Horse haemoglobin phenotyping by agarose gel isoelectric focusing comparison of Thoroughbreds with other Equidae

By using isoelectric focusing in thin agarose slab gels 1049 Thoroughbred, 82 Nooitgedachter, 45 Percheron and 244 horses of other breeds were examined. The numbers of other Equidae tested were 107 donkeys, 50 mules, 4 common zebras (Equus burchelli boehmi) and 8 mountain zebras (Equus zebra hartmannae). Phenotypic data are presented for all tested animals and gene frequencies are calculated for the horses.     

Anthrax outbreak among Grevy's zebra (Equus grevyi) in Samburu, Kenya

An anthrax outbreak occurred in the Wamba area of southern Samburu, Kenya, between December 2005 and March 2006. The outbreak affected equids including the endangered Grevy's zebras (Equus grevyi), plain zebras (Equis Burchelli) and donkeys (Equus asinus). Most of the deaths were localized in Nkaroni area just west of Wamba town. The diagnosis of anthrax was rapidly confirmed by bacteriological methods. The relevant government departments, including the Kenya Wildlife Service and Veterinary Department, and other stakeholders were promptly informed. Fifty‐three Grevy's zebra and 26 plains zebras died from anthrax. An equal number (eighteen) of adult male and female Grevy's zebras succumbed to the disease. The outbreak affected immature and mature individuals equally. The dead plain zebras included fifteen adult females, two adult males and nine immature individuals. The Veterinary Department responded by vaccinating livestock while Kenya Wildlife Service vaccinated 620 Grevy's zebras within southern Samburu. Examination of sites at which carcasses of animals which succumbed to the disease were burnt, revealed that unsupervised burning did not eliminate anthrax spores in 42% of the cases (n = 14). There is an urgent need to incorporate strategic wildlife disease monitoring in the struggle to save Grevy's zebras and other endangered species.     

Mass spectral analysis of domestic and wild equine apoA-I and A-II: detection of unique dimeric forms of apoA-II

In pigs, humans, chimpanzees and probably other great apes, a cysteine at residue 6 enables apolipoprotein A-II to form a homodimer. However, the apoA-IIs of other primates, lacking a cysteine residue, are monomeric. We have already reported that horse apoA-IIs form homodimers due also to a cysteine at residue 6. In this study, we wanted to determine whether other equine apoA-IIs might be monomeric. The high density lipoproteins were ultracentrifugally isolated from the plasmas of a horse (Equus caballus), a donkey (Equus asinus) and five wild equines: two types of zebras (Equus zebra hartmannae and Equus zebra quagga boehmi), a Przewalski's horse (Equus przewalskii), a Somali ass (Equus africanus somalicus) and a kiang (Equus kiang holdereri). Using liquid chromatography with electrospray-ionization mass spectrometry, we were able to obtain accurate values for the molecular masses of apoA-I and apoA-II. Homodimeric apoA-IIs were observed in each of the animals studied. The donkey had unique dimers, consisting of the proapolipoprotein A-II linked by a disulfide bond either to a mature apoA-II monomer or another proapoA-II. In addition, our data indicate that small amounts of apoA-I and apoA-II apparently are acylated.     

Effects of Single‐ and Mixed‐Species Group Composition on the Flight Initiation Distances of Plains and Grevy's Zebras

Zebras, as prey species, attend to the behavior of nearby conspecifics and heterospecifics when making decisions to flee from predators. Plains zebras (Equus quagga) and Grevy's zebras (E. grevyi) frequently form mixed‐species groups in zones where their ranges overlap in Kenya. Although anecdotal observations suggest that Plains zebras are more flighty around humans than Grevy's zebras are, this has not been empirically confirmed, and relatively little is known about how they may influence each other's flight behavior. We addressed these questions by examining the flight initiation distances (FIDs) of Plains and Grevy's zebras in single‐species and mixed‐species groups from an approaching human. One target individual per group was approached steadily on foot, with start distance, alert distance, and FID recorded from this target. Using start distance and alert distance separately as covariates, 22 Plains zebras in single‐species groups exhibited a significantly longer mean FID than 15 Grevy's zebras in single‐species groups. The FIDs of 7 Plains zebras and 5 Grevy's zebras tested in mixed‐species groups were virtually equivalent and intermediate to those of Plains and Grevy's zebras in single‐species groups, suggesting a bidirectional moderating influence of heterospecifics on risk assessment. This effect was most pronounced for Plains zebras in mixed‐species groups that exhibited an FID that was significantly shorter than that of Plains zebras in single‐species groups. Our findings underscore the importance of recognizing that related equids may be differently impacted by anthropogenic stress.     

