Ancient DNA Clarifies the Evolutionary History of American Late Pleistocene Equids

Hippidions are past members of the equid lineage which appeared in the South American fossil record around 2.5 Ma but then became extinct during the great late Pleistocene megafaunal extinction. According to fossil records and numerous dental, cranial, and postcranial characters, Hippidion and Equus lineages were expected to cluster in two distinct phylogenetic groups that diverged at least 10 MY, long before the emergence of the first Equus. However, the first DNA sequence information retrieved from Hippidion fossils supported a striking different phylogeny, with hippidions nesting inside a paraphyletic group of Equus. This result indicated either that the currently accepted phylogenetic tree of equids was incorrect regarding the timing of the evolutionary split between Hippidion and Equus or that the taxonomic identification of the hippidion fossils used for DNA analysis needed to be reexamined (and attributed to another extinct South American member of the equid lineage). The most likely candidate for the latter explanation is Equus (Amerhippus) neogeus. Here, we show by retrieving new ancient mtDNA sequences that hippidions and Equus (Amerhippus) neogeus were members of two distinct lineages. Furthermore, using a rigorous phylogenetic approach, we demonstrate that while formerly the largest equid from Southern America, Equus (Amerhippus) was just a member of the species Equus caballus. This new data increases the known phenotypic plasticity of horses and consequently casts doubt on the taxonomic validity of the subgenus Equus (Amerhippus).     

Morphological convergence in Hippidion and Equus (Amerhippus) South American equids elucidated by ancient DNA analysis

Unusual equids named hippidions inhabited South America for more than 2 MY (million years). Like many other animals they succumbed to the worldwide climatic change that occurred 10 KY (thousand years) ago and completely disappeared during the great late Pleistocene megafaunal extinction. According to fossil records and numerous dental, cranial, and postcranial characters, Hippidion and Equus lineages are known to have diverged prior to 10 MY. Some equid bones from Rio Verde and Ultima Esperanza (Patagonia, Chile) dating back to the late Pleistocene period (8-13 KY) have been identified as Hippidion saldiasi, while a few teeth have been assigned to Equus. Six samples of those remains have been obtained from the Zoological Museum of Amsterdam for ancient DNA analysis to try to place Hippidion in the evolutive tree of Perissodactyla. Two samples of Hippidion and one sample of Equus yielded 241-394 bp of the mtDNA control region and 172-296 bp of the cytochrome b gene. Unexpectedly, all the sequences clustered deep inside the Equus genus, casting doubt on the initial identification of the bones. For paleontologists, one of the striking and classical diagnostic characters of Hippidion is their extremely short and massive metapodials, a probable locomotory adaptation to the Andine steep slopes. However, our DNA analysis reveals that a very Hippidion-like metapod might also have been possessed by another South American equid, i.e., Equus (Amerhippus), an interpretation supported by complementary anatomical observations. This adaptive convergence between members of the two South American equid genera may lead paleontologists to limb bone misidentification.     

Late Pleistocene and Holocene equid remains from Israel

Equid remains–mostly isolated teeth–from archaeological sites in Israel are described. Particular attention is paid to dental enamel fold morphology and criteria are established tor separating several Old World Quaternary equid species. Eauus hydruntinus , which is here considered a zebra rather than an ass, was present until 12,000 bp in northern Israel, while at the same time E-asinus/hemionus inhabited the arid regions in the south. Inirequent remains of E. caballus are also described. By 4000 years ago ass, probably the domestic form, was present in northern Israel.     

African origins of modern asses as seen from paleontology and DNA: what about the Atlas wild ass?

