The genomic history and global expansion of domestic donkeys.

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 Old World Equus and origin of the zebra-ass clade.

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.

Genomics and the Evolutionary History of Equids.

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.

Genetic diversity, evolution and selection in the major histocompatibility complex DRB and DQB loci in the family Equidae.

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.

Perissodactyla (Rhinocerotidae and Equidae) from Kanapoi.

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.

Up-regulation of human immune system function by Donkey's Milk

Donkey's milk represents a good alternative to human milk because of its chemical characteristics similar to those of human's. In present study, the pro- and anti-inflammatory effects of donkey's milk were evaluated on peripheral blood mononuclear cells (PBMCs). PBMCs were isolated from 12 young and 12 aged normal subjects. PBMCs were cultured with or without the optimal and non-cytotoxic dose of pasteurized donkey's milk, and polymyxin B was used to inhibit the possible endotoxin contamination. Following 18 hours incubation, culture supernatants were harvested to measure the secreted Tumor necrosis factor-α (TNF-α), Interleukin-6 (IL-6), Interleukin-8 (IL-8) and Interleukin-10 (IL-10) by ELISA. Donkey's milk significantly increased TNF-α (p= 0.01), IL-8 (p< 0.0001), IL-6 (p< 0.0001) and IL-10 (p= 0.01) levels in PBMCs. In addition, the levels of IL-6 (p= 0.002), IL-8 (p= 0.002) and TNF-α (p= 0.002) from aged subjects were significantly higher compared with young subjects. In contrast with these data, the level of IL-10 was markedly reduced from aged subjects (p= 0.02). Considering the immune-potentiation effects of donkey's milk, it is suggested investigating milk as a beneficial dietary component for up-regulating the immune response in aged people

Factors associated with the distribution of natural Toxoplasma gondii infection among equids in Northeastern Brazil.

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.

Molecular identification of late and terminal Pleistocene Equus ovodovi from northeastern China.

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.

Factors associated with the distribution of natural Toxoplasma gondii infection among equids in Northeastern Brazil / Fatores associados à distribuição da infecção por Toxoplasma gondii em equídeos naturalmente infectados no Nordeste do Brasil

Abstract 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.

Resumo O objetivo deste estudo foi determinar a frequência e os fatores associados a Toxoplasma gondii em equídeos naturalmente infectados no Nordeste do Brasil. Amostras de soro de 569 equídeos (528 cavalos, 8 asnos e 33 muares) foram submetidas a reação de imunofluorescência indireta. Modelos lineares generalizados foram utilizados na avaliação dos fatores associados. Dos 569 animais amostrados, 118 (30,6%) soropositivos eram de área rural e 14 (26,42%) perteciam a áreas urbanas (p>0,05). Observaram-se animais soropositivos em 95% das fazendas e em todos os municípios. Asininos/muares como hospedeiro, presença de gatos domésticos e ratos na fazenda, ingestão de água de lagoa e criação de caprinos permaneceram no modelo final como fatores associados à infecção. Medidas de prevenção, como evitar a presença de gatos domésticos próximos aos locais de criação, de pastejo e fontes de água dos animais, devem ser adotadas. A ampla distribuição de animais positivos sinaliza a possibilidade de infecção também em outros animais domésticos, bem como em humanos pelo ambiente contaminado. Ressalta-se a possibilidade de que asininos e muares permaneçam com títulos detectáveis por mais tempo, justificando a prevalência encontrada, sendo necessários estudos para confirmar este possibilidade.

Genetic diversity and maternal origin of Northeast African and South American donkey populations.

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 FST values showed a high genetic differentiation between Peruvian and Brazilian donkey populations (FST  = 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).

Origin and spread of Thoroughbred racehorses inferred from complete mitochondrial genome sequences: Phylogenomic and Bayesian coalescent perspectives.

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.

Improved de novo genomic assembly for the domestic donkey.

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.

A southern African origin and cryptic structure in the highly mobile plains zebra.

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.

Mitochondrial DNA genetic diversity in six Italian donkey breeds (Equus asinus).

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.

Mechanics of evolutionary digit reduction in fossil horses (Equidae).

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.

Taming the late Quaternary phylogeography of the Eurasiatic wild ass through ancient and modern DNA.

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.

Mitochondrial DNA analyses revealed low genetic diversity in the endangered Indian wild ass Equus hemionus khur.

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.

High polymorphism at microsatellite loci in the Chinese donkey.

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.

[Literature review of the influences on error rates when identifying equids with transponder and hot-iron branding]. / Literaturübersicht zu den Einflussfaktoren auf die Fehlerwahrscheinlichkeiten bei der Identifikation von Equiden durch Transponder und Heißbrand.

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.

Genetic relationships among American donkey populations: insights into the process of colonization.

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.