sun, M. Joseph, C. Stringfield, S. Thisdell, J. Staley, D. Workman-Malcolm, L. Borenstein, E. Lehnkering, P. Ryan, J. Soukup, A. Nitta, and J. Flood.  2002.  Human exposure following  Mycobacterium tuberculosis infection of multiple animal species in a metropolitan zoo.  Emerg. Infect. Dis. 8: 1290-1293.
17.Payeur, J.B., J.L. Jarnagin, J.G. Marquardt, and D.L. Whipple.  2002.  Mycobacterial isolations in captive elephants in the United States.  Ann. N.Y. Acad. Sci. 969: 256-258.
18.Shojaei, H., J.G. Magee, R. Freeman, M. Yates, N.U. Horadagoda, and M. Goodfellow.  2000. Mycobacterium elephantis sp. nov., a rapidly growing non-chromogenic Mycobacterium isolated from an elephant.  Int. J. Syst. Evol. Microbiol.  50: 1817-1820.
19.Stratton, C.W., D.B. Phelps, and L.B. Reller.  1978.  Tuberculoid tenosynovitis and carpal tunnel syndrome caused by Mycobacterium szulgai.  Am. J. Med.  65: 349-351.
20.Tappe, D., P. Langmann, M. Zilly, H. Klinker, B. Schmausser, and M. Frosch.  2004.  Osteomyelitis and skin ulcers caused by Mycobacterium szulgai in an AIDS patient.  Scand. J. Infect. Dis. 36: 883-885.
21.Tortoli, E., G. Besozzi, C. Lacchini, V. Penati, M.T. Simonetti, and S. Emler.  1998.  Pulmonary infection due to Mycobacterium szulgai, case report and review of the literature.  Eur. Respir. J.  11: 975-977.

Nishihara, H., Satta, Y., Nikaido, M., Thewissen, J.G., Stanhope, M.J., Okada, N., 2005. A retroposon analysis of Afrotherian phylogeny
600. Molecular Biology and Evolution 22, 1823-1833.
Abstract: Recent comprehensive studies of DNA sequences support the monophyly of Afrotheria, comprising elephants, sirenians (dugongs and manatees), hyraxes, tenrecs, golden moles, aardvarks, and elephant shrews, as well as that of Paenungulata, comprising elephants, sirenians, and hyraxes. However, phylogenetic relationships among paenungulates, as well as among nonpaenungulates, have remained ambiguous. Here we applied an extensive retroposon analysis to these problems to support the monophyly of aardvarks, tenrecs, and golden moles, with elephant shrews as their sister group. Regarding phylogenetic relationships in Paenungulata, we could characterize only one informative locus, although we could isolate many insertions specific to each of three lineages, namely, Proboscidea, Sirenia, and Hyracoidea. These data prompted us to reexamine phylogenetic relationships among Paenungulata using 19 nuclear gene sequences resulting in three different analyses, namely, short interspersed element (SINE) insertions, nuclear sequence analyses, and morphological cladistics, supporting different respective phylogenies. We concluded that these three lineages diverged very rapidly in a very short evolutionary period, with the consequence that ancestral polymorphism present in the last common ancestor of Paenungulata results in such incongruence. Our results suggest the rapid fixation of many large-scale morphological synapomorphies for Tethytheria; implications of this in relation to the morphological evolution in Paenungulata are discussed

Okello, J.B., Wittemyer, G., Rasmussen, H.B., Douglas-Hamilton, I., Nyakaana, S., Arctander, P., Siegismund, H.R., 2005. Noninvasive genotyping and Mendelian analysis of microsatellites in African savannah elephants. J. Hered. 96, 679-687.
Abstract: We obtained fresh dung samples from 202 (133 mother-offspring pairs) savannah elephants (Loxodonta africana) in Samburu, Kenya, and genotyped them at 20 microsatellite loci to assess genotyping success and errors. A total of 98.6% consensus genotypes was successfully obtained, with allelic dropout and false allele rates at 1.6% (n = 46) and 0.9% (n = 37) of heterozygous and total consensus genotypes, respectively, and an overall genotyping error rate of 2.5% based on repeat typing. Mendelian analysis revealed consistent inheritance in all but 38 allelic pairs from mother-offspring, giving an average mismatch error rate of 2.06%, a possible result of null alleles, mutations, genotyping errors, or inaccuracy in maternity assignment. We detected no evidence for large allele dropout, stuttering, or scoring error in the dataset and significant Hardy-Weinberg deviations at only two loci due to heterozygosity deficiency. Across loci, null allele frequencies were low (range: 0.000-0.042) and below the 0.20 threshold that would significantly bias individual-based studies. The high genotyping success and low errors observed in this study demonstrate reliability of the method employed and underscore the application of simple pedigrees in noninvasive studies. Since none of the sires were included in this study, the error rates presented are just estimates

Raubenheimer, E.J., Ngwenya, S.P., 2005. The role of ivory in the survival of the African elephant
510. SADJ. 60, 426, 430.
Abstract: The unique chequered pattern of polished ivory has created a perverted commercial demand for elephant tusks. The morphologic basis of the pattern, which makes ivory a sought after product for the manufacturing of works of art, is discussed. Chemical analyses of ivory holds great potential in tracing the source of illegally harvested tusks and exposing poorly managed elephant sanctuaries. The impact of uncontrolled ivory hunting on the population genetics of the African elephant is briefly reviewed

Roca, A.L., Georgiadis, N., O'Brien, S.J., 2005. Cytonuclear genomic dissociation in African elephant species
650. Nat. Genet. 37 , 96-100.
Abstract: African forest and savanna elephants are distinct species separated by a hybrid zone. Because hybridization can affect the systematic and conservation status of populations, we examined gene flow between forest and savanna elephants at 21 African locations. We detected cytonuclear dissociation, indicative of different evolutionary histories for nuclear and mitochondrial genomes. Both paternally (n = 205 males) and biparentally (n = 2,123 X-chromosome segments) inherited gene sequences indicated that there was deep genetic separation between forest and savanna elephants. Yet in some savanna locales distant from present-day forest habitats, many individuals with savanna-specific nuclear genotypes carried maternally transmitted forest elephant mitochondrial DNA. This extreme cytonuclear dissociation implies that there were ancient episodes of hybridization between forest females and savanna males, which are larger and reproductively dominant to forest or hybrid males. Recurrent backcrossing of female hybrids to savanna bulls replaced the forest nuclear genome. The persistence of residual forest elephant mitochondria in savanna elephant herds renders evolutionary interpretations based on mitochondrial DNA alone misleading and preserves a genomic record of ancient habitat changes.

Roca, A.L., O'Brien, S.J., 2005. Genomic inferences from Afrotheria and the evolution of elephants
558. Curr. Opin. Genet. Dev. 15, 652-659.
Abstract: Recent genetic studies have established that African forest and savanna elephants are distinct species with dissociated cytonuclear genomic patterns, and have identified Asian elephants from Borneo and Sumatra as conservation priorities. Representative of Afrotheria, a superordinal clade encompassing six eutherian orders, the African savanna elephant was among the first mammals chosen for whole-genome sequencing to provide a comparative understanding of the human genome. Elephants have large and complex brains and display advanced levels of social structure, communication, learning and intelligence. The elephant genome sequence might prove useful for comparative genomic studies of these advanced traits, which have appeared independently in only three mammalian orders: primates, cetaceans and proboscideans

Saragusty, J., Hildebrandt, T.B., Natan, Y., Hermes, R., Yavin, S., Goeritz, F., Arav, A., 2005. Effect of egg-phosphatidylcholine on the chilling sensitivity and lipid phase transition of Asian elephant (Elephas maximus) spermatozoa. Zoo Biology 24, 233-245.
Abstract: This study was conducted in an effort to improve our understanding of the response of Asian elephant (Elephas maximus, Em) spermatozoa to chilling.  Semen was collected from two elephant bulls by means of the manual rectal stimulation method. Five out of seven semen collections were deemed to be suitable for use based on motility (ranging from 20% to 60%) and membrane integrity. We evaluated the chilling sensitivity by incubating the sperm with a fluorescent dye (5-carboxyfluorescein diacetate (cFDA)) at 16 degrees C, 12 degrees C, 4 degrees C, and 22 degrees C (control). Cells with an intact membrane retained the dye and were identified as viable. The membrane lipid phase transition (LPT) temperature curve was determined with a Fourier transform infrared (FTIR) spectrometer connected to an FTIR microscope.  The LPT center, T-m, was determined by statistical analysis. The LPT and T-m were also assessed in fresh spermatozoa and spermatozoa incubated with egg yolk or egg-phosphatidylcholine (EPC) liposomes at 16 degrees C, 12 degrees C, 4 degrees C, and 26 degrees C (control). The results show that the membrane integrity of spermatozoa incubated at 16 degrees C, 12 degrees C, and 4 degrees C decreased by 39%, 62%, and 67%, respectively, compared to the control. The LPT temperatures were between room temperature (26 degrees C) and 10 degrees C, with Tm at 14-16 degrees C. The T-m for sperm incubated with liposomes or egg-yolk extender was below the measured range (2 degrees C). Chilling sensitivity was found at a wide range of temperatures and transition temperatures, suggesting the presence of a wide variety of fatty acids (FAs) in the membrane with a high ratio of saturated-to-polyunsaturated FAs. Here we show that the protection  afforded  by the presence of egg yolk or liposomes in the extender is  accomplished by  shifting the T. to below the 4 degrees C point at which chilled semen  is  maintained for transport, or the point at which fast freezing begins to  minimize cellular damage.

Steinetz, B.G., Brown, J.L., Roth, T.L., Czekala, N., 2005. Relaxin concentrations in serum and urine of endangered species: correlations with physiologic events and use as a marker of pregnancy
596. Ann. N. Y. Acad. Sci. 1041, 367-378.
Abstract: Many mammalian species are facing extinction due to problems created by human encroachment, agriculture, pollution, and willful slaughter. Among those at risk are the Asian and African elephant, Sumatran rhinoceros, and giant panda. Conservation groups try to save species in the wild by preserving habitat and limiting animal-human conflicts, often with limited success. Another alternative is to preserve the extant gene pool through captive breeding as a hedge against extinction. Measurement of circulating reproductive hormones is impractical for most wildlife species; determination of urinary or fecal hormone metabolites provides a more viable approach. To aid breeding management, one important tool is the ability to diagnose and monitor pregnancy, especially in species with long gestations (e.g., rhinos over 15 mo and elephants over 20 mo). Unfortunately, measuring progestins often is not useful diagnostically, because concentrations are similar during at least part of the pregnancy and the nonpregnant luteal phase in some species (e.g., elephants, rhinoceroses, and giant pandas). As serum relaxin reliably distinguishes between pregnancy and pseudopregnancy in bitches, relaxin measurement might also provide a method for detecting a successful pregnancy in endangered species. Appropriate immunoassay reagents have enabled the estimation of relaxin concentrations in the serum of elephants and rhinos and the determination of pregnancy establishment and the outcome. Relaxin was also detected in panda serum and urine. However, the extreme variability of the time between observed mating and parturition and the confounding factors of delayed implantation, pseudopregnancy, and frequent fetal resorptions made it impossible to use the panda relaxin data as a specific marker of pregnancy

Vidya, T.N., Fernando, P., Melnick, D.J., Sukumar, R., 2005. Population differentiation within and among Asian elephant (Elephas maximus) populations in southern India
675. Heredity 94, 71-80.
Abstract: Southern India, one of the last strongholds of the endangered Asian elephant (Elephas maximus), harbours about one-fifth of the global population. We present here the first population genetic study of free-ranging Asian elephants, examining within- and among-population differentiation by analysing mitochondrial DNA (mtDNA) and nuclear microsatellite DNA differentiation across the Nilgiris-Eastern Ghats, Anamalai, and Periyar elephant reserves of southern India. Low mtDNA diversity and 'normal' microsatellite diversity were observed. Surprisingly, the Nilgiri population, which is the world's single largest Asian elephant population, had only one mtDNA haplotype and lower microsatellite diversity than the two other smaller populations examined. There was almost no mtDNA or microsatellite differentiation among localities within the Nilgiris, an area of about 15,000 km2. This suggests extensive gene flow in the past, which is compatible with the home ranges of several hundred square kilometres of elephants in southern India. Conversely, the Nilgiri population is genetically distinct at both mitochondrial and microsatellite markers from the two more southerly populations, Anamalai and Periyar, which in turn are not genetically differentiated from each other. The more southerly populations are separated from the Nilgiris by only a 40-km-wide stretch across a gap in the Western Ghats mountain range. These results variably indicate the importance of population bottlenecks, social organization, and biogeographic barriers in shaping the distribution of genetic variation among Asian elephant populations in southern India

Vidya, T.N.C., Sukumar, K., 2005. Social organization of the Asian elephant (Elephas maximus) in southern India inferred from microsatellite DNA. J Ethol 23, 205-210.
Abstract: Social organization of the Asian elephant (Elephas maximus) is not well understood in the absence of long-term studies of identified individuals. Adult Asian elephant females and their young offspring of both sexes form matriarchal groups, with pubertal males dispersing from natal groups, but whether these social groups represent families and whether males show locational or social dispersal were unknown. Using nuclear microsatellite loci amplified from dung-extracted DNA of free-ranging elephants in a large southern Indian population, we demonstrate that female-led herds comprise closely related individuals that are indeed families, and that males exhibit non-random locational dispersal.

Vidya, T.N.C., Sukumar, R., 2005. Amplification success and feasibility of using microsatellite loci amplified from dung to population genetic studies of the Asian elephant (Elephas maximus). Current Science 88, 489-492.
Abstract: The use of microsatellite DNA markers in large mammal conservation genetics has been limited by the logistic difficulty in obtaining optimal sources of DNA from free-ranging animals. Here, we show high amplification success of microsatellite DNA from elephant dung samples collected under field conditions. Amplification success depended on how fresh the sample was when collected, and on the sensitivity of primers at individual loci. No significant allelic dropout was observed. We also examined the loci for selective neutrality, independent inheritance, of loci, and the probability of identity of individuals to confirm their utility in studies of population genetic structure.

Walker, C.L., Stewart, E.A., 2005. Uterine fibroids: the elephant in the room. Science 308, 1589-1592.
Abstract: Uterine fibroids (leiomyomas) have historically been viewed as important chiefly as the major indication for hysterectomy. As new therapies are developed, the heterogeneity of this disease becomes therapeutically relevant. An awareness of the role of genetics, the extracellular matrix, and hormones in tumor etiology is key to understanding this disease. 