As a contribution to the still open debate on the multiple african origins of the domesticated donkey, this work focuses on the wild ass of the Maghreb. It follows the recent paleontological review of the equids from Lac Karâr, a middle Pleistocene site in Northern Algeria, where most of the faunal remains were attributed to wild asses (Equus africanus Heuglin and Fitzinger, 1866). The morphometric resemblance between the equid of Lac Karâr, and Equus melkiensis Bagtach, Hadjouis and Eisenmann, 1984 which might correspond to the equid commonly referred to, as the Atlas wild ass (vernacular name) and, by descent, to Equus tabeti Arambourg, 1970, the only subspecies of ass from the early Pleistocene in Northern Africa, highlights the need for extensive biomolecular and radiometric studies on this wild and robust ass, endemic to the Maghreb. This is especially important given that progress in extracting ancient DNA from equids has been demonstrated by several recent studies and that remains of wild asses are still being uncovered in many late Pleistocene to Holocene (Neolithic) deposits in the Maghreb. The results from such studies could substantially improve the knowledge on the origin of asses. In particular, these analyses could shed light on the, as yet, undetermined clade 2 which may originate from the wild ass of the Maghreb as recently hypothesized by several authors. The archaeological and genetic data reviewed in this paper focuses on the ancient range of the Atlas wild ass mainly from sites in the northern part of the Maghreb; it contributes to current debates regarding this subspecies as one of the possible ancestors of modern asses. In addition, the paper identifies directions for further genetic researches on the extant donkey of the Maghreb.     

Hidden diversity in bent‐winged bats (Chiroptera: Miniopteridae) of the Western Palaearctic and adjacent regions: implications for taxonomy

The taxonomic status of bent‐winged bats (Miniopterus) in the Western Palaearctic and adjacent regions is unclear, particularly in some areas of the eastern Mediterranean, Middle East and Arabia. To address this, we analysed an extensive collection of museum materials from all principal parts of this distribution range, i.e. North Africa, Europe and southwest Asia, using morphological (skull) and genetic approaches (mitochondrial DNA). Linear and geometric morphometric analysis of cranial and dental characteristics, together with molecular phylogeny, suggested that Miniopterus populations comprise four separate species: (1) M. schreibersii sensu strictissimo (s.str.) – occurring in Europe, coastal Anatolia, Levant, Cyprus, western Transcaucasia, and North Africa; (2) M. pallidus – occurring in inland Anatolia, Jordan, eastern Transcaucasia, Turkmenistan, Iran and southern Afghanistan (Kandahar); (3) a Miniopterus sp. – recorded from Nangarhar province in eastern Afghanistan, which we tentatively assign to M. cf. fuliginosus; and (4) a Miniopterus sp. with Afro‐tropic affinities confirmed from south‐western Arabia and Ethiopia, which we tentatively name M. cf. arenarius. The latter two species are well differentiated by skull morphology, while M. pallidus possesses very similar skull morphology to M. schreibersii. The results also suggest the existence of a possible new taxon (subspecies) within M. schreibersii s.str. inhabiting the Atlas Mountains of Morocco. © 2012 The Linnean Society of London     

The case of an arctic wild ass highlights the utility of ancient DNA for validating problematic identifications in museum collections

Museum collections are essential for reconstructing and understanding past biodiversity. Many museum specimens are, however, challenging to identify. Museum samples may be incomplete, have an unusual morphology, or represent juvenile individuals, all of which complicate accurate identification. In some cases, inaccurate identification can lead to false biogeographic reconstructions with cascading impacts on paleontological and paleoecological research. Here, we analyzed an unusual Equid mandible found in the Far North of the Taymyr peninsula that was identified morphologically as Equus hemionus, an ancestor of present‐day Asiatic wild asses. If correct, this identification represents the only finding of a putative Late Pleistocene hemione in the Arctic region, and is therefore critical to understanding wild ass evolution and paleoecology. To confirm the accuracy of this specimen's taxonomic assignment, we used ancient DNA and mitochondrial hybridization capture to identify and place this specimen in the larger equid phylogeny. We find that the specimen is actually a member of E. caballus, the ancestor of domestic horses. Our study demonstrates the utility of ancient DNA to validate morphological identification, in particular of incomplete, otherwise problematic, or taxonomically unusual museum specimens.     

