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Donkeys (Equus asinus) have been used extensively in agriculture and transportations since their domestication, ca. 5000-7000 years ago, but the increased mechanization of the last century has largely spoiled their role as burden animals, particularly in developed countries. Consequently, donkey breeds and population sizes have been declining for decades, and the diversity contributed by autochthonous gene pools has been eroded. Here, we examined coding-region data extracted from 164 complete mitogenomes and 1392 donkey mitochondrial DNA (mtDNA) control-region sequences to (i) assess worldwide diversity, (ii) evaluate geographical patterns of variation, and (iii) provide a new nomenclature of mtDNA haplogroups. The topology of the Maximum Parsimony tree confirmed the two previously identified major clades, i.e. Clades 1 and 2, but also highlighted the occurrence of a deep-diverging lineage within Clade 2 that left a marginal trace in modern donkeys. Thanks to the identification of stable and highly diagnostic coding-region mutational motifs, the two lineages were renamed as haplogroup A and haplogroup B, respectively, to harmonize clade nomenclature with the standard currently adopted for other livestock species. Control-region diversity and population expansion metrics varied considerably between geographical areas but confirmed North-eastern Africa as the likely domestication center. The patterns of geographical distribution of variation analyzed through phylogenetic networks and AMOVA confirmed the co-occurrence of both haplogroups in all sampled populations, while differences at the regional level point to the joint effects of demography, past human migrations and trade following the spread of donkeys out of the domestication center. Despite the strong decline that donkey populations have undergone for decades in many areas of the world, the sizeable mtDNA variability we scored, and the possible identification of a new early radiating lineage further stress the need for an extensive and large-scale characterization of donkey nuclear genome diversity to identify hotspots of variation and aid the conservation of local breeds worldwide., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Migliore et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Donkeys transformed human history as essential beasts of burden for long-distance movement, especially across semi-arid and upland environments. They remain insufficiently studied despite globally expanding and providing key support to low- to middle-income communities. To elucidate their domestication history, we constructed a comprehensive genome panel of 207 modern and 31 ancient donkeys, as well as 15 wild equids. We found a strong phylogeographic structure in modern donkeys that supports a single domestication in Africa ~5000 BCE, followed by further expansions in this continent and Eurasia and ultimately returning to Africa. We uncover a previously unknown genetic lineage in the Levant ~200 BCE, which contributed increasing ancestry toward Asia. Donkey management involved inbreeding and the production of giant bloodlines at a time when mules were essential to the Roman economy and military.

Evolution of the genus Equus has been a matter of long debate with a multitude of hypotheses. Currently, there is no consensus on either the taxonomic content nor phylogeny of Equus. Some hypotheses segregate Equus species into three genera, Plesippus, Allohippus and Equus. Also, the evolutionary role of European Pleistocene Equus stenonis in the origin of the zebra-ass clade has been debated. Studies based on skull, mandible and dental morphology suggest an evolutionary relationship between North American Pliocene E. simplicidens and European and African Pleistocene Equus. In this contribution, we assess the validity of the genera Plesippus, Allohippus and Equus by cladistic analysis combined with morphological and morphometrical comparison of cranial anatomy. Our cladistic analysis, based on cranial and postcranial elements (30 taxa, 129 characters), supports the monophyly of Equus, denies the recognition of Plesippus and Allohippus and supports the derivation of Equus grevyi and members of the zebra-ass clade from European stenonine horses. We define the following evolutionary steps directly relevant to the phylogeny of extant zebras and asses: E. simplicidens-E. stenonis-E. koobiforensis-E. grevyi -zebra-ass clade. The North American Pliocene species Equus simplicidens represents the ancestral stock of Old World Pleistocene Equus and the zebra-ass clade. Our phylogenetic results uphold the most recent genomic outputs which indicate an age of 4.0-4.5 Ma for the origin and monophyly of Equus.

The equid family contains only one single extant genus, Equus , including seven living species grouped into horses on the one hand and zebras and asses on the other. In contrast, the equine fossil record shows that an extraordinarily richer diversity existed in the past and provides multiple examples of a highly dynamic evolution punctuated by several waves of explosive radiations and extinctions, cross-continental migrations, and local adaptations. In recent years, genomic technologies have provided new analytical solutions that have enhanced our understanding of equine evolution, including the species radiation within Equus ; the extinction dynamics of several lineages; and the domestication history of two individual species, the horse and the donkey. Here, we provide an overview of these recent developments and suggest areas for further research.

Background: The mammalian Major Histocompatibility Complex (MHC) is a genetic region containing highly polymorphic genes with immunological functions. MHC class I and class II genes encode antigen-presenting molecules expressed on the cell surface. The MHC class II sub-region contains genes expressed in antigen presenting cells. The antigen binding site is encoded by the second exon of genes encoding antigen presenting molecules. The exon 2 sequences of these MHC genes have evolved under the selective pressure of pathogens. Interspecific differences can be observed in the class II sub-region. The family Equidae includes a variety of domesticated, and free-ranging species inhabiting a range of habitats exposed to different pathogens and represents a model for studying this important part of the immunogenome. While equine MHC class II DRA and DQA loci have received attention, the genetic diversity and effects of selection on DRB and DQB loci have been largely overlooked. This study aimed to provide the first in-depth analysis of the MHC class II DRB and DQB loci in the Equidae family., Results: Three DRB and two DQB genes were identified in the genomes of all equids. The genes DRB2, DRB3 and DQB3 showed high sequence conservation, while polymorphisms were more frequent at DRB1 and DQB1 across all species analyzed. DQB2 was not found in the genome of the Asiatic asses Equus hemionus kulan and E. h. onager. The bioinformatic analysis of non-zero-coverage-bases of DRB and DQB genes in 14 equine individual genomes revealed differences among individual genes. Evidence for recombination was found for DRB1, DRB2, DQB1 and DQB2 genes. Trans-species allele sharing was identified in all genes except DRB1. Site-specific selection analysis predicted genes evolving under positive selection both at DRB and DQB loci. No selected amino acid sites were identified in DQB3., Conclusions: The organization of the MHC class II sub-region of equids is similar across all species of the family. Genomic sequences, along with phylogenetic trees suggesting effects of selection as well as trans-species polymorphism support the contention that pathogen-driven positive selection has shaped the MHC class II DRB/DQB sub-regions in the Equidae.

