Molecular Evolution of Cytochrome b of Subterranean Mole Rats, Spalax ehrenbergi Superspecies, in Israel

We describe the molecular evolution of cytochrome b of blind subterranean mole rats. We examined 12 individuals for nucleotide differences in the region of 402 base pairs of mitochondrial cytochrome b. Each individual represents a different population from the entire ecological and speciational range of the four chromosomal species in Israel (2n= 52, 54, 58, and 60) belonging to the Spalax ehrenbergi superspecies. Our results indicate the following. (i) There are seven first-position transitional differences, compared to 34 variable third positions, with no observed second-position substitutions. (ii) A maximum of four amino acids differences occurs across the range. (iii) Within-species diversity increases southward. Only 1 autoapomorphic substitution characterizes either 2n= 52 or 2n= 54, but 6–11 substitutions characterize 2n= 58, and 9–13 substitutions characterize 2n= 60. (iv) Both parsimony and maximum-likelihood trees suggest two monophyletic groups: (a) 2n= 52 and 54, and (b) 2n= 58 and 60, as identified earlier by other protein and DNA markers. (v) Mitochondrial cytochrome b heterogeneity is significantly correlated with climatic factors (rainfall) and biotic factors (body size and allozymes). We hypothesize that two selective regimes direct cytochrome b evolution in the S. ehrenbergi superspecies: (i) purifying selection in the flooded, mesic, hypoxic northern range of 2n= 52 and 54 and (ii) diversifying selection in the climatically spatiotemporal, xeric, and variable southern range of 2n= 58 and 60. Thus, the molecular evolution of mitochondrial cytochrome b in S. ehrenbergi is explicable by opposite selective stresses across the range of S. ehrenbergi in Israel, associated with the ecological adaptive radiation of the complex. Received: 23 October 1998 / Accepted: 2 May 1999     

Adaptive variation of pelage color within and between species of the subterranean mole rat (Spalax ehrenbergi) in Israel

Summary Color notations of dorsal pelage were analyzed in 451 adult subterranean mole rats, comprising 19 populations and 4 chromosomal species (2n=52, 54, 58 and 60) of the Spalax ehrenbergi superspecies in Israel. In addition, the color notations of soil samples from the collection sites were measured. In an attempt to evaluate the degree of correlation between pelage color of mole rats and the local soil color, each color was compared both macro- and microgeographically. The macrogeographic comparisons were among populations of the 4 chromosomal species, and the microgeographic comparisons were among pairs of geographically neighboring subpopulations which occur under similar climatic conditions but differ in soil type and color. The results indicate that 1. no differences in pelage color notation exist between the sexes; 2. pelage of mole rats is generally characterized by an over-all grayish color, but its shades vary regionally and locally in accordance with varying soil color; 3. a positive correlation was found between pelage and local soil colors in the macrogeographic analysis and this correlation was verified in a microgeographic comparison; 4. specimens from populations of the northern chromosomal species (2n=52, 54) mainly inhabit terra rossa and basalt soils, respectively, with reddish brown tones, and they tend to have a more reddish shade, whereas animals from the central species (2n=58), which inhabits mainly dark soils including alluvial clays, terra rossa and brown rendzina and the souther species 2n=60, which occurs mainly in light soil types such as pale rendzina, sandy loams and loess, tend to have a more yellowish shade. The general appearance of specimens from 2n=58 was dark gray while that of 2n=60 specimens was light gray; 5. mole rats living in xeric environments (particularly 2n=60, but also 2n=54) are lighter than those which live in mesic environments (2n=52, 58). The results support the conclusion that pelage color of strictly subterranean mole rats is subject to selection pressures of overground predation of disharmonious types with their background soil color. The possibility that the pelage color variation and patterns also contributes to better thermoregulation while mole rats are above ground is discussed.     

Ribosomal DNA non-transcribed spacer polymorphism in subterranean mole rats: genetic differentiation,environmental correlates and phylogenetic relationships

Summary An analysis is presented of genetic differentiation in the non-transcribed spacers of ribosomal DNA (NTS rDNA). Diversity, environmental correlates and the phylogenetic relationships are examined within and between species of the actively speciating subterranean mole rat, superspeciesSpalax ehrenbergi (2n=52, 54, 58, 60) in Israel. This analysis is based on a previous study of the geographic distribution of restriction fragment length polymorphisms of NTS rDNA. Here we present results indicating that NTS rDNA diversity exists mostly (66%) within populations, while 20% is between populations within species, and 14% between species. Multivariate discriminant analysis succeeded in separating 10 of the 13 populations (77%) into their correct chromosomal species, on the basis of the combination of three NTS rDNA repetypes. The phylogenetic relationships suggest that the complex involves two pairs of closely related species (2n=52–54 and 2n=58–60). NTS rDNA diversity, as well as the decrease southward in frequency of repetype C, are correlated with climatic factors of humidity and temperature. These data are discussed in terms of the evolutionary forces of migration and selection which may cause NTS rDNA differentiation. Climatic selection appears to be the major differentiating factor of NTS rDNA.     