Valsequillo biostratigraphy. III: Equid ecospecies in Paleoindian sites

Greater precision in North American Pleistocene equid taxonomy makes it now possible to exploit the ubiquitous horse remains in Paleoindian sites as ecological index-fossils. The horses of Central Mexico and the Southern Plains can be sorted by tooth size alone, except for two rare large horses of the Southern Plains. The species endemic to these grasslands and south to Central Mexico are Equus pacificus (large), E. conversidens (small), E. francisci (smallest). The Southern Plains were also occupied by a specialized grazer E. excelsus (Burnet and Sandia caves) and E. occidentalis (Dry and Sandia caves). West of the Rocky Mountains E. occidentalis was dominant. East of the Mississippi River two woodland species are found: E. fraternus and E. littoralis.     

Karyotypic relationships of horses and zebras: results of cross-species chromosome painting

Complete sets of chromosome-specific painting probes, derived from flow-sorted chromosomes of human (HSA), Equus caballus (ECA) and Equus burchelli (EBU) were used to delineate conserved chromosomal segments between human and Equus burchelli, and among four equid species, E. przewalskii (EPR), E. caballus, E. burchelli and E. zebra hartmannae (EZH) by cross-species chromosome painting. Genome-wide comparative maps between these species have been established. Twenty-two human autosomal probes revealed 48 conserved segments in E. burchelli. The adjacent segment combinations HSA3/21, 7/16p, 16q/19q, 14/15, 12/22 and 4/8, presumed ancestral syntenies for all eutherian mammals, were also found conserved in E. burchelli. The comparative maps of equids allow for the unequivocal characterization of chromosomal rearrangements that differentiate the karyotypes of these equid species. The karyotypes of E. przewalskii and E. caballus differ by one Robertsonian translocation (ECA5 = EPR23 + EPR24); numerous Robertsonian translocations and tandem fusions and several inversions account for the karyotypic differences between the horses and zebras. Our results shed new light on the karyotypic evolution of Equidae.     

Quo vadis eohippus? The systematics and taxonomy of the early Eocene equids (Perissodactyla)

The systematics and taxonomy of early Eocene equids are investigated. A paraphyletic sequence of equid taxa is recovered from a phylogenetic analysis of 40 taxa and 121 characters. This analysis supports the identification of Hyracotherium as a primitive equoid and its restriction to the genotype, Hyracotherium leporinum . Sifrhippus gen. nov. is erected for the sister taxon of all other equids, Hyracotherium sandrae Gingerich. Minippus gen. nov., the next more-derived equid clade, is erected for two small equids, M. index Cope and M. jicarillai nov. species. Arenahippus gen. nov. is erected for the next three sequentially more-derived equid taxa, A. grangeri Kitts, A. aemulor Gingerich, and A. pernix Marsh. The genus Xenicohippus , which is the next more-derived equid clade, is redefined to include X. craspedotum Cope. Eohippus Marsh, the next more-derived equid taxon, is resurrected for E. angustidens Cope. Pliolophus , the only early Eocene equid from Europe, is identified as the sister taxon to Protorohippus , a sequence of successively more-derived equid taxa consisting of P. montanum Wortman and P. venticolum Cope. Protorohippus venticolum is identified as the sister taxon of Orohippus . Systemodon Cope is resurrected for S. tapirinum Cope. This taxon has historically been placed within Hyracotherium yet this analysis allies it with Cymbalophus near the base of the perissodactyl radiation. © 2002 The Linnean Society of London. Zoological Journal of the Linnean Society , 134 , 141–256.     