The Kanapoi collection of Rhinocerotidae, first studied by Hooijer and Patterson (1972), now consists of 25 specimens and substantial reinterpretation of their affinities is made here. Kanapoi post-dates the extinction of Brachypotherium and the whole collection belongs to the Dicerotini. It is important because it includes the type-specimen of Diceros praecox, a species that remains poorly known, but looks slightly larger and more primitive than the modern 'black' rhino, Diceros bicornis. A second species is probably ancestral to the modern 'white' rhino, Ceratotherium simum; it looks identical to the Pleistocene North African Ceratotherium mauritanicum, of which Ceratotherium efficax is probably a synonym. The evolution of the Dicerotini in Africa can be regarded as an increasing divergence in diet and related morphofunctional adaptations in the two lineages. The co-occurrence at Kanapoi of both Diceros and Ceratotherium, with distinct dietary preferences, suggests some habitat heterogeneity, although the low sample size prevents robust paleoecological conclusions. The Equidae are also rare and consist mostly of isolated teeth. I take the most parsimonious option of tentatively including all of them in a single species, whose identification is left open. Dental features of eastern African Pliocene to Pleistocene hipparions may reflect increasing adaptation to grazing., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The aim of this study was to determine the frequency and factors associated with Toxoplasma gondii in naturally infected equids in northeastern Brazil. Serum samples from 569 equids (528 horses, 8 mules and 33 donkeys) were subjected to the indirect fluorescent antibody test. Generalized linear models were used to evaluate associated factors. Among the 569 animals sampled, 118 (30.6%) living in rural areas and 14 (26.42%) in urban areas were seropositive (p>0.05). Seropositive animals were observed on 95% of the farms and in all the municipalities. Donkeys/mules as the host, presence of domestic cats and rats on the farm, ingestion of lagoon water and goat rearing remained in the final model as factors associated with infection. Preventive measures such as avoiding the presence of domestic cats close to rearing areas, pastures and sources of water for the animals should be adopted. The wide-ranging distribution of positive animals also indicated that infection in other domestic animals and in humans, through the contaminated environment, was possible. It should be highlighted that there was the possibility that donkeys and mules would continue to have detectable titers for longer, thus explaining the prevalence found. Further studies are needed to confirm this possibility.

To investigate the mtDNA variation and origin of maternal lineages in South American donkeys and to reassess the domestication of donkeys in northeast Africa, we analyzed sequences (489 bp of the D-loop) from 323 domestic donkeys sampled from Peru, Brazil, Ethiopia and Egypt. Altogether, the 323 sequences displayed 53 different haplotypes (45 in Ethiopia, 14 in Egypt, eight in Peru and six in Brazil). Among the four populations, Egyptian donkeys possessed the highest haplotype diversity (0.910 ± 0.032), followed by Brazilian donkeys (0.879 ± 0.060). The Clade I haplotypes dominated in Peruvian donkeys (65%), whereas Clade II haplotypes dominated in Brazilian donkeys (67%). Estimates of F ST values showed a high genetic differentiation between Peruvian and Brazilian donkey populations (F ST  = 0.4066), which could be explained by the complex introduction history of South American donkeys. Phylogeographic analysis indicates that northeast Africa could be the most probable domestication center for Clade I donkeys. Analysis of molecular variance confirmed a weak genetic structure in domestic donkey populations among four continents (Europe, Asia, Africa and South America)., (© 2019 Stichting International Foundation for Animal Genetics.)

The extant diversity of horses (family Equidae) represents a small fraction of that occurring over their evolutionary history. One such lost lineage is the subgenus Sussemionus, which is thought to have become extinct during the Middle Pleistocene. However, recent molecular studies and morphological analysis have revealed that one of their representatives, E. ovodovi, did exist in Siberia during the Late Pleistocene. Fossil materials of E. ovodovi have thus far only been found in Russia. In this study, we extracted DNA from three equid fossil specimens excavated from northeastern China dated at 12,770-12,596, 29,525-28,887 and 40,201-38,848 cal. yBP, respectively, and retrieved three near-complete mitochondrial genomes from the specimens. Phylogenetic analyses cluster the Chinese haplotypes together with previously published Russian E. ovodovi, strongly supporting the assignment of these samples to this taxon. The molecular identification of E. ovodovi in northeastern China extends the known geographical range of this fossil species by several thousand kilometers to the east. The estimated coalescence time of all E. ovodovi haplotypes is approximately 199 Kya, with the Chinese haplotypes coalescing approximately 130 Kya. With a radiocarbon age of 12,770-12,596 cal. yBP, the youngest sample in this study represents the first E. ovodovi sample dating to the terminal Pleistocene, moving the extinction date of this species forwards considerably compared to previously documented fossils. Overall, comparison of our three mitochondrial genomes with the two published ones suggests a genetic diversity similar to several extant species of the genus Equus., Competing Interests: The authors have declared that no competing interests exist.

The Thoroughbred horse breed was developed primarily for racing, and has a significant contribution to the qualitative improvement of many other horse breeds. Despite the importance of Thoroughbred racehorses in historical, cultural, and economical viewpoints, there was no temporal and spatial dynamics of them using the mitogenome sequences. To explore this topic, the complete mitochondrial genome sequences of 14 Thoroughbreds and two Przewalski's horses were determined. These sequences were analyzed together along with 151 previously published horse mitochondrial genomes from a range of breeds across the globe using a Bayesian coalescent approach as well as Bayesian inference and maximum likelihood methods. The racing horses were revealed to have multiple maternal origins and to be closely related to horses from one Asian, two Middle Eastern, and five European breeds. Thoroughbred horse breed was not directly related to the Przewalski's horse which has been regarded as the closest taxon to the all domestic horses and the only true wild horse species left in the world. Our phylogenomic analyses also supported that there was no apparent correlation between geographic origin or breed and the evolution of global horses. The most recent common ancestor of the Thoroughbreds lived approximately 8,100-111,500 years ago, which was significantly younger than the most recent common ancestor of modern horses (0.7286 My). Bayesian skyline plot revealed that the population expansion of modern horses, including Thoroughbreds, occurred approximately 5,500-11,000 years ago, which coincide with the start of domestication. This is the first phylogenomic study on the Thoroughbred racehorse in association with its spatio-temporal dynamics. The database and genetic history information of Thoroughbred mitogenomes obtained from the present study provide useful information for future horse improvement projects, as well as for the study of horse genomics, conservation, and in association with its geographical distribution., Competing Interests: The authors have declared that no competing interests exist.

