Mitochondrial DNA of the extinct quagga: Relatedness and extent of postmortem change

Sequences are reported for portions of two mitochondrial genes from a domestic horse and a plains zebra and compared to those published for a quagga and a mountain zebra. The extinct quagga and plains zebra sequences are identical at all silent sites, whereas the horse sequence differs from both of them by 11 silent substitutions. Postmortem changes in quagga DNA may account for the two coding substitutions between the quagga and plains zebra sequences. The hypothesis that the closest relative of the quagga is the domestic horse receives no support from these data. From the extent of sequence divergence between horse and zebra mitochondrial DNAs (mtDNAs), as well as from information about the fossil record, we estimate that the mean rate of mtDNA divergence in Equus is similar to that in other mammals, i.e., roughly 2% per million years.     

High variation and very low differentiation in wide ranging plains zebra (Equus quagga): insights from mtDNA and microsatellites

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.     

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

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

Palaeogenomics

In many ways, palaeogenomics began when the first ancient DNA sequence was reported. This first sequence was derived from a stuffed museum specimen of the quagga, an extinct mammal related to the zebra. Unspecified and unselected DNA was extracted from the quagga specimen, cloned into a bacterial library, and then sequenced. It took another 17 years and the development of PCR before two independent groups successfully sequenced the complete mitochondrial genomes from several extinct moa species. Only 4 years later, using the original approach of cloning nonspecific ancient DNA extract and shotgun sequencing, the first ancient nuclear DNA sequences were determined, this time from the extinct cave bear. Since these early successes, palaeogenomics has rapidly expanded, because of both technological development and increasing interest in ancient DNA research. New methods, developed since the cave bear sequence was reported, have produced nuclear DNA on a megabase scale from two extinct species, the mammoth and the Neanderthal, our closest relative. For both species, low-coverage genome-sequencing projects have been proposed. It is likely that these will be successful, given the rapid technical development in sequencing techniques. This review carefully examines both the promise and the current limitations of palaeogenomic analyses for both mitochondrial and nuclear DNA.     

Phylogenetic analysis of the microbial populations in the wild herbivore gastrointestinal tract: insights into an unexplored niche

At present, there is little information on the phylogenetic diversity of microbial species that inhabit the gastrointestinal tracts of wildlife. To increase understanding in this area, we initiated a characterization of the bacterial diversity in the digestive tracts of three wild African ruminant species namely eland (Taurotragus oryx), Thompson's gazelle (Gazella rufifrons) and Grant's gazelle (Gazella granti), together with a domesticated ruminant species, zebu cattle (Bos indicus), and a non-ruminant species, zebra (Equus quagga). Bacterial diversity was analysed by PCR amplification, sequencing and phylogenetic analysis of 16S ribosomal DNA (rDNA) sequences. A total of 252 full-length 16S rDNA sequences averaging 1,500 base pairs (bp) in length, and an additional 27 partial sequences were obtained and subject to phylogenetic analysis. Using a 98% criterion for similarity, all except for one of the sequences were derived from distinct phylotypes. At least 24 distinct operational taxonomic units (OTU's) could be identified, with the majority of these sequences representing hitherto uncharacterized species and genera. The sequences were generally affiliated with four major bacterial phyla, the majority being members of the Firmicutes (low G+C Gram-positives) related to the genera Clostridium and Ruminococcus. By contrast, with earlier studies using 16S rDNA sequences to assess biodiversity in Bos taurus dairy cattle, Gram-negative bacteria in the Bacteroidales (Prevotella-Bacteroides group) were poorly represented. The lack of redundancy in the 16S rDNA dataset from the five African ungulate species, and the presence of novel sequences not previously described from the gastrointestinal tract of any animal species, highlights the level of diversity that exists in these ecosystems and raises the question as to the functional role of these species in the gastrointestinal tract.     

Seasonal and demographic factors influencing gastrointestinal parasitism in ungulates of Etosha National Park

Host-parasite dynamics can be strongly affected by seasonality and age-related host immune responses. We investigated how observed variation in the prevalence and intensity of parasite egg or oocyst shedding in four co-occurring ungulate species may reflect underlying seasonal variation in transmission and host immunity. This study was conducted July 2005-October 2006 in Etosha National Park, Namibia, using indices of parasitism recorded from 1,022 fecal samples collected from plains zebra (Equus quagga), springbok (Antidorcas marsupialis), blue wildebeest (Connochaetes taurinus), and gemsbok (Oryx gazella). The presence and intensity of strongyle nematodes, Strongyloides spp. and Eimeria spp. parasites, were strongly seasonal for most host-parasite combinations, with more hosts infected in the wet season than the dry season. Strongyle intensity in zebra was significantly lower in juveniles than adults, and in springbok hosts, Eimeria spp. intensity was significantly greater in juveniles than adults. These results provide evidence that acquired immunity is less protective against strongyle nematodes than Eimeria spp. infections. The seasonal patterns in parasitism further indicate that the long dry season may limit development and survival of parasite stages in the environment and, as a result, host contact and parasite transmission.     