Mitochondrial Phylogenomics of Modern and Ancient Equids

The genus Equus is richly represented in the fossil record, yet our understanding of taxonomic relationships within this genus remains limited. To estimate the phylogenetic relationships among modern horses, zebras, asses and donkeys, we generated the first data set including complete mitochondrial sequences from all seven extant lineages within the genus Equus. Bayesian and Maximum Likelihood phylogenetic inference confirms that zebras are monophyletic within the genus, and the Plains and Grevy’s zebras form a well-supported monophyletic group. Using ancient DNA techniques, we further characterize the complete mitochondrial genomes of three extinct equid lineages (the New World stilt-legged horses, NWSLH; the subgenus Sussemionus; and the Quagga, Equus quagga quagga). Comparisons with extant taxa confirm the NWSLH as being part of the caballines, and the Quagga and Plains zebras as being conspecific. However, the evolutionary relationships among the non-caballine lineages, including the now-extinct subgenus Sussemionus, remain unresolved, most likely due to extremely rapid radiation within this group. The closest living outgroups (rhinos and tapirs) were found to be too phylogenetically distant to calibrate reliable molecular clocks. Additional mitochondrial genome sequence data, including radiocarbon dated ancient equids, will be required before revisiting the exact timing of the lineage radiation leading up to modern equids, which for now were found to have possibly shared a common ancestor as far as up to 4 Million years ago (Mya).     

Retention of solute and particle markers in the digestive tract of captive Somali wild asses (<Emphasis Type="Italic">Equus africanus somaliensis</Emphasis>)

In contrast to the domestic horse, whose digestive physiology has been thoroughly investigated, knowledge on the digestive physiology of wild equids is scarce. Comparisons between the domestic horse and the domestic donkey suggest that wild asses might achieve higher digestibilities. This could derive from longer retention times or a greater difference in the mean retention time (MRT) of particles vs. fluid (the selectivity factor (SF)). Here, we measured MRT of a solute (fluid; MRT_solute) and a particle (<2 mm; MRT_particle) marker in five captive male Somali wild asses (Equus africanus somaliensis) fed a diet of 95% grass hay. At a mean dry matter intake of 94 ± 3 g kg^?0.75 day^?1, MRT_solute was 33.3 ± 5.4 h and MRT_particle 39.6 ± 3.9 h, resulting in a SF of 1.21 ± 0.14. For their food intake, Somali wild asses appeared to have slightly higher MRT_particle than expected based on domestic equid data, in contrast to Grevy zebras (Equus grevyi), potentially indicating higher capacities of the digestive tract. However, considering data on domestic horses, donkeys, and zebra, there was no evident difference in the SF of wild equids compared to domestic ones. Together with an absence of reported anatomical differences in the digestive tract of wild and domestic equids, the data suggest a general similarity in the digestive physiology of equid species that contrasts with the diversity in the digestive physiology of ruminants, and that might be one contributing factor to a lack of sympatric, niche-differentiated equid species.     

A survey of equid mitochondrial DNA: Implications for the evolution, genetic diversity and conservation of Equus

The evolution, taxonomy and conservation of the genus Equuswere investigated by examining the mitochondrial DNA sequences of thecontrol region and 12S rRNA gene. The phylogenetic analysis of thesesequences provides further evidence that the deepest node in thephylogeny of the extant species is a divergence between twolineages; one leading to the ancestor of modern horses (E.ferus, domestic and przewalskii) and the other to thezebra and ass ancestor, with the later speciation events of the zebrasand asses occurring either as one or more rapid radiations, or withextensive secondary contact after speciation. Examination of the geneticdiversity within species suggested that two of the E. hemionussubspecies (E. h. onager and E. h. kulan) onlyrecently diverged, and perhaps, are insufficiently different to beclassified as separate subspecies. The genetic divergence betweendomestic and wild forms of E. ferus (horse) and E.africanus (African ass) was no greater than expected within anequid species. In E. burchelli (plains zebra) there was anindication of mtDNA divergence between populations increasing withdistance. The implications of these results for equid conservation arediscussed and recommendations are made for conservation action.     

Evolution, systematics, and phylogeography of pleistocene horses in the new world: a molecular perspective

The rich fossil record of horses has made them a classic example of evolutionary processes. However, while the overall picture of equid evolution is well known, the details are surprisingly poorly understood, especially for the later Pliocene and Pleistocene, c. 3 million to 0.01 million years (Ma) ago, and nowhere more so than in the Americas. There is no consensus on the number of equid species or even the number of lineages that existed in these continents. Likewise, the origin of the endemic South American genus Hippidion is unresolved, as is the phylogenetic position of the "stilt-legged" horses of North America. Using ancient DNA sequences, we show that, in contrast to current models based on morphology and a recent genetic study, Hippidion was phylogenetically close to the caballine (true) horses, with origins considerably more recent than the currently accepted date of c. 10 Ma. Furthermore, we show that stilt-legged horses, commonly regarded as Old World migrants related to the hemionid asses of Asia, were in fact an endemic North American lineage. Finally, our data suggest that there were fewer horse species in late Pleistocene North America than have been named on morphological grounds. Both caballine and stilt-legged lineages may each have comprised a single, wide-ranging species.     