Donkeys and horses share a common ancestor dating back to about 4 million years ago. Although a high-quality genome assembly at the chromosomal level is available for the horse, current assemblies available for the donkey are limited to moderately sized scaffolds. The absence of a better-quality assembly for the donkey has hampered studies involving the characterization of patterns of genetic variation at the genome-wide scale. These range from the application of genomic tools to selective breeding and conservation to the more fundamental characterization of the genomic loci underlying speciation and domestication. We present a new high-quality donkey genome assembly obtained using the Chicago HiRise assembly technology, providing scaffolds of subchromosomal size. We make use of this new assembly to obtain more accurate measures of heterozygosity for equine species other than the horse, both genome-wide and locally, and to detect runs of homozygosity potentially pertaining to positive selection in domestic donkeys. Finally, this new assembly allowed us to identify fine-scale chromosomal rearrangements between the horse and the donkey that likely played an active role in their divergence and, ultimately, speciation.

Donkeys have played an important role in agricultural land practices and in human historical periods of recent past and, still today, are used as a working power in several world areas. The objective of this study was to identify genetic variability in six Italian donkey breeds using mtDNA D-loop. Fifteen haplotypes, grouped in three haplogroups, were identified. The genetic indices were informative and showed a high population genetic variability. The results of AMOVA analyses based on geographic structuring of Italian populations highlighted that the majority of the observed variance is due to differences among samples within breeds. Comparison among Italian haplotypes and mtDNA D-loop sequences belonging to European domestic and Ethiopian donkeys and wild asses, clearly define two clades referred to Nubian lineage. The results can be useful to complement safeguard planes for donkey breeds that are considered to extinction endangered.

The plains zebra (Equus quagga) is an ecologically important species of the African savannah. It is also one of the most numerous and widely distributed ungulates, and six subspecies have been described based on morphological variation. However, the within-species evolutionary processes have been difficult to resolve due to its high mobility and a lack of consensus regarding the population structure. We obtained genome-wide DNA polymorphism data from more than 167,000 loci for 59 plains zebras from across the species range, encompassing all recognized extant subspecies, as well as three mountain zebras (Equus zebra) and three Grevy's zebras (Equus grevyi). Surprisingly, the population genetic structure does not mirror the morphology-based subspecies delineation, underlining the dangers of basing management units exclusively on morphological variation. We use demographic modelling to provide insights into the past phylogeography of the species. The results identify a southern African location as the most likely source region from which all extant populations expanded around 370,000 years ago. We show evidence for inclusion of the extinct and phenotypically divergent quagga (Equus quagga quagga) in the plains zebra variation and reveal that it was less divergent from the other subspecies than the northernmost (Ugandan) extant population.

The Indian wild ass Equus hemionus khur, belonging to ass-like equid branch, inhabits the dry and arid desert of the Little Rann of Kutch, Gujarat. The E. h. khur is the sole survivor of Asiatic wild ass species/subspecies in South Asia. To provide first ever insights into the genetic diversity, phylogeny, and demography of the endangered Indian wild ass, we sampled 52 free-ranging individuals from the Little Rann of Kutch by using a non-invasive methodology. The sequencing of 230 bp in cytochrome b (Cyt b) and displacement loop (D-loop) region revealed that current ∼4000 extant population of Indian wild ass harbours low genetic diversity. Phylogenetic analyses confirmed that E. h. khur, E. h. onager, and E. h. kulan belong to a single strict monophyletic clade. Therefore, we suggest the delimitation of the five E. hemionus subspecies in vogue to a single species E. hemionus. The application of molecular clock confirmed that the Asiatic wild ass had undergone diversification 0.65 Million years ago. Demographic measurements assessed using a Bayesian skyline plot demonstrated decline in the maternal effective population size of the Indian wild ass during different periods; these periods coincided with the origin and rise of the Indus civilization in the northwest of the Indian subcontinent during the Neolithic. In conclusion, maintaining high genetic diversity in the existing isolated population of 4000 Indian wild asses inhabiting the wild ass sanctuary is important compared with subspecies preservation alone.

Digit reduction is a major trend that characterizes horse evolution, but its causes and consequences have rarely been quantitatively tested. Using beam analysis on fossilized centre metapodials, we tested how locomotor bone stresses changed with digit reduction and increasing body size across the horse lineage. Internal bone geometry was captured from 13 fossil horse genera that covered the breadth of the equid phylogeny and the spectrum of digit reduction and body sizes, from Hyracotherium to Equus To account for the load-bearing role of side digits, a novel, continuous measure of digit reduction was also established-toe reduction index (TRI). Our results show that without accounting for side digits, three-toed horses as late as Parahippus would have experienced physiologically untenable bone stresses. Conversely, when side digits are modelled as load-bearing, species at the base of the horse radiation through Equus probably maintained a similar safety factor to fracture stress. We conclude that the centre metapodial compensated for evolutionary digit reduction and body mass increases by becoming more resistant to bending through substantial positive allometry in internal geometry. These results lend support to two historical hypotheses: that increasing body mass selected for a single, robust metapodial rather than several smaller ones; and that, as horse limbs became elongated, the cost of inertia from the side toes outweighed their utility for stabilization or load-bearing., (© 2017 The Author(s).)