Mitochondrial Phylogenomics of Modern and Ancient Equids

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

Bloodmeal analysis in Culicoides midges collected near horses,donkeys and zebras in the Eastern Cape,South Africa

An upsurge in African horse sickness (AHS) in the Eastern Cape, South Africa, from 2006 led to an epidemiological reassessment of the disease there. Light trapping surveys carried out near horses, donkeys and zebras in 2014–2016 collected 39 species of Culicoides midge (Diptera: Ceratopogonidae) that are potential vectors of AHS. To establish if these midges fed on equids, DNA sequences were obtained from the gut contents of 52 female midges (35 freshly blood‐fed, 13 gravid and four parous), representing 11 species collected across 11 sites. Culicoides leucostictus fed on all three equids. Culicoides bolitinos, Culicoides imicola and Culicoides magnus fed on both horses and donkeys. Culicoides onderstepoortensis fed on donkeys, and Culicoides similis and Culicoides pycnostictus fed on zebras. Bloodmeals from cows, pigs, warthogs, impalas and a domestic dog were also identified in various species, but none of the midges tested had fed on birds. These results contribute to knowledge of the vectorial capacity of several species of Culicoides with regard to AHS in the Eastern Cape and point to potential reservoir hosts, of which donkeys, zebras and domestic dogs have previously been found to harbour AHS. Blood‐fed midges were also obtained throughout winter, indicating the potential for endemic AHS in the province.     

Extant and fossil Equus (Mammalia, Perissodactyla) skulls: a morphometric definition of the subgenus Equus

A sample of 225 skulls belonging to all extant species of Equus and of 33 fossil skulls of ages comprised between 3.4 Ma and 0.7 Ma was studied by multivariate analyses of size, shape, and form on four axial lengths. A simple bivariate discrimination using the overall palatal length and the cranial length allows an almost complete discrimination between modern (extant and young) equids (subgenus Equus ) and old plesippine and stenonine forms. The modern pattern, possibly related to a bigger brain case, seems to appear less than 1.5 Ma ago in fossil species that can already be recognized as closely related to the extant Grevy's zebras, plains zebras, and horses. This does not support separate branchings of Equus species from a common plesippine stem.     

Molecular phylogeny and evolution of the Perissodactyla

The evolution of perissodactyls (rhinoceroses, tapirs, and horses) has been well studied primarily because of their extensive fossil record. Nevertheless, controversy persists regarding relationships of some of the extant taxa, reflecting inconsistencies between molecular and morphological studies. Here we examine the phylogenetic relationships of 16 living perissodactyl species by concatenating two mitochondrial and nine nuclear genes, and we estimate their divergence times using a relaxed Bayesian molecular clock approach. Our analyses recovered the monophyly of the suborders Ceratomorpha and Hippomorpha, and the families Rhinoceratidae, Tapiridae, and Equidae. We supported the early divergence of the Indian rhinoceros in the late Oligocene (26 Mya) relative to the Sumatran and African rhinoceroses, and the split of caballine (domestic horse and Przewalski's wild horse) and noncaballine equids (zebras and African and Asiatic asses) in the Pliocene (4 Mya). An important implication of this study is that Equus asinus, the African wild ass was found to be the sister taxon of Asiatic asses and zebras, diverging from the common ancestor with caballine horses 2 Mya. Rates of chromosome rearrangements were also evaluated in perissodactyls, placing a notably high rate of variation amongst equids, particularly within the zebra clade. The robust phylogenetic results presented here are relevant in terms of understanding the evolutionary history of this highly threatened group of mammals. © 2011 The Linnean Society of London, Zoological Journal of the Linnean Society, 2011, 163 , 1289–1303.     