The Social System of Feral Asses (Equus asinus)

Feral asses (Equus asinus) were studied in a desert range in southeastern California, USA. They formed small, highly unstable groups of varied sex and age composition. Older ♂♂ tended to be solitary and some were seasonally territorial. Home ranges of marked individuals overlapped and averaged 32 km². The territory of a marked ♂ occupied 0.5 km². The social and spatial organizations of the study population are compared with other feral ass populations and with other equid species. Loose social organization is viewed as well-adapted to the low carrying capacity of deserts, and the territorial system similarly adaptive when precipitation is periodic.     

Chromosomal distribution of the telomere sequence (TTAGGG)(n) in the Equidae

Telomeres are a class of repetitive DNA sequences that are located at chromosome termini and that act to stabilize the chromosome ends. The rapid karyotypic evolution of the genus Equus has given rise to ten taxa, all with different diploid chromosome numbers. Using fluorescence in situ hybridization (FISH) we localized the mammalian telomere sequence, (TTAGGG)(n), to the chromosomes of nine equid taxa. TTAGGG signal was located at chromosome termini in all species, however additional signal was seen at interstitial sites on some chromosomes in the Burchell's zebra, Equus quagga burchelli, the Hartmann's zebra, Equus zebra hartmannae, and at large heterochromatin-associated regions on the chromosomes of the donkey, Equus asinus. The interstitial signal in the zebras may be a relic of an ancient telomere-telomere fusion and mark the point at which two ancestral chromosomes may have fused. For the donkey, the heterochromatin-associated signal may represent degenerate telomere-like satellite sequences and identify a second type of satellite DNA for this taxon.     

Morphological and mitochondrial DNA characters for identification and phylogenetic analysis of the myiasis-causing flesh fly Wohlfahrtia magnifica and its relatives, with a description of Wohlfahrtia monegrosensis sp. n. Wyatt & Hall

Abstract.  Wohlfahrtia magnifica (Schiner) (Diptera: Sarcophagidae) is a major cause of traumatic myiasis in livestock in Central and Eastern Europe and in countries bordering the Mediterranean. The present study explored the utility of external body characters, genitalia characters and mitochondrial DNA characters for identification of this and related species in the subfamily Paramacronychiinae. Sequence analyses of the 3' terminal 273 bp of the mitochondrial cytochrome b gene revealed two lineages of W. magnifica , one from Spain and France and the other from the rest of Eurasia, differing by only two base pairs. Phylogenetic analysis of cytochrome b showed that W. magnifica and Wohlfahrtia vigil Walker were sister species; this conclusion was not contradicted by a phylogenetic analysis of the morphological characters. Based on cytochrome b , the genetic distance between specimens of W. vigil from Europe and North America was sufficiently large to justify the recognition of more than one species. A new species, Wohlfahrtia monegrosensis , from northern Spain, was described, based on morphology and cytochrome b . A unique combination of external body characters of males or females were diagnostic for W. magnifica , the W. vigil group and Wohlfahrtia bella , but only the genitalia characters were diagnostic for all nine species studied.     