Taxonomic over-splitting of extinct or endangered taxa, due to an incomplete knowledge of both skeletal morphological variability and the geographical ranges of past populations, continues to confuse the link between isolated extant populations and their ancestors. This is particularly problematic with the genus Equus. To more reliably determine the evolution and phylogeographic history of the endangered Asiatic wild ass, we studied the genetic diversity and inter-relationships of both extinct and extant populations over the last 100,000 years, including samples throughout its previous range from Western Europe to Southwest and East Asia. Using 229 bp of the mitochondrial hypervariable region, an approach which allowed the inclusion of information from extremely poorly preserved ancient samples, we classify all non-African wild asses into eleven clades that show a clear phylogeographic structure revealing their phylogenetic history. This study places the extinct European wild ass, E. hydruntinus, the phylogeny of which has been debated since the end of the 19th century, into its phylogenetic context within the Asiatic wild asses and reveals recent mitochondrial introgression between populations currently regarded as separate species. The phylogeographic organization of clades resulting from these efforts can be used not only to improve future taxonomic determination of a poorly characterized group of equids, but also to identify historic ranges, interbreeding events between various populations, and the impact of ancient climatic changes. In addition, appropriately placing extant relict populations into a broader phylogeographic and genetic context can better inform ongoing conservation strategies for this highly-endangered species.

To reveal the genetic diversity and phylogenetic relationships between Chinese donkey breeds, 415 individuals representing ten breeds were investigated using ten microsatellite markers. The observed number of alleles, mean effective number of alleles (NE), mean expected heterozygosity (HE), and polymorphic information content (PIC) of each breed and polymorphic locus were analyzed. The results showed that seven (HTG7, HTG10, AHT4, HTG6, HMS6, HMS3, and HMS7) of ten microsatellite loci were polymorphic. The mean PIC, HE, and NE of seven polymorphic loci for the ten donkey breeds were 0.7679, 0.8072, and 6.0275, respectively. These results suggest that domestic Chinese donkey breeds possess higher levels of genetic diversity and heterozygosity than foreign donkeys. A neighbor-joining tree based on Nei's standard genetic distance showed that there was close genetic distance among Xinjiang, Qingyang, Xiji, and Guanzhong donkey breeds. In addition, Mongolia and Dezhou donkey breeds were placed in the same category. The phylogenetic tree revealed that the genetic relationships between Chinese donkey breeds are consistent with their geographic distribution and breeding history.

This study presents the first insights into the genetic diversity and structure of the American donkey metapopulation. The primary objectives were to detect the main structural features underlying variability among American donkey populations, identify boundaries between differentiated gene pools, and draw the main colonization pathways since the introduction of donkeys into America in the 15th century. A panel of 14 microsatellite markers was applied for genotyping 350 American donkeys from 13 countries. The genetic structure of this metapopulation was analysed using descriptive statistics and Bayesian model-based methods. These populations were then compared to a database containing information on 476 individuals from 11 European breeds to identify the most likely ancestral donor populations. Results showed the presence of two distinct genetic pools, with confluence of the two in Colombia. The southern pool showed a unique genetic signature subsequent to an older founder event, but lacked any significant influence of modern gene flow from Europe. The northern pool, conversely, may have retained more ancestral polymorphisms and/or have experienced modern gene flow from Spanish breeds. The Andalusian and, to a lesser extent, the Catalan breeds have left a more pronounced footprint in some of the American donkey populations analysed., (© 2015 Blackwell Verlag GmbH.)

Although equids have had to be tagged with a transponder since 2009, breeding associations in Germany disagree as to which method is best suited for identification (with or without hot iron branding). Therefore, the aim of this systematic literature review was to gain an overview of how effective identification is using transponders and hot iron branding and as to which factors influence the success of identification. Existing literature showed that equids can be identified by means of transponders with a probability of 85-100%, whereas symbol brandings could be identified correctly in 78-89%, whole number brandings in 0-87% and single figures in 37-92% of the readings, respectively. The successful reading of microchips can be further optimised by a correctly operated implantation process and thorough training of the applying persons. affect identification with a scanner. The removal of transponders for manipulation purposes is virtually impossible. Influences during the application of branding marks can hardly, if at all, be standardised, but influence the subsequent readability relevantly. Therefore, identification by means of hot branding cannot be considered sufficiently reliable. Impaired quality of identification can be reduced during reading but cannot be counteracted. Based on the existing studies it can be concluded that the transponder method is the best suited of the investigated methods for clearly identifying equids, being forgery-proof and permanent. It is not to be expected that applying hot branding in addition to microchips would optimise the probability of identification relevantly.

Understanding why animal societies take on the form that they do has benefited from insights gained by applying social network analysis to patterns of individual associations. Such analyses typically aggregate data over long time periods even though most selective forces that shape sociality have strong temporal elements. By explicitly incorporating the temporal signal in social interaction data we re-examine the network dynamics of the social systems of the evolutionarily closely-related Grevy's zebras and wild asses that show broadly similar social organizations. By identifying dynamic communities, previously hidden differences emerge: Grevy's zebras show more modularity than wild asses and in wild asses most communities consist of solitary individuals; and in Grevy's zebras, lactating females show a greater propensity to switch communities than non-lactating females and males. Both patterns were missed by static network analyses and in general, adding a temporal dimension provides insights into differences associated with the size and persistence of communities as well as the frequency and synchrony of their formation. Dynamic network analysis provides insights into the functional significance of these social differences and highlights the way dynamic community analysis can be applied to other species.

All extant equid species are grouped in a single genus - Equus. Among those, ass-like equids have remained particularly unstudied and their phylogenetic relations were poorly understood, most probably because they inhabit extreme environments in remote geographic areas. To gain further insights into the evolutionary history of ass-like equids, we have used a non-invasive sampling approach to collect representative fecal samples of extant African and Asiatic ass-like equid populations across their distribution range and mitochondrial DNA (mtDNA) sequencing analyses to examine intraspecific genetic diversity and population structure, and to reconstruct phylogenetic relations among wild ass species/subspecies. Sequence analyses of 410 base pairs of the fast evolving mtDNA control region identified the Asiatic wild ass population of Kalamaili (China) as the one displaying the highest diversity among all wild ass populations. Phylogenetic analyses of complete cytochrome b sequences revealed that African and Asiatic wild asses shared a common ancestor approximately 2.3Mya and that diversification in both groups occurred much latter, probably driven by climatic events during the Pleistocene. Inferred genetic relationships among Asiatic wild ass species do not support E. kiang monophyly, highlighting the need of more extensive studies in order to clarify the taxonomic status of species/subspecies belonging to this branch of the Equus phylogeny. These results highlight the importance of re-assessing the evolutionary history of ass-like equid species, and urge to extend studies at the population level to efficiently design conservation and management actions for these threatened species., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Hippidions were equids with very distinctive anatomical features. They lived in South America 2.5 million years ago (Ma) until their extinction approximately 10 000 years ago. The evolutionary origin of the three known Hippidion morphospecies is still disputed. Based on palaeontological data, Hippidion could have diverged from the lineage leading to modern equids before 10 Ma. In contrast, a much later divergence date, with Hippidion nesting within modern equids, was indicated by partial ancient mitochondrial DNA sequences. Here, we characterized eight Hippidion complete mitochondrial genomes at 3.4-386.3-fold coverage using target-enrichment capture and next-generation sequencing. Our dataset reveals that the two morphospecies sequenced (H. saldiasi and H. principale) formed a monophyletic clade, basal to extant and extinct Equus lineages. This contrasts with previous genetic analyses and supports Hippidion as a distinct genus, in agreement with palaeontological models. We date the Hippidion split from Equus at 5.6-6.5 Ma, suggesting an early divergence in North America prior to the colonization of South America, after the formation of the Panamanian Isthmus 3.5 Ma and the Great American Biotic Interchange., (© 2015 The Author(s) Published by the Royal Society. All rights reserved.)