Wooding, F.B., Stewart, F., Mathias, S., Allen, W.R., 2005. Placentation in the African elephant, Loxodonta africanus: III. Ultrastructural and functional features of the placenta
598. Placenta 26, 449-470.
Abstract: Successful transfer of nutrients to the elephant fetus during pregnancy relies on a variety of placental modifications. Our light and electron microscopical investigations show that the structure is endotheliochorial from implantation to term, with unicellular, never syncytial trophoblast. Light and electron microscope immunocytochemistry shows the restriction of the glucose transporter 1 isoform to the basolateral surfaces of the trophoblast, with the glucose transporter 3 restricted to the apical plasmalemma of the trophoblast. Glucose transport to the fetus therefore requires a sequential use of both isoforms. Light and electron microscope cytochemistry indicate the presence of iron deposits only in the haemophagous zones confirming their iron transport function. No trophoblast areas with high concentrations of Calcium binding protein, specialised for Calcium transport were found. In situ hybridisation demonstrated the presence of IGF-II mRNA in the trophoblast from the earliest stage, with TGFbeta1 and HGF-SF mRNA expressed subsequently but only IGF-II and HGF mRNA present in the second half of pregnancy. The results are briefly discussed in terms of placental growth and function and indicate that the elephant placenta is another example of a unique solution to the variety of problems posed by a resident fetus

Yokoyama, S., Takenaka, N., Agnew, D.W., Shoshani, J., 2005. Elephants and human color-blind deuteranopes have identical sets of visual pigments
626. Genetics 170, 335-344.
Abstract: Being the largest land mammals, elephants have very few natural enemies and are active during both day and night. Compared with those of diurnal and nocturnal animals, the eyes of elephants and other arrhythmic species, such as many ungulates and large carnivores, must function in both the bright light of day and dim light of night. Despite their fundamental importance, the roles of photosensitive molecules, visual pigments, in arrhythmic vision are not well understood. Here we report that elephants (Loxodonta africana and Elephas maximus) use RH1, SWS1, and LWS pigments, which are maximally sensitive to 496, 419, and 552 nm, respectively. These light sensitivities are virtually identical to those of certain "color-blind" people who lack MWS pigments, which are maximally sensitive to 530 nm. During the day, therefore, elephants seem to have the dichromatic color vision of deuteranopes. During the night, however, they are likely to use RH1 and SWS1 pigments and detect light at 420-490 nm

Yokoyama, S., Takenaka, N., Agnew, D.W., Shoshani, J., 2005. Elephants and human color-blind deuteranopes have identical sets of visual pigments. Genetics 170, 335-344.
Abstract: Being the largest land mammals, elephants have very few natural enemies and are acti ve during both day and night. Compared with those of diurnal and nocturnal animals, the eyes of elephants and other arrhythmic species, such as many ungulates and large carnivores, must function in both the bright light of day and dim light of night. Despite their fundamental importance, the roles of photosensitive molecules, visual pigments, in arrhythmic vision are not well understood. Here we report that elephants (Loxodonta africana and Elephas maximus) use RH1, SWS1, and LWS pigments, which are maximally sensitive to 496, 419, and 552 nm, respectively. These light sensitivities are virtually identical to those of certain "color-blind" people who lack MWS pigments, which are maximally sensitive to 530 nm. During the day, therefore, elephants seem to have the dichromatic color vision of deuteranopes. During the night, however, they are likely to use RH1 and SWS1 pigments and detect light at 420-490 nm.

Agatsuma, T., Rajapakse, R.P., Kuruwita, V.Y., Iwagami, M., Rajapakse, R.C., 2004. Molecular taxonomic position of the elephant schistosome, Bivitellobilharzia nairi, newly discovered in Sri Lanka
745. Parasitol. Int. 53, 69-75.
Abstract: Bivitellobilharzia nairi (Mudaliar and Ramanujachar, 1945) Dutt and Srivastava, 1955 was first recorded in India. A number of adult worm specimens of this schistosome species were recovered from a domestic elephant, which died in 1999 in Sri Lanka. This is the first report of this schistosome from Sri Lanka. In the present study, in order to clarify the phylogenetic relationship with other species of schistosomes, sequences from the second internal transcribed spacer (ITS2) of the ribosomal gene repeat, part of the 28S ribosomal RNA gene (28S), and part of the mitochondrial cytochrome c oxidase subunit 1 (CO1) gene from B. nairi were analyzed. Two intraspecific variations were seen within 13 individuals in the ITS2 region. In the CO1 region of the mitochondrial DNA, there were four haplotypes in the nucleotide sequences and two haplotypes in the amino acid sequences. Phylogenetic analysis using the nuclear DNA showed that B. nairi was basal to all of species of the genus Schistosoma. The 28S tree also showed that the mammalian lineage was monophyletic. However, phylogenetic analysis using the mitochondrial DNA showed that B. nairi was nested within the genus Schistosoma. The taxonomical position for this species as well as the contradiction between the results from the nuclear and mitochondrial genes were discussed

Debruyne, R., 2004. [Contribution of molecular phylogeny and morphometrics to the systematics of African elephants]
590. J. Soc. Biol. 198, 335-342.
Abstract: African elephants are conventionally classified as a single species: Loxodonta africana (Blumenbach 1797). However, the discovery in 1900 of a smaller form of the African elephant, spread throughout the equatorial belt of this land, has given rise to a debate over the relevance of a second species of elephant in Africa. The twentieth century has not provided any definite answer to this question. Actually, recent molecular analyses have sustained this issue by advocating either a division of forest elephants into a valid species, or their inclusion as a subspecies of L. africana. Our work initiated at the National Museum of Natural History of Paris provides new molecular (mitochondrial) and morphological (and morphometrical) evidence making it possible to propose a comprehensive phylogenetic hypothesis. It appears that there is no conclusive argument to keep forest elephants (cyclotis form) and savannah elephants (africana form) apart in two distinct species. A high level of mitochondrial introgression between the two forms, as well as a continuum in the morphology of the skulls of the two morphotypes rather suggests that, despite an ancient division, these two taxa freely interbreed wherever their ranges intersect. We thus adopt a conservative systematic position in considering these two forms as two subspecies, respectively: L. africana africana, the savannah elephant, and L. africana cyclotis, the forest elephant. We finally discuss the conservation topic in the light of this systematic framework

Greenwood, A.D., Englbrecht, C.C., MacPhee, R.D., 2004. Characterization of an endogenous retrovirus class in elephants and their relatives
667. BMC. Evol. Biol. 4, 38.
Abstract: BACKGROUND: Endogenous retrovirus-like elements (ERV-Ls, primed with tRNA leucine) are a diverse group of reiterated sequences related to foamy viruses and widely distributed among mammals. As shown in previous investigations, in many primates and rodents this class of elements has remained transpositionally active, as reflected by increased copy number and high sequence diversity within and among taxa. RESULTS: Here we examine whether proviral-like sequences may be suitable molecular probes for investigating the phylogeny of groups known to have high element diversity. As a test we characterized ERV-Ls occurring in a sample of extant members of superorder Uranotheria (Asian and African elephants, manatees, and hyraxes). The ERV-L complement in this group is even more diverse than previously suspected, and there is sequence evidence for active expansion, particularly in elephantids. Many of the elements characterized have protein coding potential suggestive of activity. CONCLUSIONS: In general, the evidence supports the hypothesis that the complement had a single origin within basal Uranotheria

Lazar, J., Rasmussen, L.E., Greenwood, D.R., Bang, I.S., Prestwich, G.D., 2004. Elephant albumin: a multipurpose pheromone shuttle
691. Chem. Biol. 11, 1093-1100.
Abstract: (Z)-7-dodecenyl acetate (Z7-12:Ac) is present in the urine of female Asian elephants (Elephas maximus) approaching ovulation and functions as a female-to-male sex pheromone. Here we show that a significant fraction of the pheromone in the urine is bound to a protein, elephant serum albumin (ESA), and provide evidence for key physiological functions of urinary ESA. Our biochemical and behavioral experiments suggest a three-fold role of ESA in pheromone signaling: (1) transporting Z7-12:Ac from serum into urine; (2) extending the presence of the pheromone in the environment without hampering detection; and (3) targeting pheromone delivery to chemosensory organs through localized release of the ligand induced by a pH change. The exploitation of albumin in pheromone transport clearly distinguishes the elephant from other mammals studied, and complements the uniqueness of elephant anatomy, physiology, and behavior

Thongtip, N., Saikhun, J., Damyang, M., Mahasawangkul, S., Suthunmapinata, P., Yindee, M., Kongsila, A., Angkawanish, T., Jansittiwate, S., Wongkalasin, W., Wajjwalkul, W., Kitiyanant, Y., Pavasuthipaisit, K., Pinyopummin, A., 2004. Evaluation of post-thaw Asian elephant (Elephas maximus) spermatozoa using flow cytometry: the effects of extender and cryoprotectant
711. Theriogenology 62, 748-760.
Abstract: Although the development of semen cryopreservation in the African elephants (Loxodonta africana) has been accomplished, effective procedures for cryopreservation of Asian elephant (Elephas maximus) spermatozoa have not been established. In the present study, we investigate the freezing methods for conservation of Asian elephant spermatozoa under field conditions and identify the most suitable freezing protocols which provide acceptable post-thaw semen quality. Semen was collected from two Asian elephant bulls (EM1 and EM2, 10 ejaculates from each bull) by manual manipulation and were assessed for volume, pH, sperm cell concentration, and progressive motility. Eight out of 20 ejaculates were of acceptable quality (progressive motility >/= 60%), and were used for cryopreservation studies. Semen were frozen in TEST + glycerol, TEST + DMSO, HEPT + glycerol, or HEPT + DMSO. The post-thaw progressive sperm motilities were assessed, and sperm cells were stained with PI and FITC-PNA for membrane and acrosomal integrity assessment using flow cytometry. Post-thaw progressive motility of spermatozoa (EM1: 42.0 +/- 4.3%; EM2: 26.0 +/- 17.3%) and the percentage of membrane and acrosome intact spermatozoa (EM1: 55.5 +/- 8.1%; EM2: 46.3 +/- 6.4%) cryopreserved in TEST + glycerol were significantly higher than (P < 0.05) those frozen in the other medium investigated choices for cryopreservation of Asian elephant spermatozoa. The data support the use of TEST + glycerol as an acceptable cryopreservation media of Asian elephant semen for the establishment of sperm banks

Wasser, S.K., Shedlock, A.M., Comstock, K., Ostrander, E.A., Mutayoba, B., Stephens, M., 2004. Assigning African elephant DNA to geographic region of origin: applications to the ivory trade
673. Proc. Natl. Acad. Sci. U. S. A 101, 14847-14852.
Abstract: Resurgence of illicit trade in African elephant ivory is placing the elephant at renewed risk. Regulation of this trade could be vastly improved by the ability to verify the geographic origin of tusks. We address this need by developing a combined genetic and statistical method to determine the origin of poached ivory. Our statistical approach exploits a smoothing method to estimate geographic-specific allele frequencies over the entire African elephants' range for 16 microsatellite loci, using 315 tissue and 84 scat samples from forest (Loxodonta africana cyclotis) and savannah (Loxodonta africana africana) elephants at 28 locations. These geographic-specific allele frequency estimates are used to infer the geographic origin of DNA samples, such as could be obtained from tusks of unknown origin. We demonstrate that our method alleviates several problems associated with standard assignment methods in this context, and the absolute accuracy of our method is high. Continent-wide, 50% of samples were located within 500 km, and 80% within 932 km of their actual place of origin. Accuracy varied by region (median accuracies: West Africa, 135 km; Central Savannah, 286 km; Central Forest, 411 km; South, 535 km; and East, 697 km). In some cases, allele frequencies vary considerably over small geographic regions, making much finer discriminations possible and suggesting that resolution could be further improved by collection of samples from locations not represented in our study

Archie, E.A., Moss, C.J., Alberts, S.C., 2003. Characterization of tetranucleotide microsatellite loci in the African Savannah Elephant (Loxodonta africana africana). Molecular Ecology Notes 3, 244-246.
Abstract: Most African elephant (Loxodonta africana africana) populations are isolated and thus threatened by a loss of genetic diversity. As a consequence, genetic analysis of African elephant populations will play an increasing role in their conservation, and microsatellite loci will be an important tool in these analyses. Previously published sets of polymorphic microsatellites developed for African elephants are all dinucleotide repeats, which are prone to typing error. Here, we characterize 11 tetranucleotide microsatellite loci in the African elephant. All loci were polymorphic in 32 faecal samples and two tissue samples from 33 individual African savannah elephants. Biology Department, Duke University, Box 90338, Durham, NC 27708, USA; eaa@duke.edu

Debruyne, R., Van Holt, A., Barriel, V., Tassy, P., 2003. Status of the so-called African pygmy elephant (Loxodonta pumilio (NOACK 1906)): phylogeny of cytochrome b and mitochondrial control region sequences. C R Biol 326, 687-697.
Abstract: Among the African elephants, it has been unanimously acknowledged that the forest elephants (cyclotis form) are peculiar, so that they have been elevated to the specific rank. The development of molecular analyses of extant Loxodonta has only focused on two forms yet: the savannah form (africana) and the forest form (cyclotis), disregarding the so-called pygmy elephants (pumilio or fransseni) the systematic status of which has been debated since their discovery. Therefore, we have sampled nine dwarfed-labelled specimens in collection and eight specimens of typical forest elephants that we compared to three savannah elephants and two Asian elephants. Because of the degraded nature of the nuclear DNA content in bone samples of old specimens, we assayed mitochondrial markers; 1961 bp of the mitochondrial genome were sequenced (over a continuous range spanning the cytochrome b gene, tRNA Thr, tRNA Pro, hypervariable region 1 and central conserved region of the control region). Pumilio and cyclotis are not sister-taxa: the phylogenetic analyses rather account for the inclusion of the so-called pygmy elephants within a monophyletic group of forest elephants sensu lato. The internal structure of this clade reveals to depend on isolation and remoteness between populations, characteristics that may have been extensively influenced by climatic variations during the Quaternary period. We conclude that the specific taxon Loxodonta pumilio (or Loxodonta fransseni) should be abandoned. FR 1541 CNRS, Service de systematique moleculaire, Museum national d'histoire naturelle, 43, rue Cuvier, 75005 Paris, France. debruyne@mnhn.fr

Eggert, L.S., Eggert, J.A., Woodruff, D.S., 2003. Estimating population sizes for elusive animals: the forest elephants of Kakum National Park, Ghana. Mol Ecol 12, 1389-1402.
Abstract: African forest elephants are difficult to observe in the dense vegetation, and previous studies have relied upon indirect methods to estimate population sizes. Using multilocus genotyping of noninvasively collected samples, we performed a genetic survey of the forest elephant population at Kakum National Park, Ghana. We estimated population size, sex ratio and genetic variability from our data, then combined this information with field observations to divide the population into age groups. Our population size estimate was very close to that obtained using dung counts, the most commonly used indirect method of estimating the population sizes of forest elephant populations. As their habitat is fragmented by expanding human populations, management will be increasingly important to the persistence of forest elephant populations. The data that can be obtained from noninvasively collected samples will help managers plan for the conservation of this keystone species. Ecology, Behavior and Evolution Section, Division of Biological Sciences, University of California, San Diego 92093-0116, USA. lori_eggert@hotmail.com