Mitochondrial evidence uncovers a refugium for the fat dormouse (Glis glis Linnaeus, 1766) in Hyrcanian forests of northern Iran

Fat dormouse is a squirrel-like rodent which is closely tied to deciduous forest ecosystem in southwestern Eurasia. As such it is a valuable indicator of forest survival in refugia during glacial-interglacial periods. Previous phylogeographic analyses uncovered divergent fat dormouse lineages in southern refugia in Italy and the Balkans, but retrieved a surprisingly low overall genetic diversity across the majority of the species’ range. We explored 812 bp long fragment of a cytochrome b (cyt b) gene in ten fat dormice from refugial Hyrcanian forests in northern Iran. We identified 10 new cyt b haplotypes, which generated a total dataset of 28 fat dormouse haplotypes. The phylogenetic reconstruction clustered the new haplotypes into the Iranian lineage which hold a sister position against all other fat dormouse haplotypes from Europe and Asia Minor. The divergence between these lineages suggests a fragmentation event of an ancestral population at 5.76 mya (95% HPD = 3.21–8.92). This early evolutionary divergence was possibly triggered in the Middle East by dramatically divergent environmental conditions at the Messinian Salinity Crisis. The divergence clearly exceeds the intraspecific divergence, and is well within the range between congeneric rodent species. We suggest a long-term persistence of the Iranian lineage in the Hyrcanian refugium which is consitent with a high number of endemics along the southern Caspian coastal areas.     

A rapid loss of stripes: the evolutionary history of the extinct quagga

Twenty years ago, the field of ancient DNA was launched with the publication of two short mitochondrial (mt) DNA sequences from a single quagga (Equus quagga) museum skin, an extinct South African equid (Higuchi et al. 1984 Nature312, 282-284). This was the first extinct species from which genetic information was retrieved. The DNA sequences of the quagga showed that it was more closely related to zebras than to horses. However, quagga evolutionary history is far from clear. We have isolated DNA from eight quaggas and a plains zebra (subspecies or phenotype Equus burchelli burchelli). We show that the quagga displayed little genetic diversity and very recently diverged from the plains zebra, probably during the penultimate glacial maximum. This emphasizes the importance of Pleistocene climate changes for phylogeographic patterns in African as well as Holarctic fauna.     

Low "penetrance" of phylogenetic knowledge in mitochondrial disease studies

An up-to-date view of the worldwide mitochondrial DNA (mtDNA) phylogeny together with an evaluation of the conservation of each site is a reliable tool for detecting errors in mtDNA studies and assessing the functional importance of alleged pathogenic mutations. However, most of the published studies on mitochondrial diseases make very little use of the phylogenetic knowledge that is currently available. This drawback has two inadvertent consequences: first, there is no sufficient a posteriori quality assessment of complete mtDNA sequencing efforts; and second, no feedback is provided for the general mtDNA database when apparently new mtDNA lineages are discovered. We demonstrate, by way of example, these issues by reanalysing three mtDNA sequencing attempts, two from Europe and another one from East Asia. To further validate our phylogenetic deductions, we completely sequenced two mtDNAs from healthy subjects that nearly match the mtDNAs of two patients, whose sequences gave problematic results.     

Evolution of ecological traits and wing morphology in Hemileuca (Saturniidae) based on a two-gene phylogeny

We present a molecular phylogeny for the genus Hemileuca (Saturniidae), based on 624 bp of mitochondrial cytochrome oxidase I (COI) and 932 bp of the nuclear gene elongation factor 1 alpha (EF1alpha). Combined analysis of both gene sequences increased resolution and supported most of the phylogenetic relationships suggested by separate analysis of each gene. However, a maximum parsimony (MP) model for just COI sequence from one sample of most taxa produced a phylogeny incongruent with EF1alpha and combined dataset analyses under either MP or ML models. Time of year and time of day during which adult moths fly corresponded strongly with the phylogeny. Although most Hemileuca are diurnal, ancestral Hemileuca probably were nocturnal, fall-flying insects. The two-gene molecular phylogeny suggests that wing morphology is frequently homoplastic. There was no correlation between the primary larval hostplants and phylogenetic placement of taxa. No phylogenetic pattern of specialization was evident for single hostplant families across the genus. Our results suggest that phenological behavioral characters may be more conserved than the wing morphology characters that are more commonly used to infer phylogenetic relationships in Lepidoptera. Inclusion of a molecular component in the re-evaluation of systematic data is likely to alter prior assumptions of phylogenetic relationships in groups where such potentially homoplastic characters have been used.