Reasons for Performing Study: Donkeys are believed to be less demonstrative of pain than ponies. Research into comparative sensory processing between these species is required to elucidate these behavioural differences., Objectives: To compare changes in the electroencephalogram (EEG) recorded during castration between donkeys and ponies., Study Design: Prospective clinical study., Methods: Six ponies and 6 donkeys were castrated under halothane anaesthesia after acepromazine premedication and thiopental anaesthetic induction. Markers were inserted into the EEG recording at the time of skin incision (skin) and emasculation (emasc) for both testicles (T1 and T2) during a closed castration. Raw EEG data were analysed and the EEG variables median frequency (F50 ), total power (Ptot ) and spectral edge frequency (F95 ) derived using standard techniques. Baseline values of F50 , Ptot and F95 for each animal were used to calculate the percentage change from baseline at T1skin, T2skin, T1emasc and T2emasc., Results: Decreased F50 values relative to baseline were observed in 4 ponies and 2 donkeys across all castration time points. In the remaining animals, the F50 value increased compared with baseline. Both donkey and pony groups showed an overall decrease in Ptot values compared with baseline at T1skin, but the magnitude of the decrease was significantly less (P = 0.004) in ponies than in donkeys. Donkeys demonstrated an overall greater increase (P = 0.05) in F95 values at T1skin relative to baseline compared with ponies., Conclusions: Electroencephalographic responses to the noxious stimulus of castration were noted in both donkeys and ponies. Donkeys demonstrated a greater change in Ptot in response to castration than ponies; thus, donkeys appear to demonstrate a cerebral cortical response to a noxious stimulus that is similar to or greater than that in ponies, suggesting that their subtle behavioural expression of pain is not due to a difference in cortical processing of noxious sensory stimuli., (© 2014 EVJ Ltd.)

Background: The donkey (Equus asinus) is an important domestic animal that provides a reliable source of protein and method of transportation for many human populations. However, the process of domestication and the dispersal routes of the Chinese donkey are still unclear, as donkey remains are sparse in the archaeological record and often confused with horse remains. To explore the maternal origins and dispersal route of Chinese donkeys, both mitochondrial DNA D-loop and cytochrome b gene fragments of 21 suspected donkey remains from four archaeological sites in China were amplified and sequenced., Results: Molecular methods of species identification show that 17 specimens were donkeys and three samples had the maternal genetic signature of horses. One sample that dates to about 20,000 years before present failed to amplify. In this study, the phylogenetic analysis reveals that ancient Chinese donkeys have high mitochondrial DNA diversity and two distinct mitochondrial maternal lineages, known as the Somali and Nubian lineages. These results indicate that the maternal origin of Chinese domestic donkeys was probably related to the African wild ass, which includes the Nubian wild ass (Equus africanus africanus) and the Somali wild ass (Equus africanus somaliensis). Combined with historical records, the results of this study implied that domestic donkeys spread into west and north China before the emergence of the Han dynasty. The number of Chinese domestic donkeys had increased primarily to meet demand for the expansion of trade, and they were likely used as commodities or for shipping goods along the Silk Road during the Tang Dynasty, when the Silk Road reached its golden age., Conclusions: This study is the first to provide valuable ancient animal DNA evidence for early trade between African and Asian populations. The ancient DNA analysis of Chinese donkeys also sheds light on the dynamic process of the maternal origin, domestication, and dispersal route of ancient Chinese donkeys.

The contemporary South American mammalian communities were determined by the emergence of the Isthmus of Panama and by the profound climatic oscillations during the Pleistocene. Horses and gomphotheres were 2 very conspicuous groups of immigrant mammals from North America that arrived in South America during the Pleistocene. The present study compiles updated data on the phylogeny, systematics and ecology of both groups in South America. The horses in South America are represented by 2 genera, Hippidion and Equus, as are the gomphotheres, represented by Cuvieronius and Stegomastodon. Both genera of horses include small (Hippidion devillei, H. saldiasi, E. andium and E. insulatus) and large forms (Equus neogeus and H. principale), which dispersed into South America using 2 different routes. The possible model for this dispersion indicates that the small forms used the Andes corridor, while larger horses dispersed through the eastern route and through some coastal areas. In the case of gomphotheres, Cuvieronius and Stegomastodon reached South America in 2 independent dispersal events, and Cuvieronius dispersed across the Andean corridor, while large Stegomastodon spread along the eastern route. Horses and gomphotheres present values of δ(13) C from woodlands to C4 grasslands. Hippidion present lower values of δ(13) C than Equus in the late Pleistocene, whereas the gomphotheres diverge from value of δ(18) O, reflecting that Cuvieronius inhabited the Andean corridor and Stegomastodon dispersed through eastern plains. The gomphothere and horse species recorded in South America became extinct around the time that humans arrived., (© 2013 International Society of Zoological Sciences, Institute of Zoology/Chinese Academy of Sciences and Wiley Publishing Asia Pty Ltd.)