Fernando, P., Vidya, T.N., Payne, J., Stuewe, M., Davison, G., Alfred, R.J., Andau, P., Bosi, E., Kilbourn, A., Melnick, D.J., 2003. DNA analysis indicates that Asian elephants are native to Borneo and are therefore a hgh priority for conservation. PLoS Biol. 1, E6 (Epub 2003 Aug 18).
Abstract: The origin of Borneo's elephants is controversial. Two competing hypotheses argue that they are either indigenous, tracing back to the Pleistocene, or were introduced, descending from elephants imported in the 16th-18th centuries. Taxonomically, they have either been classified as a unique subspecies or placed under the Indian or Sumatran subspecies. If shown to be a unique indigenous population, this would extend the natural species range of the Asian elephant by 1300 km, and therefore Borneo elephants would have much greater conservation importance than if they were a feral population. We compared DNA of Borneo elephants to that of elephants from across the range of the Asian elephant, using a fragment of mitochondrial DNA, including part of the hypervariable d-loop, and five autosomal microsatellite loci. We find that Borneo's elephants are genetically distinct, with molecular divergence indicative of a Pleistocene colonisation of Borneo and subsequent isolation. We reject the hypothesis that Borneo's elephants were introduced. The genetic divergence of Borneo elephants warrants their recognition as a separate evolutionary significant unit. Thus, interbreeding Borneo elephants with those from other populations would be contraindicated in ex situ conservation, and their genetic distinctiveness makes them one of the highest priority populations for Asian elephant conservation. Center for Environmental Research and Conservation, Columbia University, New York, New York, United States of America. pf133@columbia.edu

Fernando, P., Vidya, T.N., Rajapakse, C., Dangolla, A., Melnick, D.J., : 2003. Reliable noninvasive genotyping: fantasy or reality? J Hered 94, 115-123.
Abstract: Noninvasive genotyping has not gained wide application, due to the notion that it is unreliable, and also because remedial measures are time consuming and expensive. Of the wide variety of noninvasive DNA sources, dung is the most universal and most widely used in studies. We have developed collection, extraction, and amplification protocols that are inexpensive and provide a high level of success in amplifying both mitochondrial and nuclear DNA from dung. Here we demonstrate the reliability of genotyping from elephant dung using these protocols by comparing results from dung-extracted DNA to results from blood-extracted DNA. The level of error from dung extractions was only slightly higher than from blood extractions, and conducting two extractions from each sample and a single amplification from each extraction was sufficient to eliminate error. Di-, tri-, and tetranucleotide loci were equally reliable, and low DNA quantity and quality and PCR inhibitors were not a major problem in genotyping from dung. We discuss the possible causes of error in genotyping with particular reference to noninvasive samples and suggest methods of reducing such error. Center for Environmental Research and Conservation, Columbia University, 1200 Amsterdam Avenue, New York, NY 10027, USA. pf133@columbia.edu

Fronicke, L., Wienberg, J., Stone, G., Adams, L., Stanyon, R., 2003. Towards the delineation of the ancestral eutherian genome organization: comparative genome maps of human and the African elephant (Loxodonta africana) generated by chromosome painting. Proc R Soc Lond B Biol Sci. 270, 1331-1340.
Abstract: This study presents a whole-genome comparison of human and a representative of the Afrotherian clade, the African elephant, generated by reciprocal Zoo-FISH. An analysis of Afrotheria genomes is of special interest, because recent DNA sequence comparisons identify them as the oldest placental mammalian clade. Complete sets of whole-chromosome specific painting probes for the African elephant and human were constructed by degenerate oligonucleotide-primed PCR amplification of flow-sorted chromosomes. Comparative genome maps are presented based on their hybridization patterns. These maps show that the elephant has a moderately rearranged chromosome complement when compared to humans. The human paint probes identified 53 evolutionary conserved segments on the 27 autosomal elephant chromosomes and the X chromosome. Reciprocal experiments with elephant probes delineated 68 conserved segments in the human genome. The comparison with a recent aardvark and elephant Zoo-FISH study delineates new chromosomal traits which link the two Afrotherian species phylogenetically. In the absence of any morphological evidence the chromosome painting data offer the first non-DNA sequence support for an Afrotherian clade. The comparative human and elephant genome maps provide new insights into the karyotype organization of the proto-afrotherian, the ancestor of extant placental mammals, which most probably consisted of 2n=46 chromosomes.  Erratum in: Proc R Soc Lond B Biol Sci. 2003 Dec 22;270(1533):2639.  Comparative Molecular Cytogenetics Section, National Cancer Institute-Genetics Branch, National Cancer Institute-Frederick, Nationa Institutes of Health, Building 560, Room 11-75, Frederick, MD 21702-1201, USA. froenickel@gmx.net

Hermes, R., Arav, A., Saragusty, J., Goeritz, F., Pettit, M., Blottner, S., Flach, E., Eshkar, G., Boardman, W., Hildebrandt, T.B. Cryopreservation of Asian elephant spermatozoa using directional freezing. Proc.Amer Assoc of Zoo Veterinarians.  264. 2003.
Ref Type: Conference Proceeding
Abstract: Male infertility and absence of males in a facility are contributing factors to the limited reproduction of Asian elephants in captivity. Subsequent transport for breeding purposes increase social stress, risks of disease transmission and management costs. Recent success in artificial insemination eliminated these obstacles only transporting the semen. However, the transport of fresh semen involves logistical<bold> </bold>difficulties: access to semen donors, consistent semen quality and preservation of the spermatozoa during transport. The use of cryo-preserved sperm for AI can partially overcome these problems and can additionally be used for the establishment of Genome Resource Banks. However, to date, attempts to cryo-preserve Asian elephant spermatozoa have failed due to its sensitivity to freezing. Aims of this study were to identify the temperature range during which spermatozoa is most sensitive to chilling injury, and to use directional freezing (DF) to reduce cell damage during the freezing process. Semen was collected from two Asian elephants by manual stimulation. DF was used for freezing sperm samples. In contrast to conventional freezing methods DF facilitated a fast cooling rate, controlled ice crystal formation and cryopreservation of large volumes. Samples extended with a variety of DMSO extenders showed post thaw motility of 30-40%. DF was able to cryo-preserve Asian Elephant spermatozoa for the first time. As DF seems to reduce cryo injury it may become of interest to optimize existing cryopreservation protocols of other endangered species, or to make cryopreservation even possible in species with cryo-sensitive spermatozoa.

Vidya, T.N.C., Kumar, V.R., Arivazhagan, C., Sukumar, R., 2003. Application of molecular sexing to free-ranging Asian elephant (Elephas maximus) populations in southern India. Current Science 85, 1074-1077.
Abstract: Selective poaching of Asian elephant (Elephas maximus) males for ivory has resulted in highly female-biased adult sex ratios, necessitating regular monitoring of population structure and demography. We demonstrate that molecular sexing from dung-extracted DNA, based on ZFX-ZFY fragment amplification and ZFY-specific BamHI site restriction, can be applied to estimate sex ratios of free-ranging Asian elephants, in addition to or instead of field demographic methods. The adult sex ratios using molecular sexing in Nagarahole and Mudumalai-Bandipur reserves during May 2001 were 1: 3.1, matching the demography-based sex ratio for the same month, and 1: 9.4, respectively.

Yang, F., Alkalaeva, E.Z., Perelman, P.L., Pardini, A.T., Harrison, W.R., O'Brien, P.C., Fu, B., 2003. Reciprocal chromosome painting among human, aardvark, and elephant (superorder Afrotheria) reveals the likely eutherian ancestral karyotype. Proc Natl Acad Sci U S A 100, 1062-1066.
Abstract: The Afrotheria, a supraordinal grouping of mammals whose radiation is rooted in Africa, is strongly supported by DNA sequence data but not by their disparate anatomical features. We have used flow-sorted human, aardvark, and African elephant chromosome painting probes and applied reciprocal painting schemes to representatives of two of the Afrotherian orders, the Tubulidentata (aardvark) and Proboscidea (elephants), in an attempt to shed additional light on the evolutionary affinities of this enigmatic group of mammals. Although we have not yet found any unique cytogenetic signatures that support the monophyly of the Afrotheria, embedded within the aardvark genome we find the strongest evidence yet of a mammalian ancestral karyotype comprising 2n = 44. This karyotype includes nine chromosomes that show complete conserved synteny to those of man, six that show conservation as single chromosome arms or blocks in the human karyotype but that occur on two different chromosomes in the ancestor, and seven neighbor-joining combinations (i.e., the synteny is maintained in the majority of species of the orders studied so far, but which corresponds to two chromosomes in humans). The comparative chromosome maps presented between human and these Afrotherian species provide further insight into mammalian genome organization and comparative genomic data for the Afrotheria, one of the four major evolutionary clades postulated for the Eutheria.

Comstock, K.E., Georgiadis, N., Pecon-Slattery, J., Roca, A.L., Ostrander, E.A., O'Brien, S.J., Wasser, S.K., 2002. Patterns of molecular genetic variation among African elephant populations. Mol Ecol 11, 2489-2498.
Abstract: The highly threatened African elephants have recently been subdivided into two species, Loxodonta africana (savannah or bush elephant) and L. cyclotis (forest elephant) based on morphological and molecular studies. A molecular genetic assessment of 16 microsatellite loci across 20 populations (189 individuals) affirms species level genetic differentiation and provides robust genotypic assessment of species affiliation. Savannah elephant populations show modest levels of phylogeographic subdivision based on composite microsatellite genotype, an indication of recent population isolation and restricted gene flow between locales. The savannah elephants show significantly lower genetic diversity than forest elephants, probably reflecting a founder effect in the recent history of the savannah species.

Nyakaana, S., Arctander, P., Siegismund, H.R., 2002. Population structure of the African savannah elephant inferred from mitochondrial control region sequences and nuclear microsatellite loci. Heredity 89, 90-98.
Abstract: Two hundred and thirty-six mitochondrial DNA nucleotide sequences were used in combination with polymorphism at four nuclear microsatellite loci to assess the amount and distribution of genetic variation within and between African savannah elephants. They were sampled from 11 localities in eastern, western and southern Africa. In the total sample, 43 haplotypes were identified and an overall nucleotide diversity of 2.0% was observed. High levels of polymorphism were also observed at the microsatellite loci both at the level of number of alleles and gene diversity. Nine to 14 alleles per locus across populations and 44 alleles in the total sample were found. The gene diversity ranged from 0.51 to 0.72 in the localities studied. An analysis of molecular variance showed significant genetic differentiation between populations within regions and also between regions. The extent of subdivision between populations at the mtDNA control region was approximately twice as high as shown by the microsatellite loci (mtDNA F(ST) = 0.59; microsatellite R(ST) = 0.31). We discuss our results in the light of Pleistocene refugia and attribute the observed pattern to population divergence in allopatry accompanied by a recent population admixture following a recent population expansion.

Roca, A.L., Georgiadis, N., Pecon-Slattery, J., O'Brien, S.J., 2002. Genetic evidence for two species of elephant in Africa. Science 293, 1473-1477.
Abstract: Elephants from the tropical forests of Africa are morphologically distinct from savannah or bush elephants. Dart-biopsy samples from 195 free-ranging African elephants in 21 populations were examined for DNA sequence variation in four nuclear genes (1732 base pairs). Phylogenetic distinctions between African forest elephant and savannah elephant populations corresponded to 58% of the difference in the same genes between elephant genera Loxodonta (African) and Elephas (Asian). Large genetic distance, multiple genetically fixed nucleotide site differences, morphological and habitat distinctions, and extremely limited hybridization of gene flow between forest and savannah elephants support the recognition and conservation management of two African species: Loxodonta africana and Loxodonta cyclotis.

Vogel, G., 2002. Ecology. African elephant species splits in two. Science 293, 1414.

Burkhardt, S., Goltz, M., Bergmann, V., Ochs, A., Weiler, H., Hentschke, J., 2001. Genetic and ultrastructural characterization of a European isolate of the fatal endotheliotropic elephant herpesvirus. Journal of General Virology 82, 475-482.
Abstract: A male Asian elephant (Elephas maximus) died at the Berlin zoological gardens in August 1998 of systemic infection with the novel endotheliotropic elephant herpesvirus (EIHV-1). This virus causes a fatal haemorrhagic disease in Asian elephants, the so-called endothelial inclusion body disease, as reported from North American zoological gardens. In the present work, EIHV-1 was visualized ultrastructurally in affected organ material. Furthermore, a gene block comprising the complete glycoprotein B (gB) and DNA polymerase (DPOL) genes as well as two partial genes was amplified by PCR-based genome walking and sequenced. The gene content and arrangement were similar to those of members of the Betaherpesvirinae. However, phylogenetic analysis with gB and DPOL consistently revealed a very distant relationship to the betaherpesviruses. Therefore, EIHV-1 may be a member of a new genus or even a new herpesvirus subfamily. The sequence information generated was used to set up a nested-PCR assay for diagnosis of suspected cases of endothelial inclusion body disease. Furthermore, it will aid in the development of antibody-based detection methods and of vaccination strategies against this fatal herpesvirus infection in the endangered Asian elephant.

Comstock, K.E., Ostrander, E.A., Wasser, S.K. A Genetic Method for Determining the Geographic Origin of Poached African Elephant Ivory. A Research Update on Elephants and Rhinos; Proceedings of the International Elephant and Rhino Research Symposium, Vienna, June 7-11, 2001.  256. 2001. Vienna, Austria, Schuling Verlag. 2001.
Ref Type: Conference Proceeding

Fernando, P., Melnick, D.J., 2001. Molecular sexing eutherian mammals. Molecular Ecology Notes 1, 350-353.
Abstract: Mammals can be molecularly sexed by polymerase chain reaction (PCR) amplification of Y chromosome fragments or coamplification of homologous fragments from both sex chromosomes, which are discriminated by size  polymorphism or Y-specific restriction digestion. Although coamplification of X and Y fragments is more reliable, size polymorphism in homologous fragments is uncommon and Y-specific restriction site identification requires screening with a battery of enzymes or cloning. Here we describe a simple approach, using 'double peaks' in the chromatogram upon direct sequencing of PCR products from males, to identify Y-specific restriction sites, and demonstrate its utility by application to a range of taxa. We demonstrate its utility by application to a range of mammalian taxa; Probocideans: Asian elephant (Elephas maximus), Perrisodactyls: Indian rhinoceros (Rhinoceros unicornis), Carnivores: domestic dog (Canis familiaris) and Primates: Tonkean macaque (Macaca tonkeana).

