Signatures of conserved and unique molecular features in Afrotheria.

Afrotheria is a clade of African-origin species with striking dissimilarities in appearance and habitat. In this study, we compared whole proteome sequences of six Afrotherian species to obtain a broad viewpoint of their underlying molecular make-up, to recognize potentially unique proteomic signatures. We find that 62% of the proteomes studied here, predominantly involved in metabolism, are orthologous, while the number of homologous proteins between individual species is as high as 99.5%. Further, we find that among Afrotheria, L. africana has several orphan proteins with 112 proteins showing < 30% sequence identity with their homologues. Rigorous sequence searches and complementary approaches were employed to annotate 156 uncharacterized protein sequences and 28 species-specific proteins. For 122 proteins we predicted potential functional roles, 43 of which we associated with protein- and nucleic-acid binding roles. Further, we analysed domain content and variations in their combinations within Afrotheria and identified 141 unique functional domain architectures, highlighting proteins with potential for specialized functions. Finally, we discuss the potential relevance of highly represented protein families such as MAGE-B2, olfactory receptor and ribosomal proteins in L. africana and E. edwardii, respectively. Taken together, our study reports the first comparative study of the Afrotherian proteomes and highlights salient molecular features.

Of elephants and errors: naming and identity in Linnaean taxonomy.

What is it to make an error in the identification of a named taxonomic group? In this article we argue that the conditions for being in error about the identity of taxonomic groups through their names have a history, and that the possibility of committing such errors is contingent on the regime of institutions and conventions governing taxonomy and nomenclature at any given point in time. More specifically, we claim that taxonomists today can be in error about the identity of taxonomic groups in a way that Carl Linnaeus (1707-1778), who is routinely cited as the "founder" of modern taxonomy and nomenclature, simply could not be. Starting from a remarkable recent study into Linnaeus's naming of Elephas maximus that led to the (putative) discovery of a (putative) nomenclatural error by him, we reconsider what it could mean to discover that Linnaeus misidentified a biological taxon in applying his taxon names. Through a further case study in Linnaean botany, we show that his practices of (re)applying names in taxonomic revisions reveal a take on determining "which taxon is which" that is strikingly different from that of contemporary taxonomists. Linnaeus, we argue, adopted a practice-based, hands-on concept of taxa as "nominal spaces" that could continue to represent the same taxon even if all its former members had been reallocated to other taxa.

Distinguishing extant elephants ivory from mammoth ivory using a short sequence of cytochrome b gene.

Trade in ivory from extant elephant species namely Asian elephant (Elephas maximus), African savanna elephant (Loxodonta africana) and African forest elephant (Loxodonta cyclotis) is regulated internationally, while the trade in ivory from extinct species of Elephantidae, including woolly mammoth, is unregulated. This distinction creates opportunity for laundering and trading elephant ivory as mammoth ivory. The existing morphological and molecular genetics methods do not reliably distinguish the source of ivory items that lack clear identification characteristics or for which the quality of extracted DNA cannot support amplification of large gene fragments. We present a PCR-sequencing method based on 116 bp target sequence of the cytochrome b gene to specifically amplify elephantid DNA while simultaneously excluding non-elephantid species and ivory substitutes, and while avoiding contamination by human DNA. The partial Cytochrome b gene sequence enabled accurate association of ivory samples with their species of origin for all three extant elephants and from mammoth. The detection limit of the PCR system was as low as 10 copy numbers of target DNA. The amplification and sequencing success reached 96.7% for woolly mammoth ivory and 100% for African savanna elephant and African forest elephant ivory. This is the first validated method for distinguishing elephant from mammoth ivory and it provides forensic support for investigation of ivory laundering cases.

Loxodonta Localizer: A Software Tool for Inferring the Provenance of African Elephants and Their Ivory Using Mitochondrial DNA.