Despite over a century of interest, the function of zebra stripes has never been examined systematically. Here we match variation in striping of equid species and subspecies to geographic range overlap of environmental variables in multifactor models controlling for phylogeny to simultaneously test the five major explanations for this infamous colouration. For subspecies, there are significant associations between our proxy for tabanid biting fly annoyance and most striping measures (facial and neck stripe number, flank and rump striping, leg stripe intensity and shadow striping), and between belly stripe number and tsetse fly distribution, several of which are replicated at the species level. Conversely, there is no consistent support for camouflage, predator avoidance, heat management or social interaction hypotheses. Susceptibility to ectoparasite attack is discussed in relation to short coat hair, disease transmission and blood loss. A solution to the riddle of zebra stripes, discussed by Wallace and Darwin, is at hand.

Most donkey and local horse breeds are vulnerable to extinction as mechanization of agriculture progress throughout the world. The present study analyzed the pedigree and herd records of the donkey Asinina de Miranda breed (RAM), identifying genealogical and human factors that may affect the breed genetic diversity in the future and suggesting suitable strategies to breed preservation, early on the conservation program. The breeding rate was very low, with a ratio of foaling/live animals of 0.23 (178/760). The estimated number of founders and ancestors contributing to the reference population was 128 and 121. The number of founder herds in the reference population was 64, with an effective number of founder herds for the reference population of 7.6. The mean age of herd owners was 65.50 ± 0.884 years, with a negative association among the herd size and owner's age (P<0.001). In contrast, the size of the herd and the ownership of a male were both positively associated (P<0.001) with the herd number of in-born foals. Both the owners' age and the herd location (RAM home region v. dispersal region) were negatively associated with the foaling number (P<0.001). The main identified risk factors were: low breeding rates; low number of males and their unequal contribution to the genetic pool; unequal contribution of the herds to genetic pool; and advanced age of herd owners.

Four groups of equids, "Anchitheriinae," Merychippine-grade Equinae, Hipparionini, and Equini, coexisted in the middle Miocene, but only the Equini remains after 16 Myr of evolution and extinction. Each group is distinct in its occlusal enamel pattern. These patterns have been compared qualitatively but rarely quantitatively. The processes influencing the evolution of these occlusal patterns have not been thoroughly investigated with respect to phylogeny, tooth position, and climate through geologic time. We investigated Occlusal Enamel Index, a quantitative method for the analysis of the complexity of occlusal patterns. We used analyses of variance and an analysis of co-variance to test whether equid teeth increase resistive cutting area for food processing during mastication, as expressed in occlusal enamel complexity, in response to increased abrasion in their diet. Results suggest that occlusal enamel complexity was influenced by climate, phylogeny, and tooth position through time. Occlusal enamel complexity in middle Miocene to Modern horses increased as the animals experienced increased tooth abrasion and a cooling climate.

Grant's zebra (Equus burchelli) are commonly kept in zoos and are subject to routine health monitoring and research studies. Recently, assays for acute phase proteins (APP) have been described in many wildlife species, and specific assays for serum amyloid A (SAA) have been well validated and studied in horses (Equus ferus caballus), in which it serves as a major APP. In the present study, serum samples from 26 Grant's zebra were subject to analysis by using assays for SAA, haptoglobin (HP), and protein electrophoresis. Reference intervals were calculated by using the robust method: SAA 1.8-31.4 mg/L and HP 0.37-1.58 mg/ml. Significant differences in SAA and HP were observed in clinically abnormal zebra; in some cases, these differences were marked and were noted in the absence of abnormal values for protein electrophoretic fractions. These data indicate that APP may be a valuable and sensitive tool in monitoring inflammation in this species.

The rich fossil record of equids has made them a model for evolutionary processes. Here we present a 1.12-times coverage draft genome from a horse bone recovered from permafrost dated to approximately 560-780 thousand years before present (kyr BP). Our data represent the oldest full genome sequence determined so far by almost an order of magnitude. For comparison, we sequenced the genome of a Late Pleistocene horse (43 kyr BP), and modern genomes of five domestic horse breeds (Equus ferus caballus), a Przewalski's horse (E. f. przewalskii) and a donkey (E. asinus). Our analyses suggest that the Equus lineage giving rise to all contemporary horses, zebras and donkeys originated 4.0-4.5 million years before present (Myr BP), twice the conventionally accepted time to the most recent common ancestor of the genus Equus. We also find that horse population size fluctuated multiple times over the past 2 Myr, particularly during periods of severe climatic changes. We estimate that the Przewalski's and domestic horse populations diverged 38-72 kyr BP, and find no evidence of recent admixture between the domestic horse breeds and the Przewalski's horse investigated. This supports the contention that Przewalski's horses represent the last surviving wild horse population. We find similar levels of genetic variation among Przewalski's and domestic populations, indicating that the former are genetically viable and worthy of conservation efforts. We also find evidence for continuous selection on the immune system and olfaction throughout horse evolution. Finally, we identify 29 genomic regions among horse breeds that deviate from neutrality and show low levels of genetic variation compared to the Przewalski's horse. Such regions could correspond to loci selected early during domestication.

Equidae is a small family which comprises horses, African and Asiatic asses, and zebras. Despite equids having diverged quite recently, their karyotypes underwent rapid evolution which resulted in extensive differences among chromosome complements in respective species. Comparative mapping using whole-chromosome painting probes delineated genome-wide chromosome homologies among extant equids, enabling us to trace chromosome rearrangements that occurred during evolution. In the present study, we performed subchromosomal comparative mapping among seven Equidae species, representing the whole family. Region-specific painting and bacterial artificial chromosome probes were used to determine the orientation of evolutionarily conserved segments with respect to centromere positions. This allowed assessment of the configuration of all fusions occurring during the evolution of Equidae, as well as revealing discrepancies in centromere location caused by centromere repositioning or inversions. Our results indicate that the prevailing type of fusion in Equidae is centric fusion. Tandem fusions of the type telomere-telomere occur almost exclusively in the karyotype of Hartmann's zebra and are characteristic of this species' evolution. We revealed inversions in segments homologous to horse chromosomes 3p/10p and 13 in zebras and confirmed inversions in segments 4/31 in African ass, 7 in horse and 8p/20 in zebras. Furthermore, our mapping results suggested that centromere repositioning events occurred in segments homologous to horse chromosomes 7, 8q, 10p and 19 in the African ass and an element homologous to horse chromosome 16 in Asiatic asses. Centromere repositioning in chromosome 1 resulted in three different chromosome types occurring in extant species. Heterozygosity of the centromere position of this chromosome was observed in the kiang. Other subtle changes in centromere position were described in several evolutionary conserved chromosomal segments, suggesting that tiny centromere repositioning or pericentric inversions are quite frequent in zebras and asses.