Fernando, P., Vidya, T.N.C., Melnick, D.J., 2001. Isolation and characterization of tri- and tetranucleotide microsatellite loci in the Asian elephant, Elephas maximus. Molecular Ecology Notes 1, 232-233.
Abstract: Asian elephants (Elephas maximus) are an endangered species. Their future survival depends on intensive conservation and management, based on in-depth knowledge of particular populations. Molecular genetic methods, especially microsatellite analysis through noninvasive sampling, provides an effective means of obtaining such information. The use of tri- and tetranucleotide microsatellite markers is advantageous in noninvasive sampling through dung analysis. Here we describe the isolation and characterization of five tri- and tetranucleotide markers in the Asian elephant. All five loci were found to be polymorphic in a sample of 20 Asian elephants from Sri Lanka.

Fleischer, R.C., Perry, E.A., Muralidharan, K., Stevens, E.E., Wemmer, C.M., 2001. Phylogeography of the Asian elephant (Elephas maximus) based on mitochondrial DNA. Evolution 55, 1882-1892.
Abstract: Populations of the Asian elephant (Elephas maximus) have been reduced in size and become highly fragmented during the past 3000 to 4000 years. Historical records reveal elephant dispersal by humans via trade and war. How have these anthropogenic impacts affected genetic variation and structure of Asian elephant populations? We sequenced mitochondrial DNA (mtDNA) to assay genetic variation and phylogeography across much of the Asian elephant's range. Initially, we compared cytochrome b sequences (cyt b) between 9 Asian and 5 African elephants and used the fossil-based age  of their separation (_5 million years ago) to obtain a rate of about 0.013  (95% CI=0.011-0.018) corrected sequence divergence per million years. We also assessed variation in part of the mtDNA control region (CR) and adjacent tRNA genes in 57 Asian elephants from 7 countries (Sri Lanka,  India, Nepal, Myanmar, Thailand, Malaysia, and Indonesia). Asian elephants had typical levels of mtDNA variation, and coalescence analyses suggested their populations were growing in the late Pleistocene. Reconstructed  phylogenies revealed 2 major clades (A and B) differing on average by HKY85/GAMMA-corrected distances of 0.020 for cyt b and 0.050 for the CR segment (corresponding to a coalescence time based on our cyt b rate of   _1.2 million years). Individuals of both major clades existed in all locations, but Indonesia and Malaysia. Most elephants from Malaysia and all from Indonesia were in well-supported, basal clades within clade A, thus supporting their status as evolutionarily significant units (ESUs). The proportion of clade A individuals decreased to the north, which could result from retention and subsequent loss of ancient lineages in long-term stable populations or, perhaps more likely, via recent mixing of 2 expanding populations that were isolated in the mid-Pleistocene. The distribution of clade A individuals appeared to be impacted by human trade in elephants among Myanmar, Sri Lanka, and India, and the subspecies and  ESU statuses of Sri Lankan elephants were not supported by molecular data.

Houck, M.L., Kumamoto, A.T., Gallagher, D.S.Jr., Benirschke, K., 2001. Comparative cytogenetics of the African elephant (Loxodonta africana) and Asiatic elephant (Elephas maximus). Cytogeneticsand Cell Genetics 93, 249-252.
Abstract: G- and C-banded karyotypes of the two extant species of the mammalian order Proboscidea are presented for the first time. Chromosome complements were 2n=56 in both Loxodonta africana and Elephas maximus. Comparisons between the species demonstrated a high level of chromosome band homology, with 26 conserved autosomal pairs. The normal diploid karyotype of L. africana had 25 acrocentric/telocentric and two metacentric/submetacentric autosomal pairs. E. maximus differed by having one less acrocentric and one additional submetacentric pair due to either a heterochromatic arm addition or deletion involving autosomal pair 27. Several acrocentric autosomes of L. africana exhibited small short arms that were absent in homologous chromosomes of E. maximus. The X chromosomes in both species were large submetacentric elements and were homologous. However, the small acrocentric Y chromosomes differed; in E. maximus it was slightly larger and had more distinct G-bands than its counterpart in L. africana. Extant Elephantidae appear to be relatively conservative in their rates of chromosomal change compared to some other mammalian families. The high-quality banded karyotypes presented here should prove useful as references for future chromosome analyses of elephant populations and in comparative cytogenetic studies with other ungulate orders.

Muller, S., Steinborn, R., Schwammer, H., Muller, M. Molecular Genetic Identification of Elephant Products. A Research Update on Elephants and Rhinos; Proceedings of the International Elephant and Rhino Research Symposium, Vienna, June 7-11, 2001.  277. 2001. Vienna, Austria, Schuling Verlag. 2001.
Ref Type: Conference Proceeding

Murphy, W.J., Eizirik, E., Johnson, W.E., Zhang, Y.P., Ryder, O.A., O'Brien, S.J., 2001. Molecular phylogenetics and the origins of placental mammals. Nature 409, 614-618.
Abstract: The precise hierarchy of ancient divergence events that led to the present assemblage of modern placental mammals has been an area of controversy among morphologists, paleontologists and molecular evolutionists. Here we address the potential weaknesses of limited character and taxon sampling in a comprehensive molecular phylogenetic analysis of 64 species sampled across all extant orders of placental mammals. We examined sequence variation in 18 homologous gene segments (including nearly 10,000 base pairs) that were selected for maximal phylogenetic informativeness in resolving the hierarchy of early mammalian divergence. Phylogenetic analyses identify four primary superordinal clades: (I) Afrotheria (elephants, manatees, hyraxes, tenrecs, aardvark and elephant shrews); (II) Xenarthra (sloths, anteaters and armadillos); (III) Glires (rodents and lagomorphs), as a sister taxon to primates, flying lemurs and tree shrews; and (IV) the remaining orders of placental mammals (cetaceans, artiodactyles, perissodactyles, carnivores, pangolins, bats and core insectivores). Our results provide new insight into the pattern of the early placental mammal radiation.

Okayama, T., Sugardjito, J., Yusuf, I. Mitochondrial DNA Analysis of Sumatran Elephants. A Research Update on Elephants and Rhinos; Proceedings of the International Elephant and Rhino Research Symposium, Vienna, June 7-11, 2001.  278-281. 2001. Vienna, Austria, Schuling Verlag. 2001.
Ref Type: Conference Proceeding

Southern, S. Molecular analysis of stress-activated proteins and genes in dolphins and whales: a new technique for monitoring environmental stress. Proc AAZV and AAAM Joint Conference.  240-242. 2001.
Ref Type: Conference Proceeding
Abstract: In the past several decades, there has been a worldwide increase in marine diseases resulting in mass mortality among all major taxa and shifts in ecologic community structures in the oceans.1 Marine mammals have experienced a pandemic of morbilliviral infections and outbreaks of diseases caused by influenza viruses, fungi and algal toxins. Many of the disease outbreaks appear to have been facilitated by increased environmental stress burden in the global marine ecosystems due to changing environmental conditions triggered by climate variability and human activities. It is imperative to develop novel health-monitoring tools that could guide the management of marine ecosystems and facilitate the conservation of key species. Our research is focused on the molecular mechanisms underlying molecular stress response in humans and cetaceans exposed to
environmental stress and disease. We have developed new techniques for detecting the molecular signature of stress based on molecular analysis of stress-activated proteins and genes in field tissue specimens.2 The detection of molecular stress signature has been applied to evaluate the impact of tuna fishery on the spotted dolphins in the Eastern Tropical Pacific, the effects of coastal pollution on the beluga whales in the St. Lawrence River, and the idiopathic population decline of the North Atlantic right whale population.

Tiedemann, R., Kurt, F., Gonnesekere, N., Gunasekera, M., Ratnasooriya, W.D. Population Dynamics in Wild-Living and Captive Asian Elephants: Demographic and Genetic Aspects. A Research Update on Elephants and Rhinos; Proceedings of the International Elephant and Rhino Research Symposium, Vienna, June 7-11, 2001.  140. 2001. Vienna, Austria, Schuling Verlag. 2001.
Ref Type: Conference Proceeding

Whitehouse, A.M., Harley, E.H. Genetic Diversity and Paternity of the Elephant Population in Addo Elephant National Park, South Africa. A Research Update on Elephants and Rhinos; Proceedings of the International Elephant and Rhino Research Symposium, Vienna, June 7-11, 2001.  146. 2001. Vienna, Austria, Schuling Verlag. 2001.
Ref Type: Conference Proceeding

Whitehouse, A.M., Harley, E.H., 2001. Post-bottleneck genetic diversity of elephant populations in South Africa,revealed using microsatellite analysis. Mol Ecol 10, 2139-2149.
Abstract: Widespread hunting had fragmented and severely reduced elephant populations in South Africa by 1900. Elephant numbers increased during the 1900s, although rates of recovery of individual populations varied. The Kruger National Park elephant population increased rapidly, to more than 6000 by 1967, with recruitment boosted by immigration from Mozambique. The Addo Elephant National Park population was reduced to 11 elephants in 1931 and remains relatively small (n = 325). Loss of genetic variation is expected to occur whenever a population goes through a bottleneck, especially when post-bottleneck recovery is slow. Variation at nine polymorphic microsatellite loci was analysed for Kruger and Addo elephants, as well as museum specimens of Addo elephants shot prior to the population bottleneck. Significantly reduced genetic variation and heterozygosity were observed in Addo in comparison to Kruger (mean alleles/locus and H(E): Addo 1.89, 0.18; Kruger 3.89, 0.44). Two alleles not present in the current Addo population were observed in the museum specimens. Addo elephants represent a genetic subset of the Kruger population, with high levels of genetic differentiation resulting from rapid genetic drift. The Kruger population is low in genetic diversity in comparison to East African elephants, confirming this population also suffered an appreciable bottleneck.

Comstock, K.E., Wasser, S.K., Ostrander, E.A., 2000. Polymorphic microsatellite DNA loci identified in the African elephant(Loxodonta africana). Mol Ecol 9, 1004-1006.

Eggert, L.S., Ramakrishnan, U., Mundy, N.I., Woodruff, D.S., 2000. Polymorphic microsatellite DNA markers in the African elephant (Loxodonta africana) and their use in the Asian elephant (Elephas maximus). Molecular Ecology 9, 2223-2225.
Abstract: Genomic DNA from 4 captive African elephants was pooled and used to produce a DNA library. The library was screened with 32P-labelled (CA)15 and (GA)15 probes. 32 microsatellites were detected, 12 of which were uninterrupted and had sufficient flanking DNA to permit design of PCR primers. These primers were used on DNA samples from 10 African savannah elephants from the Frozen Zoo of the Zoological Society of San Diego. Three primer sets amplified monomorphic sequences, 3 did not give a usable product. The number of alleles for the other 6 loci ranged from 3 to 11. The 6 primers that gave polymorphic products were used on samples from 12 Asian elephants from the Frozen Zoo; 1 locus failed to amplify, the others yielded 2-4 alleles. The primers were also tested on DNA extracted from dung samples of 86 African forest elephants from Kakum National Park (Ghana). One set of primers did not amplify a sequence in the forest elephants, another was monomorphic. The remaining 4 loci had expected heterozygosities of 0.521 to 0.760 and observed heterozygosities of 0.377 to 0.747. The nucleotide sequences reported have been deposited with the GenBank database with the accession numbers AF311670-AF311675.

Fernando, P., Lande, R., 2000. Molecular genetic and behavioral analysis of social organization in the Asian elephant (Elephas maximus). Behavioral Ecology and Sociobiology 48, 84-89.
Abstract: We report on the genetic evaluation and behavioural study of social organization in the Asian elephant (Elephas maximus). Although Asian elephants and African elephants (Loxodonta africana) were previously thought to have similar social organizations, our results demonstrate a substantial difference in the complexity and structure of Asian elephant social groupings from that described for African savanna elephants. Photographic cataloguing of individuals, radio telemetry, and behavioural observations in Ruhuna National Park, Sri Lanka, enabled us to assign associated females and young to four groups with overlapping ranges. Genetic sampling of individuals from the four groups in Ruhuna National Park and three other groups in surrounding areas, conducted through PCR amplification and sequencing of mitochondrial DNA from dung, supported the matriarchal nature of female groups and the lack of inter-group transfer of females. Behaviourally and genetically, the identified social groups were best described as "family groups". We did not find any evidence for the existence of social groups of higher complexity than family groups.

Fernando, P., Pfrender, M.E., Encalada, S.E., Lande, R., 2000. Mitochondrial DNA variation, phylogeography and population structure of the Asian elephant. Heredity 84, 362-372.
Abstract: We report the first genetic analysis of free-ranging Asian elephants (Elephas maximus). We sampled 118 elephants from Sri Lanka, Bhutan/North India, and Laos/Vietnam by extracting DNA from dung, PCR amplifying and sequencing 630 nucleotides of mtDNA, including part of the variable left domain of the control region. Comparison with African elephant (Loxodonta africana) sequences indicated a relatively slow molecular clock in the Proboscidea with a sequence divergence of _1%/million years. Genetic diversity within Asian elephants was low, suggesting a small long-term effective population size. 17 haplotypes were identified within Asian elephants, which clustered into 2 well-differentiated assemblages with an estimated Pliocene divergence of 2.5-3.5 million years ago. The 2 assemblages showed incomplete geographical partitioning, suggesting allopatric divergence and secondary admixture. On the mainland, little genetic differentiation was observed between elephant populations of Bhutan and India or Laos and Vietnam. A significant difference in haplotype frequencies but relatively weak subdivision was observed between the Bhutan-India and Laos-Vietnam regions. Significant genetic differentiation was observed between the mainland and Sri Lanka and between northern, mid-latitude and southern regions in Sri Lanka.