Illegal hunting is a major threat to the elephants of Africa, with more elephants killed by poachers than die from natural causes. DNA from tusks has been used to infer the source populations for confiscated ivory, relying on nuclear genetic markers. However, mitochondrial DNA (mtDNA) sequences can also provide information on the geographic origins of elephants due to female elephant philopatry. Here, we introduce the Loxodonta Localizer (LL; www.loxodontalocalizer.org), an interactive software tool that uses a database of mtDNA sequences compiled from previously published studies to provide information on the potential provenance of confiscated ivory. A 316 bp control region sequence, which can be readily generated from DNA extracted from ivory, is used as a query. The software generates a listing of haplotypes reported among 1917 African elephants in 24 range countries, sorted in order of similarity to the query sequence. The African locations from which haplotype sequences have been previously reported are shown on a map. We demonstrate examples of haplotypes reported from only a single locality or country, examine the utility of the program in identifying elephants from countries with varying degrees of sampling, and analyze batches of confiscated ivory. The LL allows for the source of confiscated ivory to be assessed within days, using widely available molecular methods that do not depend on a particular platform or laboratory. The program enables identification of potential regions or localities from which elephants are being poached, with capacity for rapid identification of populations newly or consistently targeted by poachers.

Update on Comparative Biology of Elephants: Factors Affecting Reproduction, Health and Welfare.

Asian (Elephas maximus) and African (Loxodonta africana) elephants serve as important keystone, umbrella and flagship species. Despite that, population numbers are declining, due mainly to poaching and habitat destruction. Understanding reproductive mechanisms is vital to effective management, particularly insurance populations in captivity, and to that end, long-term biological databases are key to understanding how intrinsic and extrinsic factors affect reproductive function at individual and population levels. Through decades of hormonal and ultrasonographic monitoring, many unique aspects of zoo elephant reproduction have been identified, including differences in luteal steroidogenic activity, follicular maturation, pituitary gonadotropin secretion, fetal development and reproductive tract anatomy. Reproductive problems also hamper captive propagation efforts, particularly those related to abnormal or lack of ovarian cyclicity. Recent large-scale, multi-institutional studies and use of epidemiological approaches have identified factors important for good welfare and reproduction, which include enrichment, feeding diversity, good elephant-keeper relations, social compatibility, exercise, and not being obese. There are notable differences in reproductive mechanisms between Asian and African elephants, as well as the factors that influence reproduction and welfare, suggesting species-targeted management approaches are needed to maximize fitness. In the first edition, we discussed reproductive function in male and female elephants. Since then, a number of significant advances have been made primarily in female elephants, which will be the focus of this updated review.

A comprehensive genomic history of extinct and living elephants.

Elephantids are the world's most iconic megafaunal family, yet there is no comprehensive genomic assessment of their relationships. We report a total of 14 genomes, including 2 from the American mastodon, which is an extinct elephantid relative, and 12 spanning all three extant and three extinct elephantid species including an ∼120,000-y-old straight-tusked elephant, a Columbian mammoth, and woolly mammoths. Earlier genetic studies modeled elephantid evolution via simple bifurcating trees, but here we show that interspecies hybridization has been a recurrent feature of elephantid evolution. We found that the genetic makeup of the straight-tusked elephant, previously placed as a sister group to African forest elephants based on lower coverage data, in fact comprises three major components. Most of the straight-tusked elephant's ancestry derives from a lineage related to the ancestor of African elephants while its remaining ancestry consists of a large contribution from a lineage related to forest elephants and another related to mammoths. Columbian and woolly mammoths also showed evidence of interbreeding, likely following a latitudinal cline across North America. While hybridization events have shaped elephantid history in profound ways, isolation also appears to have played an important role. Our data reveal nearly complete isolation between the ancestors of the African forest and savanna elephants for ∼500,000 y, providing compelling justification for the conservation of forest and savanna elephants as separate species.

Automated detection of low-frequency rumbles of forest elephants: A critical tool for their conservation.

African forest elephants (Loxodonta cyclotis) occupy large ranges in dense tropical forests and often use far-reaching vocal signals to coordinate social behavior. Elephant populations in Central Africa are in crisis, having declined by more than 60% in the last decade. Methods currently used to monitor these populations are expensive and time-intensive, though acoustic monitoring technology may offer an effective alternative if signals of interest can be efficiently extracted from the sound stream. This paper proposes an automated elephant call detection algorithm that was tested on nearly 4000 h of field recordings collected from five forest clearings in Central Africa, including sites both inside protected areas and in logging concessions. Recordings were obtained in different seasons, years, and under diverse weather conditions. The detector achieved an 83.2% true positive rate when the false positive rate is 5.5% (approximately 20 false positives per hour). These results suggest that this algorithm can enable analysis of long-term recording datasets or facilitate near-real-time monitoring of elephants in a wide range of settings and conditions.