The enzyme glutamine synthetase (GS; glutamate-ammonia ligase, EC 6.3.1.2) plays an important role in the nitrogen metabolism of fish. In this study the GS activity and the corresponding genes were examined to understand how they are regulated in zebrafish in response to hyperammonemic stress during a 72 h simulated transport. Whole body ammonia levels, the activity of the enzyme GS and the mRNA expression of the splice variants of three paralogues of glul, glutamine synthetase gene (glula, glulb and glulc) were examined in brain, liver and kidney of zebrafish. Whole body ammonia reached significantly higher levels by 48 h, while brain showed higher levels as early as 24 h, compared to the values at the start of the transport. The GS activities in brain, liver and kidney were significantly higher at the end of 72 h transport than those at the start. However, only the expression of mRNA of glulb-002 and glulb-003 were significantly upregulated during the simulated transport. In silico analysis of the putative promoter regions of glul paralogues revealed glucocorticoid receptor binding sites. However, glucocorticoid response elements of glulb were not different. The up-regulation of GS enzyme activity and hitherto unreported mRNA expression of glul paralogues during zebrafish transport indicate a physiological response of fish to ammonia., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Short divergence times and processes such as incomplete lineage sorting and species hybridization are known to hinder the inference of species-level phylogenies due to the lack of sufficient informative genetic variation or the presence of shared but incongruent polymorphism among taxa. Extant equids (horses, zebras, and asses) are an example of a recently evolved group of mammals with an unresolved phylogeny, despite a large number of molecular studies. Previous surveys have proposed trees with rather poorly supported nodes, and the bias caused by genetic introgression or ancestral polymorphism has not been assessed. Here we studied the phylogenetic relationships of all extant species of Equidae by analyzing 22 partial mitochondrial and nuclear genes using maximum likelihood and Bayesian inferences that account for heterogeneous gene histories. We also examined genetic signatures of lineage sorting and/or genetic introgression in zebras by evaluating patterns of intraspecific genetic variation. Our study improved the resolution and support of the Equus phylogeny and in particular the controversial positions of the African wild ass (E. asinus) and mountain zebra (E. zebra): the African wild ass is placed as a sister species of the Asiatic asses and the mountain zebra as the sister taxon of Grevy's and Burchell's zebras. A shared polymorphism (indel) detected among zebra species in the Estrogen receptor 1 gene was likely due to incomplete lineage sorting and not genetic introgression as also indicated by other mitochondrial (Cytochrome b) and nuclear (Y chromosome and microsatellites) markers. Ancestral polymorphism in equids might have contributed to the long-standing lack of clarity in the phylogeny of this highly threatened group of mammals., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Comparative painting has provided a wealth of useful information and helped to reconstruct the pathways of karyotype evolution within major eutherian phylogenetic clades. New data have come from gene localizations, BAC mapping and high throughout sequencing projects that enrich and provide new details of genome evolution. Extensive research on perissodactyl genomes has revealed not only increased rates of chromosomal rearrangements, but also an exceptionally high number of centromere repositioning events in equids. Here were combined new physical mapping, comparative painting and genome sequencing data to refine the putative ancestral karyotype maps and to revise the previously proposed scenario of perissodactyl karyotype evolution., (Copyright © 2012 S. Karger AG, Basel.)

Multiple cysts ranging from 2-111 mm were noted bilaterally in the ovulation fossa of 11 mature Miranda jennets. These ovulation fossa inclusion cysts (OFICs) were lined by a simple low to columnar epithelium that included many ciliated cells. Although most cases were incidental findings, two of the jennets were presented with reduced fertility. Extensive cyst formation could have been responsible for the reproductive problems because they replaced most of the ovarian parenchyma. Due to their close proximity to the ovulation fossa, the OFICs may have mechanically interfered with passage of eggs into the oviduct. OFICs are histologically common in equids, but are reported uncommonly as gross lesions in either mares or jennets. Ovarian inclusion cysts are associated with neoplasia in women; however, these OFICs showed no evidence of epithelial hyperplasia or cellular atypia and no evidence of independent growth, therefore they were considered to be non-neoplastic. The bilateral occurrence and high incidence of OFICs in Miranda jennets, a breed with limited genetic variability, suggests that the lesion has a genetic causation., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Although horses and donkeys belong to the same genus, their genetic characteristics probably result in specific proteomes and post-translational modifications (PTM) of proteins. Since PTM can alter protein properties, specific PTM may contribute to species-specific characteristics. Therefore, the aim of the present study was to analyse differences in serum protein profiles of horses and donkeys as well as mules, which combine the genetic backgrounds of both species. Additionally, changes in PTM of the protein transthyretin (TTR) were analysed. Serum protein profiles of each species (five animals per species) were determined using strong anion exchanger ProteinChips® (Bio-Rad, Munich, Germany) in combination with surface-enhanced laser desorption ionisation-time of flight MS. The PTM of TTR were analysed subsequently by immunoprecipitation in combination with matrix-assisted laser desorption ionisation-time of flight MS. Protein profiling revealed species-specific differences in the proteome, with some protein peaks present in all three species as well as protein peaks that were unique for donkeys and mules, horses and mules or for horses alone. The molecular weight of TTR of horses and donkeys differed by 30 Da, and both species revealed several modified forms of TTR besides the native form. The mass spectra of mules represented a merging of TTR spectra of horses and donkeys. In summary, the present study indicated that there are substantial differences in the proteome of horses and donkeys. Additionally, the results probably indicate that the proteome of mules reveal a higher similarity to donkeys than to horses.

In a recent paper, Eronen et al. (2010; hereafter EEFJ) observe differences in occlusal morphology, tooth crown height, and mesowear pattern between populations of the Miocene tridactyl equid Anchitherium from Spain and Germany, proposing that Spanish Anchitherium underwent adaptive evolution to local or regional arid conditions. However, these authors do not take into account the actual diversity of Iberian representatives of Anchitherium, or the fact that the Spanish fossils cover a wider temporal and geographical range than those from Germany. For these reasons, we suggest that their subsequent statistical work should be reconsidered., (© 2011 The Author(s). Evolution© 2011 The Society for the Study of Evolution.)