Brain size diversity in adaptation and speciation of subterranean mole rats

We have tested brain size diversity and encephalization in the actively speciating subterranean mole rats of the Spalax ehrenbergi superspecies in Israel. Our sample involved 171 individuals comprising 12 populations and 4 chromosomal species (2n = 52, 54, 58 and 60) distributed parapatrically from the northern Mediterranean region southward (2n = 52, 54→+58→60) into increasingly more arid and unpredictable climatic regimes, approaching the Negev Desert. Our results indicate that relative brain size and encephalization are highest in 2n = 60 as compared with 2n = 52, 54 and 58. We hypothesize that this pattern is adaptive and molded by natural selection. Brain evolution and higher encephalization in the S. ehrenbergi complex appears to be associated with increasing ecological stresses of aridity and climatic unpredictability.     

Habitat selection in evolving mole rats

Summary The actively speciating four chromosomal species of fossorial mole rats of the Spalax ehrenbergi complex in Israel (2n=52, 58, 54 and 60) which inhabit an increasingly arid environment in this order were tested to determine their habitat preference. The testing apparatus simulated four climatic regimes based on temperature and humidity combinations corresponding to the climatic origins of the four chromosomal species: coolhumid, cool-dry, warm-humid and warm-dry, respectively. The tests involved 175 adults comprising all four chromosome species and representing 10 populations. Out of the 139 analyzed animals 88% selected the warm cages and only 12% selected the cool cages. The four karyotype progressively preferred the warm-dry cage in the following order: 53, 59, 60 and 72% for 2n=58, 52, 54 and 60 respectively, largely in accord with their increasingly arid climatic origins. Even larger differences were found in populations within karyotypes in accord with the local climatic variation within a karyotype range.The results of our analysis indicate that the chromosomal species and populations select their climatic habitat in accord with the climatic conditions of their geographic localities. The humidity index appears to be the prime differentiator of habitat selection and may have been a substantial ecological factor in species differentiation and distribution of the Spalax ehrenbergi complex in Israel.     

Adaptive physiological variation in nonshivering thermogenesis and its significance in speciation

Summary Nonshivering thermogenesis (NST) magnitude was studied in the four chromosomal species of subterranean mole rats of theSpalax ehrenbergi superspecies in Israel. The four species show a distribution pattern which correlates with increasing aridity. The 2n=52 species inhabits the cold and humid regions, 2n=54 cold and dry, 2n-58 warm and humid, and 2n=60 the more arid regions in which temperatures fluctuate daily and annually. NST was measured as the ratio between maximal oxygen due to noradrenaline injection and minimal measured in anaesthetized animals.The chromosomal species 2n=60 from semiarid and arid habitats has the highest NST value. This fact emerges from the low Basal Metabolic Rate (BMR) of this species relative to all the other three species. A linear correlation was found between NST magnitude and the average daily range of temperature during June and September.We conclude that speciation of theS. ehrenbergi complex in Israel has thermoregulatory correlates such as heat production by NST, among others. The level of NST appears to be an adaptive physiological characteristic in the ecological speciation of subterranean mole rats.     

Morphometrics of speciating mole rats: Adaptive differentiation in ecological speciation

The morphometrics of subterranean mole rats, Spalax ehrenbergi superspecies in Israel, were studied. A total of 42 skull and body variables of 327 adults from 44 populations across the ranges of the 4 chromosomal species (2n = 52, 54, 58 and 60) were measured. The results showed: a. in general, significantly higher morphological values in males than in females; b. significant interspecific quantitative diffferences in 15 skull variables of males, and in 3 of females; but no qualitative diagnosis of any of the species; c. an intercorrelation between most skull variables at different orgamsmal levels, and a uniform and complete correlation with weight; d. a statistical discrimination in both metric and nonmetric multivariate analyses between most species pairs; e. an indication of high morphological similarity based on the relatively small multivariate Mahalanobis distances of dissimilarity between species; f. a possible explanation for a significant part of the variance in most skull and body variables resulting from a combination of temperature variables and water availability; g. a declination in size in skull and body variables between northern and southern species; h. the order of speciation events to be 2n = (54, 52)→58→60. Based on these results we conclude: 1. no morphological breaks are associated with the speciation of the S. ehrenbergi complex, and morphological differentiation is quantitative rather than qualitative between the species: 2. morphological diversity is significantly explained by climatic selection, and is therefore adaptive and explicable on even very low selective pressures over evolutionary time: 3. the morphospecies seem unable to reliably reflect the biological species in the S. ehrenbergi complex, and morphological differentiation, at least in this superspecies, evolves in a gradual rather than a punctual way: this would conform with Neo-Darwinian evolutionary theory.     