Suwattana, D., Koykul, W., Mahasawangkul, S., Kanchanapangka, S., Joerg, H., Stranzinger, G., 2000. The GTG-banded karyotype and telomere FISH in Asian elephants (Elephas maximus). Veterinarni Medicina 45, 10-11.
Abstract: Lymphocyte cultures were performed for chromosome preparations from 70 Asian elephants (Elephas maximus) held captive in the province of Lampang, Thailand. The elephant karyotype was demonstrated using Giemsa staining and GTG banding techniques. The diploid number (2n) was 56 or 28 chromosome pairs, consisting of 6 pairs of bi-armed chromosomes and 21 telocentric chromosome pairs. The chromosomes were classified and numbered according to size, centromere position and banding patterns. Chromosome 1 exhibited a secondary constriction on the proximal third of the p-arm whereas chromosomes 2- 6 showed faint bands probably due to their heterochromatic constitution. The X chromosome was medium submetacentric with distinctive bands while the Y was a small telocentric chromosome with a dark band around the centromere. An idiogram of a total 216 bands was established from the GTG-bands which will be useful for physical gene mapping. Telomeres of the Asian elephant chromosomes were localized using a fluorescein-conjugated peptide nucleic acid (PNA) probe containing a telomeric sequence (TTAGGG). Fluorescence in situ hybridization (FISH) revealed the locations of four spots of telomeres on all chromosomes including the sex chromosomes. Our observations demonstrated that telomeric DNA sequences have been well conserved in the Asian elephants and similar to those shown in almost all vertebrates.

Thomas, M.G., Hagelberg, E., Jones, H.B., Yang, Z., Lister, A.M., 2000. Molecular and morphological evidence on the phylogeny of the Elephantidae. Proceedings of the Royal Society of London SeriesB, Biological-Sciences 267:1561, 2493-2500.
Abstract: The African and Asian elephants and the mammoth diverged ca. 4-6 million years ago and their phylogenetic relationship has been controversial. Morphological studies have suggested a mammoth-Asian elephant relationship, while molecular studies have produced conflicting results. We obtained cytochrome b sequences of up to 545 base pairs from five mammoths, 14 Asian and eight African elephants. A high degree of polymorphism is detected within species. With a dugong sequence used as the outgroup, parsimony and maximum-likelihood analyses support a mammoth-African elephant clade. As the dugong is a very distant outgroup, we employ likelihood analysis to root the tree with a molecular clock, and use bootstrap and Bayesian analyses to quantify the relative support for different topologies. The analyses support the mammoth-African elephant relationship, although other trees cannot be rejected. Ancestral polymorphisms may have resulted in gene trees differing from the species phylogeny. Examination of morphological data, especially from primitive fossil members, indicates that some supposed synapomorphies between the mammoth and Asian elephant are variable, others convergent or autapomorphous. A mammoth-African elephant relationship is not excluded. Our results highlight the need, in both morphological and molecular phylogenetics, for multiple markers and close attention to within-taxon variation and outgroup selection.

Tiedemann, R., Hardy, O., Vekemans, X., Milinkovitch, M.C., 2000. Higher impact of female than male migration on population structure in large mammals. Mol Ecol 9, 1159-1163.
Abstract: We simulated large mammal populations using an individual-based stochastic model under various sex-specific migration schemes and life history parameters from the blue whale and the Asian elephant. Our model predicts that genetic structure at nuclear loci is significantly more influenced by female than by male migration. We identified requisite comigration of mother and offspring during gravidity and lactation as the primary cause of this phenomenon. In addition, our model predicts that the common assumption that geographical patterns of mitochondrial DNA (mtDNA) could be translated into female migration rates (Nmf) will cause biased estimates of maternal gene flow when extensive male migration occurs and male mtDNA haplotypes are included in the analysis.  

Greenwood, A.D., Paabo, S., 1999. Nuclear insertion sequences of mitochondrial DNA predominate in hair but not in blood of elephants. Molecular Ecology 8, 133-137.
Abstract: It was found that PCR primers that amplified a segment of the mitochondrial control region from blood samples from African and Asian elephants mainly amplified integrated nuclear copies of mitochondrial DNA in hair samples. It is concluded that, in some species under some circumstances, DNA from hair may yield unreliable results.

Greenwood, A.D., Capelli, C., Possnert, G., Paabo, S., 1999. Nuclear DNA sequences from late Pleistocene megafauna. Mol Biol Evol 16, 1466-1473.
Abstract: We report the retrieval and characterization of multi- and single-copy nuclear DNA sequences from Alaskan and Siberian mammoths (Mammuthus primigenius). In addition, a nuclear copy of a mitochondrial gene was recovered. Furthermore, a 13,000-year-old ground sloth and a 33,000-year-old cave bear yielded multicopy nuclear DNA sequences. Thus, multicopy and single-copy genes can be analyzed from Pleistocene faunal remains. The results also show that under some circumstances, nucleotide sequence differences between alleles found within one individual can be distinguished from DNA sequence variation caused by postmortem DNA damage. The nuclear sequences retrieved from the mammoths suggest that mammoths were more similar to Asian elephants than to African elephants.

Holt, W.V., Pickard, A.R., 1999. Role of reproductive technologies and genetic resource banks in animal conservation. Rev Reprod 4, 143-150.
Abstract: In combination with modem reproductive technologies, there is potential to use frozen and stored germplasm (genetic resource banks) to support conservation measures for the maintenance of genetic diversity in threatened species. However, turning this idea into reality is a complex process, requiring interdisciplinary collaboration and clearly defined goals. As the number of species deserving the attention of conservation scientists is overwhelmingly large, yet detailed knowledge of reproductive physiology is restricted to relatively few of them, choosing which species to conserve is one of the most difficult issues to be tackled. Besides the direct application of technologically advanced reproductive procedures, modern approaches to non-invasive endocrine monitoring play an important role in optimizing the success of natural breeding programmes. Through the analysis of urine and faecal samples, this type of technology provides invaluable management information about the reproductive status of diverse species. For example, it is possible to diagnose pregnancy and monitor oestrous cycles in elephants and rhinos without causing stress through restraint for sample collection. In this review, we identify the potential contribution of reproductive biology and genetic resource banks to animal conservation, but also highlight the complexity of issues determining the extent to which this potential can be achieved.

Nyakaana, S., Arctander, P., 1999. Population genetic structure of the African elephant in Uganda based on variation at mitochondrial and nuclear loci: evidence for male-biased gene flow. Mol Ecol 8, 1105-1115.
Abstract: A drastic decline has occurred in the size of the Uganda elephant population in the last 40 years, exacerbated by two main factors; an increase in the size of the human population and poaching for ivory. One of the attendant consequences of such a decline is a reduction in the amount of genetic diversity in the surviving populations due to increased effects of random genetic drift. Information about the amount of genetic variation within and between the remaining populations is vital for their future conservation and management. The genetic structure of the African elephant in Uganda was examined using nucleotide variation of mitochondrial control region sequences and four nuclear microsatellite loci in 72 individuals from three localities. Eleven mitochondrial DNA (mtDNA) haplotypes were observed, nine of which were geographically localized. We found significant genetic differentiation between the three populations at the mitochondrial locus while three out of the four microsatellite loci differentiated KV and QE, one locus differentiated KV and MF and no loci differentiated MF and QE. Expected heterozygosity at the four loci varied between 0.51 and 0.84 while nucleotide diversity at the mitochondrial locus was 1.4%. Incongruent patterns of genetic variation within and between populations were revealed by the two genetic systems, and we have explained these in terms of the differences in the effective population sizes of the two
genomes and male-biased gene flow between populations.

Waddell, P.J., Cao, Y., Hauf, J., Hasegawa, M., 1999. Using novel phylogenetic methods to evaluate mammalian mtDNA, including amino acid-invariant sites-LogDet plus site stripping, to detect internal conflicts in the data, with special reference to the positions of hedgehog, armadillo, and elephant. Syst Biol 48, 31-53.
Abstract: We look at the higher-order phylogeny of mammals, analyzing in detail the complete mtDNA sequences of more than 40 species. We test the support for several proposed superordinal relationships. To this end, we apply a number of recently programmed methods and approaches, plus better-established methods. New pairwise tests show highly significant evidence that amino acid frequencies are changing among nearly all the genomes studied when unvaried sites are ignored. LogDet amino acid distances, with modifications to take into account invariant sites, are combined with bootstrapping and the Neighbor Joining algorithm to account for these violations of standard models. To weight the more slowlyevolving sites, we exclude the more rapidly evolving sites from the data by using "site stripping". This leads to changing optimal trees with nearly all methods. The bootstrap support for many hypotheses varies widely between methods, and few hypotheses can claim unanimous support from these data. Rather, we uncover good evidence that many of the earlier branching patterns in the placental subtree could be incorrect, including the placement of the root. The tRNA genes, for example, favor a split between the group hedgehog, rodents, and primates versus all other sequenced placentals. Such a grouping is not ruled out by the amino acid sequence data. A grouping of all rodents plus rabbit, the old Glires hypothesis, is also feasible with stripped amino acid data, and rodent monophyly is also common. The elephant sequence allows confident rejection of the older taxon Ferungulata (Simpson, 1945). In its place, the new taxa Scrotifera and Fereuungulata are defined. A new likelihood ratio test is used to detect differences between the optimal tree for tRNA versus that for amino acids. While not clearly significant as made, some results indicate the test is tending towards significance with more general models of evolution. Individual placement tests suggest alternative positions for hedgehog and elephant. Congruence arguments to support elephant and armadillo together are striking, suggesting a superordinal group composed of Xenarthra and African endemic mammals, which in turn may be near the root of the placental subtree. Thus, while casting doubt on some recent conclusions, the analyses are also unveiling some interesting new possibilities.

Burk, N.E., Messer, L.A., Ernst, C.W., Rothschild, M.F., 1998. Identification of sequence tagged sites in the Asian and African elephant. Animal Biotechnology 9, 155-160.
Abstract: PCR was used to identify sequence-tagged sites in Asian and African elephants. Heterologous primers were designed from sequences available in other species. Fragments of 141 bp for retinoic acid receptor-beta and 327 bp for leptin receptor were obtained by amplifying genomic Asian and African elephant DNA. The leptin receptor fragment included an intron of 164 bp. A 417 bp fragment for melatonin receptor 1a was obtained from Asian elephants only. All PCR products were sequenced and comparison computations were made at the nucleotide and amino acid levels with sequences available in the GenBank database. The nucleotide sequences were identical for Asian and African elephants for retinoic acid receptor-beta and differed by only 3 bp for leptin receptor. Deduced amino acid sequences were identical for both sequence-tagged sites in the 2 species. The sequences from elephants were relatively similar to those of other mammals and less similar to those of fowls. The nucleotide sequences have been deposited in the GenBank database with accession numbers U95047, U95050 and U95048 for melatonin receptor 1a, retinoic acid receptor-beta and leptin receptor of Asian elephants and U95051 and U95049 for retinoic acid receptor beta and leptin receptor of African elephants respectively.

Chen, Y.Z., Liu, R.Q., Nai, W.H., Wang, J.H., He, C.H., Bao, Y.F., Lei, W.H., 1998. The chromosomes of the Asian elephant. Zoological Research 19, 93-94.

Gilmore, J.A., McGann, L.E., Ashworth, E., Acker, J.P., Raath, J.P., Bush, M., Critser, J.K., 1998. Fundamental cryobiology of selected African mammalian spermatozoa and its role in biodiversity preservation through the development of genome resource banking. Anim Reprod Sci 53, 277-197.
Abstract: Fundamental cryobiological characteristics of spermatozoa from threatened or endangered species must be determined for successful cryopreservation techniques to be established. In this study, spermatozoa from four diverse species, impala (Aepyceros melampus), wart hog (Phacochoerus aethiopicus), elephant (Loxodonta africana), and lion (Panthera leo), were collected by electroejaculation or epididymal aspiration. Spermatozoal plasma membrane permeability to water (hydraulic conductivity, Lp) and the osmotically inactive fraction of the sperm cell (Vb) were determined from each species. Changes in cell volume were measured over time using an electronic particle counter. A Kedem-Katchalsky membrane transport model was used to theoretically characterize the data to determine Lp and Vb for each species. In addition to determining plasma membrane characteristics, spermatozoa were also studied to determine their sensitivity to low temperatures and to permeating cryoprotectant solutes. Cells maintained at room temperature (20-22 degrees C) were slowly or rapidly exposed to cold temperatures (1-4 degrees C), and percent motility was estimated to determine the sensitivity of the cells to cooling. Spermatozoa were also in media containing 1 M glycerol, dimethyl sulfoxide or ethylene glycol, and percent motility was measured at 15, 30 and 60 min intervals to determine the sensitivity of the cells to the cryoprotectant agent over time. Results indicate that sperm motility is significantly effected by decreased temperatures and the presence of cryoprotectant agents.

Gunasena, K.T., Lakey, J.R., Villines, P.M., Bush, M., Raath, C., Critser, E.S., McGann, L.E., Critser, J.K., 1998. Antral follicles develop in xenografted cryopreserved African elephant (Loxodonta africana) ovarian tissue. Anim Reprod Sci 53, 265-275.
Abstract: The preservation of germ plasm from endangered species could augment captive breeding programs aimed at maintaining genetic diversity. Mammalian female germ plasm (oocytes) is extremely difficult to collect and cryopreserve; however, a promising alternative is the cryopreservation of ovarian tissue. In the present study, athymic nude (nu/nu) Balb/C mice were used to evaluate in vivo viability of cryopreserved ovarian tissue from Institute of Cancer Research genotype (ICR) mice or elephants. Female mice were ovariectomized prior to transplant of cryopreserved-thawed ovarian tissue from ICR mice (n=4) or elephants (n=6). Control mice were sham operated (n=4) or ovariectomized (n=5). Transplants were in the ovarian bursa, enabling in vivo ovulation and pregnancies from allografts. Vaginal cytology was monitored daily, and the intervals between andduration of epithelial cells present in smears were evaluated. Appearance of epithelial cells in sham-operated and allografted mice were at intervals of 4.3+/-0.6 and 3.3+/-0.5 days, lasting for 1.4+/-0.1 and 1.6+/-0.2 days, respectively. Sporadic incidence of epithelial cells in ovariectomized animals occurred at longer intervals (8.6+/-3.8 days). Females with xenografted elephant ovarian tissue demonstrated epithelial cells in vaginal smears at intervals of 4.5+/-1.0 days, for 2.5+/-0.5 days duration, which was significantly longer than the other groups (P < 0.05). Histological evaluation of tissues at the time of epithelial cells in smears demonstrated well-developed antral follicles, although oocytes were of poor morphological appearance or only cumulus-like complexes were seen. The nude mouse model is effective for assessing cryopreserved ovarian tissue xenograft function which can support the development of antral follicles.

Noro, M., Masuda, R., Dubrovo, I.A., Yoshida, M.C., Kato, M., 1998. Molecular phylogenetic inference of the woolly mammoth Mammuthus primigenius, based on complete sequences of mitochondrial cytochrome b and 12S ribosomal RNA genes. J Mol Evol 46, 314-326.
Abstract: Complete sequences of cytochrome b (1,137 bases) and 12S ribosomal RNA (961 bases) genes in mitochondrial DNA were successfully determined from the woolly mammoth (Mammuthus primigenius), African elephant (Loxodonta africana), and Asian elephant (Elephas maximus). From these sequence data, phylogenetic relationships among three genera were examined. Molecular phylogenetic trees reconstructed by the neighbor-joining and the maximum parsimony methods provided an identical topology both for cytochrome b and 12S rRNA genes. These results support the "Mammuthus-Loxodonta" clade, which is contrary to some previous morphological reports that Mammuthus is more closely related to Elephas than to Loxodonta.