Ivory species identification using electrophoresis-based techniques.

Despite continuous conservation efforts by national and international organizations, the populations of the three extant elephant species are still dramatically declining due to the illegal trade in ivory leading to the killing of elephants. A requirement to aid investigations and prosecutions is the accurate identification of the elephant species from which the ivory was removed. We report on the development of the first fully validated multiplex PCR-electrophoresis assay for ivory DNA analysis that can be used as a screening or confirmatory test. SNPs from the NADH dehydrogenase 5 and cytochrome b gene loci were identified and used in the development of the assay. The three extant elephant species could be identified based on three peaks/bands. Elephas maximus exhibited two distinct PCR fragments at approximate 129 and 381 bp; Loxodonta cyclotis showed two PCR fragments at 89 and 129 bp; and Loxodonta africana showed a single fragment of 129 bp. The assay correctly identified the elephant species using all 113 ivory and blood samples used in this report. We also report on the high sensitivity and specificity of the assay. All single-blinded samples were correctly classified, which demonstrated the assay's ability to be used for real casework. In addition, the assay could be used in conjunction with the technique of direct amplification. We propose that the test will benefit wildlife forensic laboratories and aid in the transition to the criminal justice system.

Development and characterization of microsatellite markers in the African forest elephant (Loxodonta cyclotis).

BACKGROUND: African elephants comprise two species, the savanna elephant (Loxodonta africana) and the forest elephant (L. cyclotis), which are distinct morphologically and genetically. Forest elephants are seriously threatened by poaching for meat and ivory, and by habitat destruction. However, microsatellite markers have thus far been developed only in African savanna elephants and Asian elephants, Elephas maximus. The application of microsatellite markers across deeply divergent lineages may produce irregular patterns such as large indels or null alleles. Thus we developed novel microsatellite markers using DNA from two African forest elephants. FINDINGS: One hundred microsatellite loci were identified in next generation shotgun sequences from two African forest elephants, of which 53 were considered suitable for testing. Twenty-three microsatellite markers successfully amplified elephant DNA without amplifying human DNA; these were further characterized in 15 individuals from Lope National Park, Gabon. Three of the markers were monomorphic and four of them carried only two alleles. The remaining sixteen polymorphic loci carried from 3 to 8 alleles, with observed heterozygosity ranging from 0.27 to 0.87, expected heterozygosity from 0.40 to 0.86, and the Shannon diversity index from 0.73 to 1.86. Linkage disequilibrium was not detected between loci, and no locus deviated from Hardy-Weinberg equilibrium. CONCLUSIONS: The markers developed in this study will be useful for genetic analyses of the African forest elephant and contribute to their conservation and management.

Elephantid Genomes Reveal the Molecular Bases of Woolly Mammoth Adaptations to the Arctic.

Woolly mammoths and living elephants are characterized by major phenotypic differences that have allowed them to live in very different environments. To identify the genetic changes that underlie the suite of woolly mammoth adaptations to extreme cold, we sequenced the nuclear genome from three Asian elephants and two woolly mammoths, and we identified and functionally annotated genetic changes unique to woolly mammoths. We found that genes with mammoth-specific amino acid changes are enriched in functions related to circadian biology, skin and hair development and physiology, lipid metabolism, adipose development and physiology, and temperature sensation. Finally, we resurrected and functionally tested the mammoth and ancestral elephant TRPV3 gene, which encodes a temperature-sensitive transient receptor potential (thermoTRP) channel involved in thermal sensation and hair growth, and we show that a single mammoth-specific amino acid substitution in an otherwise highly conserved region of the TRPV3 channel strongly affects its temperature sensitivity.

Elephant natural history: a genomic perspective.