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.     

Population genetic history of the dreissenid mussel invasions: expansion patterns across North America

This study tests population genetic patterns across the Eurasian dreissenid mussel invasions of North America—encompassing the zebra mussel Dreissena polymorpha (1986 detection) and the quagga mussel D. rostriformis bugensis (detected in 1990, which now has largely displaced the former in the Great Lakes). We evaluate their source-spread relationships and invasion genetics using 9–11 nuclear microsatellite loci for 583 zebra mussels (21 sites) and 269 quagga mussels (12 sites) from Eurasian and North American range locations, with the latter including the Great Lakes, Mississippi River basin, Atlantic coastal waterways, Colorado River system, and California reservoirs. Additionally, mtDNA cytochrome b gene sequences are used to verify species identity. Our results indicate that North American zebra mussels originate from multiple non-native northern European populations, whereas North American quagga mussels trace to native estuaries in the Southern Bug and Dnieper Rivers. Invasive populations of both species show considerable genetic diversity and structure (zebra F _ST = 0.006–0.263, quagga F _ST = 0.008–0.267), without founder effects. Most newer zebra mussel populations have appreciable genetic diversity, whereas quagga mussel populations from the Colorado River and California show some founder effects. The population genetic composition of both species changed over time at given sites; with some adding alleles from adjacent populations, some losing them, and all retaining closest similarity to their original composition. Zebra mussels from Kansas and California appear genetically similar and assign to a possible origin from the St. Lawrence River, whereas quagga mussels from Nevada and California assign to a possible origin from Lake Ontario. These assignments suggest that overland colonization pathways via recreational boats do not necessarily reflect the most proximate connections. In conclusion, our microsatellite results comprise a valuable baseline for resolving present and future dreissenid mussel invasion pathways.     

Toxoplasma gondii antibodies in free-living African mammals.

Twelve species of free-living African mammals from Kenya, Tanzania, Uganda and Zambia were tested for antibodies to Toxoplasma gondii using the indirect hemagglutination test. Of 157 animals sampled, 20 (13%) were seropositive. T. gondii antibodies were detected in Burchell's zebra, (Equus burchelli), hippopotamus (Hippopotamus amphibius), African elephant (Loxodonta africana), defassa waterbuck (Kobus defassa), lion (Panthera leo), and rock hyrax (Procavia capensis), The highest titers were found in elephants, two having titers of 1:4096 and one of 1:8192. These results are discussed in relation to the maintenance of T. gondii among African wildlife.     

Morphological and physiological aspects of digestive processes in the graminivorous primate Theropithecus gelada-a preliminary study

Hindgut fermentation has been suggested to contribute significantly to the digestive process in the gelada (Theropithecus gelada). We therefore hypothesized that in an in vitro fermentation test (Hohenheim gas test, using gas production as measure of microbial digestion) inoculum based on fresh gelada feces would degrade grass to a similar degree as zebra (Equus burchelli chapmani) feces and to a higher degree than that of hamadryas baboons (Papio hamadryas). Additionally, morphology of gelada tongue, salivary glands, stomach, and intestine were examined in this study. Gas production was measured between 4 and 96 hr using animal feces incubated with 200 mg of air-dry hay or mixed concentrate sample. For grass hay, 12-hr gas production was as follows: T. gelada (19.9 ml)>Papio (18.4 ml)>Equus (15.7 ml). After 24 hr, gas production changed: Papio (35.1 ml)>T. gelada (31.9 ml)>Equus (27.9 ml). At 96 hr, Papio was unexpectedly the most effective species with the highest gas production (53.1 ml)>zebra (51.2 ml)>gelada (49.4 ml). With a concentrate standard, 12-hr gas production was as follows: T. gelada (38.5 ml)>Equus (36.8 ml) = Papio (36.4 ml). At 24 hr, gas production differed: Papio (51.7 ml)>Equus (47.0 ml) = T. gelada (46.8 ml). At 96 hr, zebra was the most effective species with the highest gas production (63.9 ml)>Papio (60 ml) = T. gelada (59.9 ml). In conclusion, the results show that the microbial population present in gelada feces is able to ferment forage and concentrate substrates in vitro, although this fermentation did not occur with the expected effectiveness. Future studies should therefore focus also on the bacteria species involved.     