Nyakaana, S., Arctander, P., 1998. Isolation and characterization of microsatellite loci in the African elephant,Loxodonta africana. Mol Ecol 7, 1436-1437.

Madsen, O., Deen, P.M., Pesole, G., Saccone, C., De Jong, W.W., 1997. Molecular evolution of mammalian aquaporin-2: further evidence that elephant shrew and aardvark join the paenungulate clade. Mol Biol Evol 14, 363-371.

Ozawa, T., Hayashi, S., Mikhelson, V.M., 1997. Phylogenetic position of mammoth and Steller's sea cow within Tethytheria demonstrated by mitochondrial DNA sequences. J Mol Evol 44, 406-413.
Abstract: Here we report DNA sequences from mitochondrial cytochrome b gene segments (1,005 base pairs per species) for the extinct woolly mammoth (Mammuthus primigenius) and Steller's sea cow (Hydrodamalis gigas) and the extant Asian elephant (Elephas maximus), the Western Indian manatee (Trichechus manatus), and the hyrax (Procavia capensis). These molecular data have allowed us to construct the phylogeny for the Tethytheria. Our molecular data resolve the trichotomy between the two species of living elephants and the mammoth and confirm that the mammoth was more closely related to the Asian elephant than to the African elephant. Our data also suggest that the sea cow-dugong divergence was likely as ancient as the dugong-manatee split, and it appears to have been much earlier (22 million years ago) than had been previously estimated (4-8 million years ago) by immunological comparison.

Yang, H., Golenberg, E.M., Shoshani, J., 1997. Proboscidean DNA from museum and fossil specimens: an assessment of ancient DNA extraction and amplification techniques. Biochem Genet 35, 165-179.

Lavergne, A., Douzery, E., Stichler, T., Catzeflis, F.M., Springer, M.S., 1996. Interordinal mammalian relationships: evidence for paenungulate monophyly is provided by complete mitochondrial 12S rRNA sequences. Mol Phylogenet Evol 6, 245-158.
Abstract: The complete mitochondrial 12S rRNA sequences of 5 placental mammals belonging to the 3 orders Sirenia, Proboscidea, and Hyracoidea are reported together with phylogenetic analyses (distance and parsimony) of a total of 51 mammalian orthologues. This 12S rRNA database now includes the 2 extant proboscideans (the African and Asiatic elephants Loxodonta africana and Elephas maximus), 2 of the 3 extant sirenian genera (the sea cow Dugong dugon and the West Indian manatee Trichechus manatus), and 2 of the 3 extant hyracoid genera (the rock and tree hyraxes Procavia capensis and Dendrohyrax dorsalis). The monophyly of the 3 orders Sirenia, Proboscidea, and Hyracoidea is supported by all kinds of analysis. There are 23 and 3 diagnostic substitutions shared by the 2 proboscideans and the 2 hyracoids, respectively, but none by the 2 sirenians. The 2 proboscideans exhibit the fastest rates of 12S rRNA evolution among the 11 placental orders studied. Based on various taxonomic sampling methods among eutherian orders and marsupial outgroups, the most strongly supported clade in our comparisons clusters together the 3 orders Sirenia, Proboscidea, and Hyracoidea in the superorder Paenungulata. Within paenungulates, the grouping of sirenians and proboscideans within the mirorder Tethytheria is observed. This branching pattern is supported by all analyses by high bootstrap percentages (BPs) and decay indices. When only one species is selected per order or suborder, the taxonomic sampling leads to a relative variation in bootstrap Paernungulata (92-99%). When each order or suborder is represented by two species, this relative variation decreased to 10% for Tethytheria (78-87%) and 3% for Paenungulata (96-99%). Two nearly exclusive synapomorphies for paenungulates are identified in the form of one transitional compensatory change, but none were detected for tethytherians. Such a robust and reliable resolution of the paenungulate node implies a long history of the common ancestors, allowing time for synapomorphies to accumulate. This observation suggests a Late Cretaceous/Early Paleocene origin for the Paenungulata.

Maliarchuk, B.A., Derevenko, M.V., Lapinskii, A.G., Solovenchuk, L.L., 1996. The use of the polymerase chain reaction in analyzing ancient DNAs (exemplified by that of the Enmynveem mammoth). Izv Akad Nauk Ser Biol 6, 681-686.
Abstract: DNA was isolated from the Enmynveem mammoth muscles, and the control region and cytochrome b gene of the mitochondrial genome were analyzed by polymerase chain reaction (PCR). The mammoth DNA was amplified by both the classical PCR (two primer system) and the single-primer PCR (spPCR) resulting in DNA fragments up to 1600 bp long. Restriction analysis of the mitochondrial cytochrome b gene was carried out. Cytochromes b genes in three Elephantidae genera were compared.

Porter, C.A., Goodman, M., Stanhope, M.J., 1996. Evidence on mammalian phylogeny from sequences of exon 28 of the von Willebrand factor gene. Mol Phylogenet Evol 5, 89-101.
Abstract: Phylogenetic relationships among 27 extant mammalian species (representing 15 placental orders) were studied using sequences of exon 28 of the gene encoding von Willebrand Factor (vWF), a glycoprotein which functions in blood clotting. Analysis of sequences coding for vWF revealed evidence for several subordinal and superordinal groupings, but the earliest branching sequence of placental mammals was left largely unresolved. Strong support was found for a monophyletic clade consisting of elephants, sea cows, hyraxes, aardvarks, and elephant shrews. This systematic placement of the elephant shrews agrees strongly with two other molecular data sets (interphotoreceptor retinoid binding protein and alpha-lens crystallins) and is consistent with analysis of fossil elephant shrews recently discovered in north Africa. Evidence from vWF sequences agrees with a number of previous molecular and morphological studies in providing strong support for the monophyly of both bats and rodents. The orders Primates, Proboscidea, Carnivora, Perissodactyla, and Artiodactyla were represented by more than one species which joined in each case to form a monophyletic order.

Rannala, B., Hartigan, J.A., 1996. Estimating gene flow in island populations. Genet Res 67, 147-158.
Abstract: A new method is presented for estimating the rate of gene flow into island populations using the distribution of alleles in samples from a number of islands. The pseudo maximum likelihood estimator (PMLE) that we derive may be applied to species with either discrete or continuous generation times. For Wright's discrete-generation island model, the method provides an estimate of theta = 2Nm where N is the (haploid) population size on each island and m is the fraction of individuals replaced by immigrants in each generation. For a continuous-generation island model, the corresponding parameter theta is the ratio of the immigration rate phi to the individual birth rate lambda. Monte Carlo simulations are used to compare the statistical properties of the PMLE with those of two alternative estimators of theta derived from Wright's F-statistics. The PMLE is shown to have greatest efficiency (least mean square error) in most cases for a wide range of sample sizes and parameter values. The PMLE is applied to estimate theta using mtDNA haplotypes and allozymes for subdivided populations of African elephants and Channel Island foxes.

Stanhope, M.J., Smith, M.R., Waddell, V.G., Porter, C.A., Shivji, M.S., Goodman, M., 1996. Mammalian evolution and the interphotoreceptor retinoid binding protein (IRBP)gene: convincing evidence for several superordinal clades. J Mol Evol 43, 83-92.
Abstract: Phylogenetic relationships of 25 mammalian species representing 17 of the 18 eutherian orders were examined using DNA sequences from a 1.2-kb region of the 5' end of exon 1 of the single-copy nuclear gene known as interphotoreceptor retinoid binding protein (IRBP). A wide variety of methods of analysis of the DNA sequence, and of the translated products, all supported a five-order clade consisting of elephant shrew (Macroscelidea)/aardvark (Tubulidentata)/and the paenungulates (hyracoids, sirenians, and elephants), with bootstrap support in all cases of 100%. The Paenungulata was also strongly supported by these IRBP data. In the majority of analyses this monophyletic five-order grouping was thefirst branch off the tree after the Edentata. These results are highly congruent with two other recent sources of molecular data. Another superordinal grouping, with similar 100% bootstrap support in all of the same wide-ranging types of analyses, was Artiodactyla/Cetacea. Other superordinal affinities, suggested by the analyses, but with less convincing support, included a Perissodactyla/Artiodactyla/Cetacea clade, an Insectivora/Chiroptera clade, and Glires (an association of rodents and lagomorphs).

Yang, H., Golenberg, E.M., Shoshani, J., 1996. Phylogenetic resolution within the Elephantidae using fossil DNA sequence from the American mastodon (Mammut americanum) as an outgroup. Proc Natl Acad Sci U S A 93, 1190-1194.
Abstract: DNA was extracted from the extinct American mastodon, the extinct woolly mammoth, and the modern Asian and African elephants to test the traditional morphologically based phylogeny within Elephantidae. Phylogenetic analyses of the aligned sequences of the mitochondrial gene cytochrome b support a monophyletic Asian elephant-woolly mammoth clade when the American mastodon is used as an outgroup. Previous molecular studies were unable to resolve the relationships of the woolly mammoth, Asian elephant, and African elephant because the sequences appear to have evolved at heterogeneous rates and inappropriate outgroups were used for analysis. The results demonstrate the usefulness of fossil molecular data from appropriate sister taxa for resolving phylogenies of highly derived or early radiating lineages. Erratum in: Proc Natl Acad Sci U S A 1996 Apr 30;93(9):4519

Bisig, D.A., Di Iorio, E.E., Diederichs, K., 1995. Crystal structure of Asian elephant (Elephas maximus) cyanometmyoglobin at 178-A resolution.  Phe29(B10) accounts for its unusual ligand binding properties. Journal of Biological Chemistry 270, 20752-20754.
Abstract: The crystal structure of Asian elephant cyano-metmyoglobin which has a glutamine instead of the usual distal site histidine has been determined to high resolution. In addition to this replacement, the substitution of a conserved leucine residue in position 29(B10) at the distal side by a phenylalanine was unambiguously identified based on the available electron density. The suspicion, that there were errors in the original sequence which has caused some confusion, is thus confirmed. Comparison with other myoglobin structures in various ligated forms reveals an essentially unchanged tertiary structure in elephant myoglobin despite the two amino acid substitutions in the heme pocket. Our current structural model shows that the N epsilon 2 atom of Gln64(E7) has moved with respect to the corresponding nitrogen position of His64(E7) in the CO complex of sperm whale myoglobin. The newly assigned residue Phe29(B10) penetrates into the distal side of the heme pocket approaching the ligand within van der Waals distance and causing a much more crowded heme pocket compared to other myoglobins. Kinetic properties of Asian elephant myoglobin, wild type, and recombinant sperm whale myoglobins are discussed in relation to the structural consequences of the two amino acid substitutions H64Q and L29F.

Fernando, P., 1995. Implications of Socio-Ecology and Genetics on the Conservation and Management of the Sri Lankan Elephant. In: Daniel, J.C. (Ed.), A Week with Elephants; Proceedings of the International Seminar on Asian Elephants. Bombay Natural History Society; Oxford University Press, Bombay, India, pp. 225-235.

Hartl, G.B., Kurt, F., Hemmer, W., Nadlinger, K., 1995. Electrophoretic and chromosomal variation in captive Asian elephants(Elephas maximus). Zoo Biology 14, 87-95.

Hauf, J., Baur, A., Chalwatzis, N., Zimmermann, F.K., Joger, U., Lazarev, P.A., 1995. Selective amplification of a mammoth mitochondrial cytochrome b fragment using an elephant-specific primer. Curr Genet 27, 486-487.

Mezhzherin, S.V., Morozov-Leonov, S.I., 1995. The genetic differentiation of mammalian taxa: their assessment by biochemical genetic markers. Zh Obshch Biol 56, 71-96.
Abstract: A review of data on genetic differentiation of mammalian taxa has been made on the basis of estimating the percent of fixed gene differences (PFD). The results substantiate the existence of evident differences in the scale of genetic divergence between taxa in different mammalian orders. Among smaller mammals (marsupials, insectivores, chiropterans, myomorph and sciuromorph rodents, african mole rats, and elephant shrews) interspecific differences within a genus involve the average of 25-40% of investigated loci. At the genetic level the value is 50-60%, whereas at the familial level the differences are beyond the resolution capacity of the method (PFD = 60-80%). Orders of larger mammals can be divided into two subgroups. One of them that includes carnivores, artiodactylans, and hystricomorph rodents is characterized by PFD values of 10-14%, 30-50%, and 69-70% at respective levels. The other subgroup composed of proboscideans, primates, pinnipeds, and toothed whales, has a low level of
genetic divergence expressed by PFD values of 0-3%, 7-36%, and 50-60% at species, generic and familial levels, respectively. Insufficiency of data on baleen whales and Perissodactyla does not allow to cluster them ultimately with any of these groups. There are three possible, but not necessarily alternative, causes for the observed differences in genetic divergence: 1) over-ranking of genera in larger mammals; 2) different paleontological age of orders; 3) unequal rates of molecular evolution.

Queralt, R., Adroer, R., Oliva, R., Winkfein, R.J., Retief, J.D., Dixon, G.H., 1995. Evolution of protamine P1 genes in mammals. Journal of Molecular Evolution 40, 601-607.
Abstract: A polymerase chain reaction-based approach was used to amplify and sequence the protamine genes of the rat, guineapig (order Rodentia), cat, bear (Carnivora), elephant (Proboscidea), horse (Perissodactyla), camel, elk, deer, moose and gazelle (Artiodactyla). The predicted amino acid sequences for these genes, together with previously reported amino acid sequences of protamine genes of humans, mice, pigs, cattle, fowls, quails and opossums, resulted in a data set of 25 P1 genes and 30 P1 amino acid sequences. A bootstrapped DNA parsimony tree of the set of protamine sequences was constructed. The results showed that protamines were amongst the most rapidly diverging proteins studied. In spite of the large differences, there were conserved motifs that were also common to birds. The C-terminus appeared to be the most variable region. The molecular evolution of P1 genes was in agreement with the expected species evolution.

Siegismund, H.R., Arctander, P., 1995. Structure of African elephant populations. J Hered 86, 467-469.
Abstract: The structure of elephant populations from east and south Africa has been analyzed by Georgiadis et al. (1994) on the basis of restriction site variation of mitochondrial DNA. They used F statistics based on identity by descent in tests for subdivision and reached the conclusion that there was a significant differentiation at the continental level, but that "populations were not significantly subdivided at the regional levels." The data were reanalyzed by Monte-Carlo permutation tests where population subdivision was tested by using F statistics based on partitioning the total haplotype diversity among populations. This resulted in identical conclusions at the continental level, but revealed in addition a significant subdivision at the regional level indicating haplotype frequency differences among the population.