Early Pleistocene Equus (Equidae,Perissodactyla) from Andersson Loc. 32 in Qixian,Shanxi, China

The Equus specimens of Andersson Loc. 32 in Qixian, Shanxi previously identified as Equus cf. sanmeniensis are rediscovered in this research. The skull has moderate size, deep nasal notch, clear preorbital fossa, undulated lateral outline, upper cheek tooth with simple fossette and incisor with incomplete cup, should be identified as Equus teilhardi. The mandible has cheek tooth with V-shaped linguaflexid, incisor with incomplete cup, moderate size, strong pli caballinid and deep ectoflexid on molar, should be identified as Equus qingyangensis. This skull is first record of a complete cranial material of E. teilhardi, reveals many important cranial features of E. teilhardi, supports anatomical comparison and phylogenetic discussion with other stenonid horses. The morphological differences between E. teilhardi and E. qingyangensis show their different niches, further explain the reason of their coexistence. Incomplete cup on incisor is an unstable feature. It has been found in several species of stenonid horses, likely a remaining feature derived from the most primitive Equus, Equus simplicidens. The small-sized stenonid horses with short limbs and incisors with incomplete cups likely have relation with E. teilhardi, such as Equus yunnanensis and Equus stehlini. E. qingyangensis has most primitive features in Eurasian stenonid horses.     

A comparison of the intestinal helminth communities of Equidae in Southern Africa

The intestinal helminth communities of 8 horses, 12 donkeys, 21 Hartmann's mountain zebras, and 44 Burchell's zebras were compared using the original data from 6 studies in South Africa and Namibia. Necropsy and worm recovery techniques were comparable between the studies. Sixty helminth species (58 nematode, 1 cestode, and 1 trematode species) were recorded. There were significant differences in the helminth community structures of the 4 Equus species. The helminth communities of the 2 closely related zebra subspecies were most similar, and they jointly shared 7 helminth species with donkeys and only 1 with horses. Geographic variation and host-mixing contributed to the helminth species composition. Multiple confamilial species infections were the norm in the donkeys and zebra subspecies, and no single-species infection was recorded for the Strongylidae. Congeneric species were commonly recorded in 3 genera (Cyathostomum, Cylicocyclus, and Cylicostephanus). The shape of the occupancy frequency distributions for the donkeys and zebra subspecies was multimodal, with no clear satellite or core modes. Despite the presence of environmental variability and comparatively low parasite-host specificity, the phylogenetic signal within Equus helminth communities remains strong.     

Unicorn “holotype” skeleton from the Late Pleistocene spotted hyena den site Sewecken-Berge (Germany)

Late Pleistocene Ice Age spotted hyena remains are described from the “Unicorn holotype skeleton” gypsum karst site Quedlinburg-Sewecken-Berge, Germany (Central Europe). The hyena population consists of adolescent to late adult individuals (96% of hyena NISP; 15% of megafauna NISP) indicating a commuting den site type. The comparisons to other European bone assemblages support hunting specialization on woolly rhinoceros (19% of NISP) and horses (27% of NISP). Specialization on bovids (Bison/Bos) can be added for this site. The megafauna contain few Eemian warm period remains of a large horse Equus ferus fossilis. Most (95%) of the megafauna is attributed to the Late Pleistocene glacial (Weichselian/Wuermian). Horse bones are dominated by distal leg elements from the smaller Przewalski horses Equus ferus przewalskii (26% of NISP). The Unicorn “holotype” skeleton originates from a composed horse skull, vertebrae and front legs, whereas the elephant remains added to this biologically not valid species must have been a straight-tusked elephant tusk.     

Identification of progesterone and 5α‐dihydroprogesterone (5α‐DHP) in zebras by two‐dimensional high‐performance liquid chromatography and their dependence on the state of reproduction