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.     

Ancient DNA Clarifies the Evolutionary History of American Late Pleistocene Equids

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

Divergent water requirements partition exposure risk to parasites in wild equids

For grazing herbivores, dung density in feeding areas is an important determinant of exposure risk to fecal‐orally transmitted parasites. When host species share the same parasite species, a nonrandom distribution of their cumulative dung density and/or nonrandom ranging and feeding behavior may skew exposure risk and the relative selection pressure parasites impose on each host. The arid‐adapted Grevy''s zebra (Equus grevyi) can range more widely than the water‐dependent plains zebra (Equus quagga), with which it shares the same species of gastrointestinal nematodes. We studied how the spatial distribution of zebra dung relates to ranging and feeding behavior to assess parasite exposure risk in Grevy''s and plains zebras at a site inhabited by both zebra species. We found that zebra dung density declined with distance from water, Grevy''s zebra home ranges (excluding those of territorial males) were farther from water than those of plains zebras, and plains zebra grazing areas had higher dung density than random points while Grevy''s zebra grazing areas did not, suggesting a greater exposure risk in plains zebras associated with their water dependence. Fecal egg counts increased with home range proximity to water for both species, but the response was stronger in plains zebras, indicating that this host species may be particularly vulnerable to the elevated exposure risk close to water. We further ran experiments on microclimatic effects on dung infectivity and showed that fewer nematode eggs embryonated in dung in the sun than in the shade. However, only 5% of the zebra dung on the landscape was in shade, indicating that the microclimatic effects of shade on the density of infective larvae is not a major influence on exposure risk dynamics. Ranging constraints based on water requirements appear to be key mediators of nematode parasite exposure in free‐ranging equids.     

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 Nooit-gedachter, 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.     

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.     

Development of a molecular diagnostic system to discriminate Dreissena polymorpha (zebra mussel) and Dreissena bugensis (quagga mussel)

A 3-primer PCR system was developed to discriminate invasive zebra (Dreissena polymorpha) and quagga (Dreissena bugensis) mussel. The system is based on: 1) universal primers that amplifies a region of the nuclear 28s rDNA gene from both species and 2) a species-specific primer complementary to either zebra or quagga mussel. The species-specific primers bind to sequences between the binding sites for the universal primers resulting in the amplification of two products from the target species and one product from the nontarget species. Therefore, nontarget products are positive amplification controls. The 3-primer system accurately discriminated zebra and quagga mussels from seven geographically distinct populations.     

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.     

Mitochondrial DNA evolution in the genus Equus

Employing mitochondrial DNA (mtDNA) restriction-endonuclease maps as the basis of comparison, we have investigated the evolutionary affinities of the seven species generally recognized as the genus Equus. Individual species' cleavage maps contained an average of 60 cleavage sites for 16 enzymes, of which 29 were invariant for all species. Based on an average divergence rate of 2%/Myr, the variation between species supports a divergence of extant lineages from a common ancestor approximately 3.9 Myr before the present. Comparisons of cleavage maps between Equus przewalskii (Mongolian wild horse) and E. caballus (domestic horse) yielded estimates of nucleotide sequence divergence ranging from 0.27% to 0.41%. This range was due to intraspecific variation, which was noted only for E. caballus. For pairwise comparisons within this family, estimates of sequence divergence ranged from 0% (E. hemionus onager vs. E. h. kulan) to 7.8% (E. przewalskii vs. E. h. onager). Trees constructed according to the parsimony principle, on the basis of 31 phylogenetically informative restriction sites, indicate that the three extant zebra species represent a monophyletic group with E. grevyi and E. burchelli antiquorum diverging most recently. The phylogenetic relationships of E. africanus and E. hemionus remain enigmatic on the basis of the mtDNA analysis, although a recent divergence is unsupported.      

Ultrastructure of filamentous microorganisms associated with zebra cyathostomes

Filamentous microorganisms were observed colonizing the cuticle of cyathostomes (Nematoda: Strongylidae) collected from the large intestine of Burchell's zebra (Equus burchelli antiquorum). Scanning (SEM) and transmission (TEM) electron microscopy revealed three filamentous microorganisms, designated as segmented, continuous multicellular, and helical. The segmented and continuous multicellular morphotypes each revealed a process of segmentation in their developmental stages, and those of the segmented filaments are demonstrated in this report. In contrast, the helical types of filaments appeared to be complexes of spiraling rodshaped organisms. In addition to these filaments, the cyathostome cuticle was also colonized by other smaller microorganisms.