Sukumar, R., 1995. Minimum Viable Populations for Asian Elephant Conservation. In: Daniel, J.C. (Ed.), A Week with Elephants; Proceedings of the International Seminar on Asian Elephants. Bombay Natural History Society; Oxford University Press, Bombay, India, pp. 279-288.

Sunderraj, S.F.W., Mishra, B.K., Johnsingh, A.J.T., 1995. Elephant Use of Rajaji - Corbett Forest Corridor, North West India. In: Daniel, J.C. (Ed.), A Week with Elephants; Proceedings of the International Seminar on Asian Elephants. Bombay Natural History Society; Oxford University Press, Bombay, India, pp. 261-269.

Ye Htut, U., 1995. Management of Wild Elephants in Myanmar. In: Daniel, J.C. (Ed.), A Week with Elephants; Proceedings of the International Seminar on Asian Elephants. Bombay Natural History Society; Oxford University Press, Bombay, India, pp. 236-241.

Zhao, X., Vyas, K., Nguyen, B.D., Rajarathnam, K., La Mar, G.N., Li, T., Phillips, G.N., Jr., Eich, R.F., Olson, J.S., Ling, J., 1995. A double mutant of sperm whale myoglobin mimics the structure and function of elephant myoglobin. J Biol Chem 270, 20763-20764.
Abstract: The functional, spectral, and structural properties of elephant myoglobin and the L29F/H64Q mutant of sperm whale myoglobin have been compared in detail by conventional kinetic techniques, infrared and resonance Raman spectroscopy, 1H NMR, and x-ray crystallography. There is a striking correspondence between the properties of the naturally occurring elephant protein and those of the sperm whale double mutant, both of which are quite distinct from those of native sperm whale myoglobin and the single H64Q mutant. These results and the recent crystal structure determination by Bisig et al. (Bisig, D. A., Di Iorio, E. E., Diederichs, K., Winterhalter, K. H., and Piontek, K. (1995) J. Biol. Chem. 270, 20754-20762) confirm that a Phe residue is present at position 29 (B10) in elephant myoglobin, and not a Leu residue as is reported in the published amino acid sequence. The single Gln64(E7) substitution lowers oxygen affinity approximately 5-fold and increases the rate of autooxidation 3-fold. These unfavorable effects are reversed by the Phe29(B10) replacement in both elephant myoglobin and the sperm whale double mutant. The latter, genetically engineered protein was originally constructed to be a blood substitute prototype with moderately low O2 affinity, large rate constants, and increased resistance to autooxidation. Thus, the same distal pocket combination that we designed rationally on the basis of proposed mechanisms for ligand binding and autooxidation is also found in nature.

Bischof, L.L., Duffield, D.A., 1994. Relatedness estimation of captive Asian elephants (Elephas maximas) by DNA fingerprinting. Zoo Biology 13, 77-82.

Georgiadis, N., Bischof, L., Templeton, A., Patton, J., Karesh, W., Western, D., 1994. Structure and history of African elephant populations: I. Eastern and southern Africa. J Hered 85, 100-104.
Abstract: Patterns of restriction site variation within mitochondrial DNA (mtDNA) of 270 individuals were used to examine the current structure of savanna elephant populations and to infer historical patterns of gene flow across eastern and southern Africa. Elephants have a complex population structure characterized by marked subdivision at the continental level (Fst = 0.39; 95% confidence interval 0.19-0.58), and isolation by distance at the regional level. However, phylogeographic analysis revealed evidence of protracted gene flow across the continent. First, one relatively derived haplotype was found at all sampling locations. Second, haplotypes representing exceptionally divergent (up to 8.3%) mitochondrial clades were found to coexist at distant (> 2,000 km) sampling locations. In the few other species characterized by sympatric individuals bearing such divergent haplotypes, all such individuals were found to coexist within limited geographical regions. Accordingly, pronounced mitochondrial divergence within populations is often attributed to ancestral isolation in allopatry, followed by secondary contact. The patterns within elephants do not accord with ancestral isolation in allopatry. Given the exceptional mobility of elephants, a geographical barrier is unlikely to have obstructed gene flow between regions for long enough to produce the observed mitochondrial divergence. Rather, the patterns are consistent with the more parsimonious hypothesis, based on neutral coalescent theory, that gene flow has maintained a sufficiently large effective population size (> 50,000 females) for representatives of clades that diverged at least 4 million years ago to have persisted by chance within a population that was subdivided, but not strictly isolated in allopatry.

Hagelberg, E., Thomas, M.G., Cook, C.E.Jr., Sher, A.V., Baryshnikov, G.F., Lister, A.M., 1994. DNA from ancient mammoth bones. Nature 370, 333-334.

Hoss, M., Paabo, S., Vereshchagin, N.K., 1994. Mammoth DNA sequences. Nature 370, 333.

Coetzee, E.M., Van-der-Bank, F.H., Critser, J.K., 1993. Allozyme variation in a wild African elephant (Loxodonta africana) population from the Kruger National Park, South Africa. Comparative Biochemistry and Physiology B-Comparative-Biochemistry 106, 109-114.
Abstract: 1. Blood, liver, heart, testis, skin, eye, muscle and kidney samples were obtained from elephants (Loxodonta africana) in the Kruger National Park during a culling programme in April 1992. 2. Gene products of 25 protein coding loci in L. africana were examined by horizontal starch-gel electrophoresis. 3. Eighteen protein coding loci (72%) displayed monomorphic gel banding patterns whereas only seven (28%) displayed polymorphic gel banding patterns. 4. Average heterozygosity values for adults, youngsters and the total population are respectively 0.058, 0.024 and 0.047. 5. Relative gene diversities within and between populations are 84% and 16% respectively. 6. Two population simulation programmes were utilized to predict the duration of the current variability present in this species, based on current genetic variation and gene transfer from one generation to the next.

Ermel, R.W., Kenny, T.P., Chen, P.P., Robbins, D.L., 1993. Molecular analysis of rheumatoid factors derived from rheumatoid synovium suggests an antigen-driven response in inflamed joints. Arthritis and Rheumatism 36, 380-388.
Abstract: Objective.  Understanding the molecular genetic basis for rheumatoid factor (RF) production is necessary to a better understanding of the etiology and pathogenesis of rheumatoid arthritis (RA).  We sought to define the genetic basis for RF in RA.  Methods.  The heavy and light chain variable region genes encoding 4 human monoclonal RF were cloned and sequenced using the polymerase chain reaction and the dideoxynucleotide chain-termination method.  Results.  The heavy and light chains of the C6 RF and the light chain of the G9 RF were encoded by 3 new RF-related Ig V-region genes.  The heavy and light chains of D5 and G4 RF's were identical: most of their mutations caused amino acid substitutions.  Conclusions.  The RF-related Ig V-region gene repertoire is large and still expanding.  The data from D5 and G4 strongly suggest that these 2 RF's arise in an antigen-driven response in rheumatoid synovium.  The presumed germline V genes for C6 may represent disease-specific RF-related V genes.

Ma, D.P., Zharkikh, A., Graur, D., VandeBerg, J.L., Li, W.H., 1993. Structure and evolution of opossum, guinea pig, and porcupine cytochrome b genes. J Mol Evol 36, 327-334.
Abstract: We have sequenced the mitochondrial cytochrome b gene from the guinea pig, the African porcupine, and a South American opossum. A phylogenetic analysis, which includes 22 eutherian and four other vertebrate cytochrome b sequences, indicates that the guinea pig and the porcupine constitute a natural clade (Hystricomorpha) that is not a sister group to the clade of mice and rats (Myomorpha). Therefore, the hypothesis that the Rodentia is paraphyletic receives additional support. The artiodactyls, the perissodactyls, and the cetaceans form a group that is separated from the primates and the rodents. The 26 sequences are used to study the structure/function relationships in cytochrome b, whose function is electron transport. Most of the amino acid residues involved in the two reaction centers are well conserved in evolution. The four histidines that are believed to ligate the two hemes are invariant among the 26 sequences, but their nearby residues are not well conserved in
evolution. The eight transmembrane domains represent some of the most divergent regions in the cytochrome b sequence. The rate of nonsynonymous substitution is considerably faster in the human and elephant lineages than in other eutherian lineages; the faster rate might be due to coevolution between cytochrome b and cytochrome c.

Sakthikumar, A., Mukundan, G., Raghunandanan, K.V., 1992. Chromosome profile of Indian elephants. In: Silas, E.G., Nair, M.K., Nirmalan, G. (Eds.), The Asian Elephant: Ecology, Biology, Diseases, Conservation and Management (Proceedings of the National Symposium on the Asian Elephant held at the Kerala Agricultural University, Trichur, India, January 1989). Kerala Agricultural University, Trichur, India, pp. 41-42.

Irwin, D.M., Kocher, T.D., Wilson, A.C., 1991. Evolution of the cytochrome b gene of mammals. J Mol Evol 32, 128-144.
Abstract: With the polymerase chain reaction (PCR) and versatile primers that amplify the whole cytochrome b gene (approximately 1140 bp), we obtained 17 complete gene sequences representing three orders of hoofed mammals (ungulates) and dolphins (cetaceans). The fossil record of some ungulate lineages allowed estimation of the evolutionary rates for various components of the cytochrome b DNA and amino acid sequences. The relative rates of substitution at first, second, and third positions within codons are in the ratio 10 to 1 to at least 33. For deep divergences (greater than 5 million years) it appears that both replacements and silent transversions in this mitochondrial gene can be used for phylogenetic inference. Phylogenetic findings include the association of (1) cetaceans, artiodactyls, and perissodactyls to the exclusion of elephants and humans, (2) pronghorn and fallow deer to the exclusion of bovids (i.e., cow, sheep, and goat), (3) sheep and goat to the exclusion of other pecorans (i.e., cow, giraffe, deer, and pronghorn), and (4) advanced ruminants to the exclusion of the chevrotain and other artiodactyls. Comparisons of these cytochrome b sequences support current structure-function models for this membrane-spanning protein. That part of the outer surface which includes the Qo redox center is more constrained than the remainder of the molecule, namely, the transmembrane segments and the surface that protrudes into the mitochondrial matrix. Many of the amino acid replacements within the transmembrane segments are exchanges between hydrophobic residues (especially leucine, isoleucine, and valine). Replacement changes at first and second positions of codons approximate a negative binomial distribution, similar to other protein-coding sequences. At four-fold degenerate positions of codons, the nucleotide substitutions approximate a Poisson distribution, implying that the underlying mutational spectrum is random with respect to position.

Sakthikumar, A., Mukundan, G., Raghunandanan, K., 1990. Chromosome profile of Indian elephants (Elephas maximus indicus). Indian Journal of Animal Sciences 60, 175-182.
Abstract: 15 elephants (7 males with tusks, 7 females and 1 male without tusks) were karyotyped. The diploid chromosome number was 56. There were 12 submetacentric and 42 acrocentric autosomes, and 2 sex chromosomes. The X-chromosome was submetacentric, and was the 4th largest chromosome; the Y-chromosome was the smallest, and was acrocentric. The arm ratio of the submetacentric chromosomes ranged from 1.38 to 4.23, and the centromeric index ranged from 0.42 to 0.19. The karyotype of the tuskless male was similar to that of the males with tusks.

Hegel, G.V., Hanichen, T., Mahnel, H., Wiesner, H., 1989. Warts (papilloma/sarcoid) in elephant. Erkrankungen der Zootiere 31, 201-205.
Abstract: Warts ( Papilloma, Sarcoid) in  Elephants  ( Hegel,G.)1989; translated from German by Gerda Martin. Papilloma virus - from the group Papova virus - is considered  an etiological agents of wart- like skin changes in cattle, sheep, mountain goat, and rabbit. (ROSENBERGER,1970; ROLLE and MAYR, 1984). Equine sarcoid (PALMER. 1985) found in horses is most likely caused by bovine papilloma virus. The alternate name is based on clinical and morphological differences in the actual papilloma. In the initial stage, the sarcoid is similar to that of the papilloma; however in later stages, tumorous decay on the surface of the epidermis, and proliferation of the mesenchymal part of the tumor in the subcutis dominate (DIET and WIESNER, 1982). Wart- like changes in the skin of elephants as described by PILASKI et al (1987, 1988), proved to be caused by Herpes virus.  Such skin changes in elephants are not rare and require treatment since size and volume of the excrescences  may cause functional disturbances in the patient. Even if the animal's  general   well being  is not impaired, the importance of esthetics and hygiene should not be disregarded in a place where there are spectators and visitors (zoo, circus). The following paper reports findings of wart- like skin changes in elephants. Observations and Therapy In the elephants  kept in the Hellabrunn  Zoo, no case of papilloma or similar skin tumors had occurred since 1972. First case: In 5-28 - 1987, a ca. 18 month old female L.a. named " Sabi" arrived In Hellabrunn. This animal had a wart- like thickening of 1 cm at the dorsal end of the trunk. After 8 weeks, more of those such skin changes appeared on trunk and lower lip without  impairment in general well being. Treatment consisted of  one daily, subcutaneous injection of 1 amp. Chelidonium D7 (DHU Chelidonium majus L.), and application of fresh ??Schoellkraut  juice dabbed onto the warts but was unsuccessful.  After a change of treatment was made: 10 drops of Thuja D4 (DHU Thuja occidentalis L) and 20 drops Acidum nitricum D12 (DHU Acidum nitricum), orally, once a day, at separate times of the day, there remained, after 2 weeks, a wart on the lower lip the size of a cherry pit, and the before mentioned  wart on the dorsal end of  the trunk had now grown to the size of a cherry. Even the strength of Thuja LM 6 (DHU Thuja occidentalis L.)  20 drops, oral, the growth of the wart on the dorsal end of the trunk, now with a diameter of 5 cm, could not be stopped: Exstirpation had to be performed. Frequent  sucking had promoted strong ulceration.  A secondary infection  had set in,  the surface showed granular  tissue exuding blood and pus.

On 10 -  6 -  1987 the growth was exstirpated and tissue was sent for virolog. and histolog. examination. In addition, tissue was removed from a fresh small wart for vaccine. During the operation the animal was immobilized (anesthetic: 0.3 ml Immobilon* (large animal Immobilon Rc* - Vet. Ltd.),  10mg Xylacin, 150 IE Hyaluronidase i. m.). There were no complications during recovery. Two  weeks post op., the first vaccination was given, followed by a second vacc. four weeks there after, of 5.0 ml, subcut.., of an auto vaccine developed by the Institute for Medical Microbiology, Dept. of Infectious and Epidemic Medicine. In February 1988, there occurred another bout with wart- like growth on the ventral part of the trunk, lower jaw, shoulders and feet, some with a diameter of 15 mm.  From the sedated young animal tissue was taken from several newly grown warts for the manufacture of auto vaccine (sedation: "Hellabrunner Mischung" / 150 IE Hyaluronidase). After 10 days, the first vaccination was given, and by the time of the second vacc." Sabi" was free of externally visible skin changes.