Because of its low levels in late pregnancy, the relationship of progesterone to pregnancy maintenance in Equidae is not obvious. This study investigated the levels of progesterone (4‐pregnane‐3,20‐dione; P4) and 5α‐dihydroprogesterone (5α‐DHP) during pregnancy in zebras in relation to reproductive state. Blood samples from female zebras (Equus burchelli, E. zebra hartmannae, E. grevyi) were taken at Dvur Kralove Zoo. Progesterone and 5α‐DHP were separated by high‐performance liquid chromatography techniques and detected by cross‐reacting antibodies. Identification of progestins was achieved by comparing the identity of peaks of the samples with a standard. In E. z. hartmannae progesterone, values reached 50 ng/mL at the beginning of pregnancy and dropped to levels below 1 ng/mL during the second half of pregnancy. In contrast, 5α‐DHP increased up to 123 and 183 ng/mL during late pregnancy in E. z. hartmannae and E. burchelli, respectively. In E. grevyi, 5α‐DHP levels of 368 ng/mL were obtained during pregnancy, whereas progesterone values were similar in pregnant and non‐pregnant individuals. These marked differences in the course of progesterone and 5α‐DHP levels point to the importance of 5α‐DHP for pregnancy maintenance in zebras. Zoo Biol 18:325–333, 1999. © 1999 Wiley‐Liss, Inc.     

Reactive responses of zebras to lion encounters shape their predator–prey space game at large scale

The predator–prey space game and the costs associated with risk effects are affected by prey 1) proactive adjustments (when prey modify their behaviour in response to an a priori assessment of the risk level) and 2) reactive adjustments (when prey have detected an immediate threat). Proactive adjustments are generally well‐studied, whereas the frequency, strength and duration of reactive adjustments remain largely unknown. We studied the space use and habitat selection of GPS‐collared zebras Equus quagga from 2 to 48 h after an encounter with lions Panthera leo. Lion–zebra encounters generally occurred close to artificial waterholes (< 1 km). Two hours after an encounter, zebras were more likely to have fled than stay when the encounter occurred in more risky bushy areas. During their flight, zebras selected grasslands more than usual, getting great visibility. Regardless of their initial response, zebras finally fled at the end of the night and reached areas located far from waterholes where encounters with lions are less frequent. The large‐scale flights (～4–5 km) of zebras led to a local zebra depression for lions. Zebras that had fled immediately after the encounter resumed their behaviour of coming close to waterholes on the following day. However, zebras that had initially stayed remained far from waterholes for an extra 24 h, remaining an elusive prey for longer. The delay in the flight decision had different short‐term consequences on the lion–zebra game. We reveal that the spatial context of the encounter shapes the immediate response of prey, and that encountering predators induces strong behavioural responses: prey flee towards distant, safer, areas and have a constrained use of key resource areas which are at the heart of the predator–prey game at larger spatio‐temporal scales. Nighttime encounters were infrequent (once every 35 days on average), zebra responses were short‐lived (< 36 h) but occurred over a large spatial scale (several km).     

Robertsonian Fusion and Centromere Repositioning Contributed to the Formation of Satellite-free Centromeres During the Evolution of Zebras

Centromeres are epigenetically specified by the histone H3 variant CENP-A and typically associated with highly repetitive satellite DNA. We previously discovered natural satellite-free neocentromeres in Equus caballus and Equus asinus. Here, through ChIP-seq with an anti-CENP-A antibody, we found an extraordinarily high number of centromeres lacking satellite DNA in the zebras Equus burchelli (15 of 22) and Equus grevyi (13 of 23), demonstrating that the absence of satellite DNA at the majority of centromeres is compatible with genome stability and species survival and challenging the role of satellite DNA in centromere function. Nine satellite-free centromeres are shared between the two species in agreement with their recent separation. We assembled all centromeric regions and improved the reference genome of E. burchelli. Sequence analysis of the CENP-A binding domains revealed that they are LINE-1 and AT-rich with four of them showing DNA amplification. In the two zebras, satellite-free centromeres emerged from centromere repositioning or following Robertsonian fusion. In five chromosomes, the centromeric function arose near the fusion points, which are located within regions marked by traces of ancestral pericentromeric sequences. Therefore, besides centromere repositioning, Robertsonian fusions are an important source of satellite-free centromeres during evolution. Finally, in one case, a satellite-free centromere was seeded on an inversion breakpoint. At 11 chromosomes, whose primary constrictions seemed to be associated with satellite repeats by cytogenetic analysis, satellite-free neocentromeres were instead located near the ancestral inactivated satellite-based centromeres; therefore, the centromeric function has shifted away from a satellite repeat containing locus to a satellite-free new position.     