We review DNA-based studies of elephants and recently extinct proboscideans. The evidence indicates that little or no nuclear gene flow occurs between African savanna elephants (Loxodonta africana) and African forest elephants (Loxodonta cyclotis), establishing that they comprise separate species. In all elephant species, males disperse, whereas females remain with their natal social group, leading to discordance in the phylogeography of nuclear and mitochondrial DNA patterns. Improvements in ancient DNA methods have permitted sequences to be generated from an increasing number of proboscidean fossils and have definitively established that the Asian elephant (Elephas maximus) is the closest living relative of the extinct woolly mammoth (Mammuthus primigenius). DNA-based methods have been developed to determine the geographic provenance of confiscated ivory in an effort to aid the conservation of elephants.

Making the impossible possible: rooting the tree of placental mammals.

Untangling the root of the evolutionary tree of placental mammals has been nearly an impossible task. The good news is that only three possibilities are seriously considered. The bad news is that all three possibilities are seriously considered. Paleontologists favor a root anchored by Xenarthra (e.g., sloths and anteater), whereas molecular evolutionists have favored the two other possible roots: Afrotheria (e.g., elephants, hyraxes, and tenrecs) and Atlantogenata (Afrotheria + Xenarthra). Now, two groups of researchers have scrutinized the largest available genomic data sets bearing on the question and have come to opposite conclusions, as reported in this issue of Molecular Biology and Evolution. Needless to say, more research is needed.

Histogenesis of the unique morphology of proboscidean ivory.

The chequered pattern (often called Schreger pattern), which can be seen by unaided eye on transverse profiles of several proboscidean tusks and which can be emphasized by the spreading pattern of the cracks or by mineral discoloration, is an autapomorph feature of the clade Elephantoidea. The pattern differs among proboscidean taxa; therefore, it allows the coarse differentiation of elephants, mammoths, and some other basal groups. Such identification methods could facilitate efforts concerned with protection of the remaining elephant populations through ivory trade restrictions, since the tooth dentine from extinct Mammuthusprimigenius and from extant Loxodontaafricana and Elephasmaximus are the most common raw materials of the ivory carvings. The aim of this study was to show the internal structure of proboscidean ivory and to revise the existing theories on the aforementioned pattern of the elephantoids with reflections on the events which lead to the development of this microstructure. Thin sections and natural crack surfaces with various orientations of M.primigenius, Elephasantiquus, Prodeinotherium, and Deinotherium tusk fragments were used to produce a three-dimensional model which explains the features on all profiles. The "phase shift" model is introduced, which assumes a sinusoid undulation of the dentinal tubules in radial profiles in the case of elephantoids. The model was confirmed by photomicrographs, scanning electron microscopic images, interpretation of natural crack surfaces, and radial displacement analysis of the dentinal tubules. The latter proved that the adjacent waves are not in the same phase. Several new nondestructive distinguishing methods are described here on the basis of the correlation between some microscopic and macroscopic features related to the Schreger pattern.

Extreme insular dwarfism evolved in a mammoth.

The insular dwarfism seen in Pleistocene elephants has come to epitomize the island rule; yet our understanding of this phenomenon is hampered by poor taxonomy. For Mediterranean dwarf elephants, where the most extreme cases of insular dwarfism are observed, a key systematic question remains unresolved: are all taxa phyletic dwarfs of a single mainland species Palaeoloxodon antiquus (straight-tusked elephant), or are some referable to Mammuthus (mammoths)? Ancient DNA and geochronological evidence have been used to support a Mammuthus origin for the Cretan 'Palaeoloxodon' creticus, but these studies have been shown to be flawed. On the basis of existing collections and recent field discoveries, we present new, morphological evidence for the taxonomic status of 'P'. creticus, and show that it is indeed a mammoth, most probably derived from Early Pleistocene Mammuthus meridionalis or possibly Late Pliocene Mammuthus rumanus. We also show that Mammuthus creticus is smaller than other known insular dwarf mammoths, and is similar in size to the smallest dwarf Palaeoloxodon species from Sicily and Malta, making it the smallest mammoth species known to have existed. These findings indicate that extreme insular dwarfism has evolved to a similar degree independently in two elephant lineages.