On 6. 6. 1988, "Sabi" fell ill again. Over night she was covered with 48 warts,  with  diameters from 2mm - 15 mm on trunk and head, and 10 more on the chest.The attempt to "ice"  the warts with liquid nitrogen was not successful. Instead, coagulation of ca. 20 of the larger warts was used. The monopolar coagulation electrode of the Erbotom F 2 (Erbe Elektromedizin) coagulates reaching deeply  into the healthy zone of the surrounding tissue. As before, tissue for the manufacture of the auto vaccine was taken, as well as  0.5 ml of blood from the ear vein for the manufacture of a "own- blood"  nosode. (Large animal, premedication: 20 mg Xylazin i.m., 20 minutes later : 0.5 ml Immobilon R (large animal Immobilon R c - Vet Ltd.) and 150 IE Hyaluronidase i.m. The following day, "Sabi" was given the "own- blood" nosode at a strength of C5 (20 drops daily).In addition, she was vacc. once again. Since "Sabi" was free of warts at the time of the second vaccination -   given 4 weeks after the first - the "own- blood" treatment was discontinued. Shortly there after, however, several new warts cropped up (diameter ca. 1 cm), so that the "own- blood"  treatments were continued. Since that time "Sabi" has had no recurrences.Second case : The Indian elephant cow (E. maximus) , named "Dirndl" , age ca. 22 years, had been kept in the box next to "Sabi" since "Sabi's" arrival. They kept trunk contact. On 5-2-1988, "Dirndl" showed on the distal trunk a substantially increased raised area ca. 2 x 2 cm oozing blood. It seemed to be an injury from a metal rope used in off limiting. The wound was disinfected and treated twice a day with chloromycetin spray with Gentian violetR (Parke Davis).  After  one week  the growth had increased substantially  and on the surface,  it had a cauliflower-like ulcerated  appearance.Upon light touch or movement of the dorsal  trunk, blood appeard spontaneously.   Four days later, the growth was exstirpated, while the animal was standing. (Sedation: 2.2 ml Hellabrunn mixture / 150 Hyaluronidase i. m.) . The attempt to close the skin of the trunk over the wound failed because the tension in that area was too great.  The surface of the wound was cauterized and treated with ChloromycetinSpray with Gentian violet R (Parke Davis). Tissue for pathological and histological examination was sent out.  One week after the operation,  the area of the wound was  highly swollem and the wound was infected. Treatment: Several times a day, an  ablution with a 0.1 % Rivanol solutionnR (Asid - 2 Aethoxy-6.9-diamin  acridinlactat)  and application of Sulfonamid-Codliver oil salve (WDT = Sulfadimidin- Sodium- cod liver oil).  In addition, analogous to "Sabi" , once daily 20 drops of "own-blood" nosode,  potency C 5  given orally. Three weeks post. op., there could be clearly distinguished a limited relapse, an area of  6 x 9 cm rising  ca. 2 cm  above the healthy skin of the trunk. The surface looked like the first growth.  It was extirpated under general anesthetic  (Premed.:80 mg Xylazin i. m., 20 min. later: 1.8 ml ImmobilionR and 150 IE Hyaluronidase). In addition, the whole wound was coagulated  by monopolar coagulation electrode as above. Daily for 4 weeks, the wound  was brushed with a 1:5 wood tar -alcohol - solution.There were no complications during recovery. After 5 weeks , all that could be seen was a ca. 1.5 cm long small scar on the skin of the trunk.

Histomorphological Findings:  Fixation with formalin, embedding in paraffin; stain: Hemalaun-Eosin, connective tissue stain  in the manner of Masson. The histomorphological  findings based on the tissue samples of "Sabi" and "Dirndl"  are the  same, and agree  with the findings of 3 other skin tumor tissue taken from elephants of different origin (tab. 1). The tumors  consist mainly of fibroplastic cells  with more or less  abundant collagen fibers and blood vessels. The boundaries from the adjacent corium and lower skin is largely indistinct.  In all larger neoplasties , the covering epidermis has been preserved  at margins only due to superficial ulceration.  Here the P. acuta aseptica diffusa borders  are irregular and strongly profiled, the epithelium is acanthoid and hyperkeratotic. The nuclei of tumor cells  are considerably anisomorphic, some have gigantic nuclei. Mitosis is frequent. Due to the ulcerated epidermis , there is deep infiltration with infectious cells. Virological findings: From the extirpated tissue taken from the African Elephant "Sabi" ca. 3 g was homogenated, in addition, the cells were "opened" by defrosting and ultrasound, and the "cleared" tissue suspension was analyzed  for free virus particles after concentration and negative-contrasting with  electron microscopy .  At the same time, small tissue samples of 2 mm  from deeper epidermis layers  were fixed as usual for the ultrahistological exam , embedded in epoxy  resin, and ultra thin slices were scanned by the electron microscope. No papilloma virus was found  in the concentrated, cell free tissue extract or  the ultrathin slices of tissue samples .No virus particle of  any kind was found.

Discussion
To  show papilloma by culturing cannot be done since  no species of this genus can  be propagated  in cell cultures  with the exception of its original host. The failed  attempt to prove their presence with the electronmicroscope does not exclude a papilloma virus etiology in tumors. When virus particles are viewed in higher concentrations, the electron microscopic  proof is successful. Using ultrahistologyical methods the particles in cell nuclei can only be found when the few cells  of specific skin cells  are in the virus propagation stage. In the case of virally induced papilloma however, a true virus propagation is not necessary. In the last few years, it was found that equine sarcoid  can be caused  by bovine papilloma virus. But it was only the genome of the virus which could be isolated by means of gene technology (ALTMANN, 1980; HAUSEN, 1980); the virus itself could not.  The oncogenetic potency of the virus in heterologic hosts , without true virus production, has been established.  A broader spectrum of hosts  for , at least , the papilloma virus in cattle seems to be the case.  And a bovine papilloma induced skin fibromatose in  (a) horse has been reported (LANCASTER, 1979). This virus can also appear in wild 'cud chewers, perhaps even carnivores. It is in part also related to the human papilloma virus. The possibility of transfer to humans (LANCASTER 1982) as well as other mammals such as elephants  has not been proved but is probable. In comparing the histological findings of the 5 skin growths with those of the viral fibropapilloma in cattle and horse (called equine sarcoid here), the relative immaturity of the tumorous tissue is evident. It compares to the so- called sarcoid in horses. The sarcoid-like structure and the indistinct  separation from healthy tissue  speaks for a virus etiology and  morphologically a relapse can be expected. This occurred in both of the clinically described cases.

A differential diagnosis excludes a Herpes virus infection, as described by PILASKI et al. (1987, 1988) in elephants on the basis of different histological findings. Inclusions could not be found in any of the cases. The warts on the elephants were clinically similar to the well known sarcoids in horses (DIETZ and WIESNER, 1982). The two sick animals were in "trunk contact" occupying adjacent boxes. Almost one year after the arrival of  "Sabi" who had warts, "Dirndl" fell sick. That points to the infectious nature of warts. The relapse after the first operation on "Dirndl" suggests that the extirpation of the growths was not complete. This may be related to the fact that the animal was standing  and only sedated. In contrast , the extirpation of the "relapse" was carried out on a fully immobilized animal and with the use of the Erbotom F 2  for coagulation including the adjacent tissue.  We know of various 'wart therapies' in human medicine with differing success. The various treatments employed in the one and one half years of "Sabi's" illness can be labled neither successful, nor unsuccessful. The use of auto vaccine which is analogous  to a "stable specific " vaccine in the treatment of papilloma in cattle, could  perhaps have triggered the recurrence of warts at the  conclusion of the vaccination treatments. That would favor the etiology of a virus 'picture.' The influence of the 'burn' or extirpation of a single or more growths which returned, in the surrounding growths cannot be determined. It remains inconclusive if the use of the "own- blood" nosode C 5 aided the successful therapy , since the necessity  to fight a recurrence had not yet occurred.

Merkt, H., Bader, H., Rath, D., Dittrich, L., 1987. An attempt to deep-freeze elephant semen. Deutsche Tierarztliche Wochenschrift 94, 488-489.

Howard, J.G., Bush, M., de Vos, V., Schiewe, M.C., Pursel, V.G., Wildt, D.E., 1986. Influence of cryoprotective diluent on post-thaw viability and acrosomal integrity of spermatozoa of the African elephant (Loxodonta africana). Journal of Reproduction and Fertility 78, 295-306.
Abstract: Electroejaculates from free-ranging African elephants were frozen to test various seminal diluents, freezing methods and thawing media on post-thaw sperm viability and structural integrity.  In Study I, each ejaculate was tested in each of 7 cryoprotective diluents.  After cooling to 5C and equilibration on ice (4C) for 120min, each aliquot was pellet frozen on solid CO₂, stored in liquid nitrogen and thawed (37C) in saline or tissue culture solution.  Amongst the diluents, post-thaw sperm motility, motility duration in vitro (37C) and acrosomal integrity were greatest when diluent BF5F was used.  Thawing medium had no effect on results.  In Study II, the optimal diluent from Study I (BF5F) was compared with the diluent SGI. Results were not affected by a 90- or a 150-min cooling-equilibrium interval in an electronic cooler (5C); however, post-thaw sperm motility rating and duration of motility in vitro were grater with the pellet than the straw container freezing method.  When the pelleting method was used, diluents BF5F and SGI provided comparable cryoprotection. Duration of post-thaw motility was enhanced 2-fold and up to 12h my maintaining thawed semen at 21 rather than 37C.  All diluents provided some protection on acrosomal integrity, but the overall proportion of intact acrosomes after thawing was markedly less in Study II, apparently as a result of the slower initial cooling rate (approx 1.5C/min) compared to that of Study I (approx 6.5C/min).  This study demonstrates the feasibility of cryopreserving semen from free-ranging African elephants and indicates that spermatozoa most effectively survive freezing when the BF5F or SGI diluent is used in conjunction with the pelleting method.

Pilaski, J., Rosen, A., Darai, G., 1986. Comparative analysis of the genomes of orthopoxviruses isolated from elephant, rhinoceros, and okapi by restriction enzymes. Brief report. Archives of Virology 88, 135-142.
Abstract: Orthopoxviruses from different zoo-kept mammalian species including Elephas maximus (8 isolates), Ceratotherium simum (1 isolate), and Okapia johnstoni (2 isolates) were characterized by restriction enzyme analysis of the viral genome. The four enzymes BamHI, MluI, NcoI, and SalI were found to be optimal for strain differentiation.

Ryder, O.A., 1986. Genetic investigations: tools for supportings breeding programme goals. International Zoo Yearbook 24/25, 157-162.

Howard, J.G., Bush, M., Schiewe, M.C., de Vos, V., Wildt, D.E., 1985. Further developments in comparative semen freezing in free-ranging African elephants. Proceedings American Association of Zoo Veterinarians 31-32.

Johnson, P.H., Olson, C.B., Goodman, M., 1985. Isolation and characterization of deoxyribonucleic acid from tissue of the woolly mammoth, Mammuthus primigenius. Comp Biochem Physiol B 81, 1045-1051.
Abstract: DNA was isolated from tissue samples of several mammoth specimens, radiocarbon dated between 10,000 and 53,000 years old. The DNA was purified by chromatography on hydroxyapatite at 60 degrees C and was characterized as a heterogeneous population of fragments ranging in size from 3000 to 200 base pairs. Thermal denaturation analysis demonstrated that approximately 25% of the DNA had a base composition similar to Asian elephant DNA calculated as 36% G + C. Preliminary analysis by nucleic acid hybridization indicated that only a small fraction of DNA isolated from mammoth tissue (2-5%) was homologous to DNA of Asian elephant, a close living relative of the mammoth. Our results provide the first definitive isolation and characterization of DNA from ancient tissue and suggest a purification strategy that will lead to preparations of DNA from mammoth tissue significantly enriched in elephant-related DNA sequences.

 1979. "Motty" -- Birth of an African/Asian elephant at Chester Zoo. Elephant 1, 36-40.

De Jong, W.W., Nuy-Terwindt, E.C., Versteeg, M., 1977. Primary structures of alpha crystallin A chains of elephant, whale, hyrax and rhinoceros. Biochim. Biophys. Acta 491, 573-580.
Abstract: As part of a study of the evolutionary development of the eye lens protein alpha-crystallin the 173 residue A chain of this protein has been studied in elephant, whale, hyrax and rhinoceros.  The primary sturctures were inferred mainly from amino acid compositions of peptides obtained by enzymatic digestions and CNBr cleavage.  The positions of substitutions, as compared to known bovine A chain, were confirmed by Edman degradation.  In accordance with the previously observed slow rate of evolution of the A chain only a small number of substitutions were found among these species.  Elephant and hyrax share a number of unique substitutions, strongly indicating a common ancestry of these two species within the mammalian class.

Jones, R.C., Bailey, D.W., Skinner, J.D., 1975. Studies on the collection and storage of semen from the African elephant, Loxodonta africana. Koedoe 18, 147-164.

Thurig, D., 1970. Karyotype and sex chromatin of the African elephant. Acta Anatomica Nipponica 75, 6-12.

Norberg, H.S., 1969. The chromosomes of the Indian female elephant (Elephas indicus Syn. E. maximus). Hereditas 63, 279-281.

Hooijer, D.A., 1967. Indo-Australian insular elephants. Genetica 38, 143-162.

Hungerford, D.A., Chandra, H.S., Snyder, R.L., Ulmer, F.A., 1966. Chromosomes of three elephants, two Asian (Elephas maximus) and one African (Loxodonta africana). Cytogenetics 5, 243-246.
Abstract: Cultured somatic cells of three elephants, male and female Asian (Elephas maximus) and female African (Loxodonta africana), had 56 chromosomes.  Karyotypes of the Asian specimens were morphologically identical, except for the sex chromosomes, and that of the African specimen closely similar. Identification of the African X was made by inference.

Rao, S.R.V., Prasad, M.R.N., 1963. The nuclear sex in the Indian elephant, Elephas maximus L. Naturwissenechaften 50, 313.

Venkatasubba, R.S.R., Prasad, M.R.N., 1963. The nuclear sex in the Indian elephant, Elephas maximus L. Naturwissenechaften 50, 313.

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