The Southern Levant is a pivotal area for the study of hominin paleoecology during the Lower Paleolithic, because of its location on the out-of-Africa dispersal route and its significant ecological diversity. Important information has been gained by archaeofaunal studies, which usually reveal that exploitation of diverse Mediterranean environments with woodlands, marshes and lake margins, represents a dominant subsistence strategy for Lower Paleolithic hominins. Here, we present new taxonomic and taphonomic data from two sites in the southern coastal plain of the Southern Levant, at the fringe of the Negev Desert: Bizat Ruhama (Early Pleistocene) and Nahal Hesi (Middle Pleistocene). The sites preserve anthropogenic faunas, with the former signaling a marrow-exploitation strategy, perhaps related to scavenging from carnivore kills, and the latter showing evidence for primary access to fleshed ungulate carcasses. The species composition of these Northern Negev sites is unique for the Levantine Lower Paleolithic in that these sites lack typical woodland and riparian species, probably indicating an open, relatively uniform environment with patchy water sources and trees, much like this semiarid region today. Bizat Ruhama and Nahal Hesi are among the only Levantine Lower Paleolithic faunas associated with such a setting, thereby widening the known spectrum of environments exploited by hominins in the region. It is suggested that the two sites, coupled with the nearby Late Pleistocene evidence, reflect a largely stable semiarid environment on the northwestern fringe of the Negev Desert throughout much of the Pleistocene., (Copyright © 2010. Published by Elsevier Ltd.)

The evolution of high-crowned molars among horses (Family Equidae) is thought to be an adaptation for abrasive diets associated with the spread of grasslands. The sharpness and relief of the worn cusp apices of teeth (mesowear) are a measure of dietary abrasion. We collected mesowear data for North American Equidae for the past 55.5 million years to test the association of molar height and dietary abrasion. Mesowear trends in horses are reflective of global cooling and associated vegetation changes. There is a strong correlation between mesowear and crown height in horses; however, most horse paleopopulations had highly variable amounts of dietary abrasion, suggesting that selective pressures for crown height may have been weak much of the time. However, instances of higher abrasion were observed in some paleopopulations, suggesting intervals of stronger selection for the evolution of dentitions, including the early Miocene shortly before the first appearance of Equinae, the horse subfamily in which high-crowned dentitions evolved.

The new subgenus of Equus, Sussemionus, is defined by peculiar dental characters so far unknown, or exceptional in late Pleistocene and extant Equus; it was in consequence assumed to be restricted to the early and middle Pleistocene. During that period, it was highly successful, ranging from North America to Ethiopia, and included dry-adapted (E. granatensis-like) and more humid-adapted (E. coliemensis-like) species. Recent molecular and osteological analyses concurred to prove its survival until ca 45 KYBP in Khakassia, southwest Siberia, Russia., (Copyright 2009 Académie des sciences. Published by Elsevier SAS. All rights reserved.)

The major histocompatibility complex genes coding for antigen binding and presenting molecules are the most polymorphic genes in the vertebrate genome. We studied the DRA and DQA gene polymorphism of the family Equidae. In addition to 11 previously reported DRA and 24 DQA alleles, six new DRA sequences and 13 new DQA alleles were identified in the genus Equus. Phylogenetic analysis of both DRA and DQA sequences provided evidence for trans-species polymorphism in the family Equidae. The phylogenetic trees differed from species relationships defined by standard taxonomy of Equidae and from trees based on mitochondrial or neutral gene sequence data. Analysis of selection showed differences between the less variable DRA and more variable DQA genes. DRA alleles were more often shared by more species. The DQA sequences analysed showed strong amongst-species positive selection; the selected amino acid positions mostly corresponded to selected positions in rodent and human DQA genes.

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.

Patterns of genetic differentiation in the plains zebra (Equus quagga) were analysed using mitochondrial DNA control region variation and seven microsatellites. The six morphologically defined subspecies of plains zebra lacked the population genetic structure indicative of distinct evolutionary units. Both marker sets showed high levels of genetic variation and very low levels of differentiation. There was no geographical structuring of mitochondrial DNA haplotypes in the phylogenetic tree, and the plains zebra showed the lowest overall differentiation recorded in any African ungulate studied so far. Arid-adapted African ungulates have shown significant regional genetic structuring in support of the Pleistocene refuge theory. This was not the case in the zebra, and the data are discussed in relation to the impact of Pleistocene climate change on a nonbovid member of the savannah ungulate community. The only other species showing a similar absence of genetic structuring is the African buffalo (Syncerus caffer), but this taxon lacks the high levels of morphological variation present in the plains zebra.

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).

Analysis of the 367 mtDNA D-loop sequences (of which 241 sequences were collected from literature) of 399 bp in 13 Chinese domestic donkey breeds revealed 96 different haplotypes with 57 polymorphic sites. The haplotype diversity and the nucleotide diversity were 0.767-0.967 and 0.014-0.032, respectively, indicating abundant genetic diversity in Chinese domestic donkeys. The Neighbor-joining tree of Chinese domestic donkey sequences was constructed with 3 Nubian wild ass sequences, 3 Somali wild ass sequences and 6 Asian wild ass sequences. Our results suggest that the maternal ancestor of Chinese domestic donkeys is highly likely to be Somali and Nubian of African wild ass instead of Asian wild ass.

Restriction endonucleases, namely BamH I, Taq I, Hae III, Rsa I and Hinc II, were used to analyze the polymorphism of partial mtDNA Cytb gene sequences from 256 horses 6 types (Thoroughbred, Sanhe, Wuzhumuqin, Xinihe, Wushen and Pony) including the imported breed, cultivated breed and local breed. The products of endonuclease digestion were run on 8% non-denaturing polyacrylamide gel electrophoresis and detected by silver staining. Results indicated BamH I and Taq I polymorphism. In all 7 restriction patterns were defected that could be sorted into 3 haplotypes, of which haplotypes I and III were the basic haplotypes. We infer that these horses came from one female ancestor through the analysis thorough one pattern, namely BamH I-B.

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.

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.