Forest elephant mitochondrial genomes reveal that elephantid diversification in Africa tracked climate transitions.

Among elephants, the phylogeographic patterns of mitochondrial (mt) and nuclear markers are often incongruent. One hypothesis attributes this to sex differences in dispersal and in the variance of reproductive success. We tested this hypothesis by examining the coalescent dates of genetic markers within elephantid lineages, predicting that lower dispersal and lower variance in reproductive success among females would have increased mtDNA relative to nuclear coalescent dates. We sequenced the mitochondrial genomes of two forest elephants, aligning them to mitogenomes of African savanna and Asian elephants, and of woolly mammoths, including the most divergent mitogenomes within each lineage. Using fossil calibrations, the divergence between African elephant F and S clade mitochondrial genomes (originating in forest and savanna elephant lineages, respectively) was estimated as 5.5 Ma. We estimated that the (African) ancestor of the mammoth and Asian elephant lineages diverged 6.0 Ma, indicating that four elephantid lineages had differentiated in Africa by the Miocene-Pliocene transition, concurrent with drier climates. The coalescent date for forest elephant mtDNAs was c. 2.4 Ma, suggesting that the decrease in tropical forest cover during the Pleistocene isolated distinct African forest elephant lineages. For all elephantid lineages, the ratio of mtDNA to nuclear coalescent dates was much greater than 0.25. This is consistent with the expectation that sex differences in dispersal and in variance of reproductive success would have increased the effective population size of mtDNA relative to nuclear markers in elephantids, contributing to the persistence of incongruent mtDNA phylogeographic patterns.

Distinguishing forest and savanna African elephants using short nuclear DNA sequences.

A more complete description of African elephant phylogeography would require a method that distinguishes forest and savanna elephants using DNA from low-quality samples. Although mitochondrial DNA is often the marker of choice for species identification, the unusual cytonuclear patterns in African elephants make nuclear markers more reliable. We therefore designed and utilized genetic markers for short nuclear DNA regions that contain fixed nucleotide differences between forest and savanna elephants. We used M13 forward and reverse sequences to increase the total length of PCR amplicons and to improve the quality of sequences for the target DNA. We successfully sequenced fragments of nuclear genes from dung samples of known savanna and forest elephants in the Democratic Republic of Congo, Ethiopia, and Namibia. Elephants at previously unexamined locations were found to have nucleotide character states consistent with their status as savanna or forest elephants. Using these and results from previous studies, we estimated that the short-amplicon nuclear markers could distinguish forest from savanna African elephants with more than 99% accuracy. Nuclear genotyping of museum, dung, or ivory samples will provide better-informed conservation management of Africa's elephants.

Complete Columbian mammoth mitogenome suggests interbreeding with woolly mammoths.

BACKGROUND: Late Pleistocene North America hosted at least two divergent and ecologically distinct species of mammoth: the periglacial woolly mammoth (Mammuthus primigenius) and the subglacial Columbian mammoth (Mammuthus columbi). To date, mammoth genetic research has been entirely restricted to woolly mammoths, rendering their genetic evolution difficult to contextualize within broader Pleistocene paleoecology and biogeography. Here, we take an interspecific approach to clarifying mammoth phylogeny by targeting Columbian mammoth remains for mitogenomic sequencing. RESULTS: We sequenced the first complete mitochondrial genome of a classic Columbian mammoth, as well as the first complete mitochondrial genome of a North American woolly mammoth. Somewhat contrary to conventional paleontological models, which posit that the two species were highly divergent, the M. columbi mitogenome we obtained falls securely within a subclade of endemic North American M. primigenius. CONCLUSIONS: Though limited, our data suggest that the two species interbred at some point in their evolutionary histories. One potential explanation is that woolly mammoth haplotypes entered Columbian mammoth populations via introgression at subglacial ecotones, a scenario with compelling parallels in extant elephants and consistent with certain regional paleontological observations. This highlights the need for multi-genomic data to sufficiently characterize mammoth evolutionary history. Our results demonstrate that the use of next-generation sequencing technologies holds promise in obtaining such data, even from non-cave, non-permafrost Pleistocene depositional contexts.