Comparative parasitological examination on sympatric equids in the Great Gobi “B” Strictly Protected Area,Mongolia

The Przewalski’s horse (Equus caballus przewalskii) became extinct in the wild during the 1960s. Based on a successful captive breeding program, Przewalski’s horses were reintroduced to the Great Gobi Part “B” strictly protected area (SPA) in SW Mongolia in the late 1990s. The Asiatic wild ass (Equus hemionus hemionus), Przewalski’s horse, and sometimes domestic horses live sympatricly in the Gobi B SPA. Previously published data demonstrates that, as a result of their different requirements and utilization of the park’s resources, their home-range size and social structure differ. Parasitological examinations in the three equid species show how the factors “home range, social structure, and resource selection” significantly impact parasitic burden. Asiatic wild asses are potentially exposed to a higher risk of parasite re-infection due to their temporal aggregation in very large groups. This study demonstrates a highly significant greater parasite load in the Asiatic wild ass for the majority of parasites evaluated (Dictyocaulus arnfieldi, Trichostrongylus axei, Strongyloides westeri, Parascaris equorum) compared to Przewalski’s horses and domestic horses in the same habitat. Domestic horses had higher parasite loads for eggs of strongylids, eggs of anoplocephalidae, and Eimeria leuckarti. The potential risk of cross infection between sympatric living equids is high, as is the cross infection between ruminants and equids. Furthermore, this study reports for the first time the occurrence of lungworms in free-ranging Przewalski’s horses. Whereas, Asiatic wild asses and Przewalski’s horses seem to cope very well with the sometimes high parasite burden, Mongolian domestic horses manifested typical parasite burden symptoms.     

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

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

Predation, sex ratios, and male competition in equids (Mammalia: Perissodactyla)

Existing data indicate that a greater preponderance of adult females rather than adult males occurs in most species of mammals. The hypothesis that such differences arise as a result of intermale reproductive competition for females (and not predation) was examined in the Equidae by comparing populations of horses ( Equus caballus ), asses ( E. asinus ), and two species of zebras ( E. zebra and E. burchelli) in predator-free, predator-rich and insular ecosystems.
Evidence is presented that: (1) sex differences in adult mortality occur; (2) they relate to the type and intensity of natural predation; and (3) asymmetries in sex ratios are most often explicable in terms of intermale reproductive competition. Exceptions are discussed and they are complicated by numerous proximate factors.     

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

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

Evolutionary movement of centromeres in horse, donkey, and zebra

Centromere repositioning (CR) is a recently discovered biological phenomenon consisting of the emergence of a new centromere along a chromosome and the inactivation of the old one. After a CR, the primary constriction and the centromeric function are localized in a new position while the order of physical markers on the chromosome remains unchanged. These events profoundly affect chromosomal architecture. Since horses, asses, and zebras, whose evolutionary divergence is relatively recent, show remarkable morphological similarity and capacity to interbreed despite their chromosomes differing considerably, we investigated the role of CR in the karyotype evolution of the genus Equus. Using appropriate panels of BAC clones in FISH experiments, we compared the centromere position and marker order arrangement among orthologous chromosomes of Burchelli's zebra (Equus burchelli), donkey (Equus asinus), and horse (Equus caballus). Surprisingly, at least eight CRs took place during the evolution of this genus. Even more surprisingly, five cases of CR have occurred in the donkey after its divergence from zebra, that is, in a very short evolutionary time (approximately 1 million years).These findings suggest that in some species the CR phenomenon could have played an important role in karyotype shaping, with potential consequences on population dynamics and speciation.     

An assessment of feral horse impacts on treeless drainage lines in the Australian Alps

The feral Horse (Equus caballus) is widespread across the Australian Alps. Feral horses degrade alpine and sub‐alpine ecosystems and damage habitat of a range of threatened species. Despite this, there is little published work to document the extent and severity of these impacts. This study investigated impacts of feral horses on treeless drainage lines at 186 sites across the Australian Alps. The study included sites in the Australian Capital Territory, New South Wales and Victoria. We assessed nine variables related to soil and stream stability and vegetation cover, which in turn influence ecosystem function and habitat quality. We found significant differences among horse‐occupied and horse‐free sites for all soil and stream stability variables assessed. For all variables assessed, the average score (and hence, condition) was worse in horse‐occupied areas. The sites in poorest condition were occupied by horses. Impacts from other mammalian herbivores species appeared to be minor. Management intervention is necessary if these impacts of feral horses are to be addressed.