Nidicolous arthropods associated with different chromosomal types of Spalax ehrenbergi Nehring*

The mole rat, Spalax ehrenbergi Nehring, is a complex group involving four chromosomal types of In = 52, 54, 58 and 60. These are clinally distributed from Mount Hermon in the north to the Negev in the south, covering humid, semihumid, semiarid and arid climates. Forty-seven breeding nests of Sehrenbergi were collected from 23 localities throughout the range and processed for nidicolous arthropods. A total of five species of fleas and 53 species of gamasid mites were collected. Their distribution in the investigated area has been analysed in relation to that of the chromosomal types of S.ehrenbergi. The following conclusions have been reached:(a) Only three of the species (two haematophagous gamasids and one fungivorous acarid) range across the whole investigated area, showing strong host specificity, (b) A correlation is apparent between the distribution of several nidicolous arthropods (fleas and gamasids) and the ‘54’ geographic region. This probably reflects geographical distribution rather than host specificity, (c) Palaearctic species reach their southern limit of distribution on Mount Hermon and the Golan Heights; possibly Ethiopian ones reach their northern limit in the Negev. (d) No direct correlation has been found between nidicolous arthropods and the chromosomal types of S. ehrenbergi. (e) Subjects for future investigation are outlined.     

Molecular genetics of Rhabdomys pumilio subspecies boundaries: mtDNA phylogeography and karyotypic analysis by fluorescence in situ hybridization

The phylogeography of the African four-striped mouse, Rhabdomys pumilio, was investigated using complete sequences of the mtDNA cytochrome b gene (1140 bp) and a combination of fluorescence in situ hybridization (FISH) and conventional cytogenetic banding techniques (G- and C-banding). Two cytotypes (2n=46 and 2n=48) were identified by cytogenetic analysis. There is no evidence of diploid number variation within populations, difference in gross chromosome morphology or of subtle interchromosomal rearrangements at levels detected by ZOO-FISH. Analysis of the mtDNA cytochrome b resulted in two major lineages that correspond roughly to the xeric and mesic biotic zones of southern Africa. One mtDNA clade comprises specimens with 2n=48 and the other representatives of two cytotypes (2n=48 and 2n=46). The mean sequence divergence (12%, range 8.3–15.6%) separating the two mtDNA clades is comparable to among-species variation within murid genera suggesting their recognition as distinct species, the prior names for which would be R. dilectus and R. pumilio. Low sequence divergences and the diploid number dichotomy within the mesic lineage support the recognition of two subspecies corresponding to R. d. dilectus (2n=46) and R. d. chakae (2n=48). Our data do not support subspecific delimitation within the nominate, R. pumilio. Molecular dating places cladogenesis of the two putative species at less than five million years, a period characterised by extensive climatic oscillations which are thought to have resulted in habitat fragmentation throughout much of the species range.     

Adaptive differentiation of body size in speciating mole rats

Summary We report the body weight of 1,653 subterranean mole rats comprising 12 populations and 4 chromosomal species (2n=52, 54, 58 and 60) of the Spalax ehrenbergi superspecies in Israel. The sample was collected from 1970 to 1985 and includes all captured animals with a minimal age of 10 months. The results indicated the following. (a) Body weight of males was significantly higher than that of females. (b) There is a southward latitudinal gradient in body size. Northern animals living in cooler and more productive mesic environments are larger than southern animals living in warmer and less productive xeric environments. (c) The interspecific differences for each sex are statistically significant. (d) Body size is negatively correlated with temperature variables, and positively correlated with plant cover (reflecting productivity or food resources) and rainy days. (e) The best predictors of body size, explaining up to 87% of the variation in size included various combinations of temperature variables and plant cover.We conclude that in both adaptation and speciation natural selection is a major agent of differentiation of body size in accordance with multiple factors, primarily temperature and food resources operating on the energetics balance.     

Origin and evolution of assortative mating in actively speciating mole rats

The origin and evolution of positive assortative mating in the actively speciating subterranean mole rats of the Spalax ehrenbergi superspecies in Israel, may be deciphered by comparing female mate preference in the laboratory between ancestral and derivative species. Estrous females of the recent derivative of speciation (chromosomal species 2n = 60) showed trimodal mate preference distribution significantly differing from a normal curve. Females consisted of three phenotypes, comprising negative, low positive, and high positive preference for homospecific males. By contrast, mate preference in encounters of ancestral species (2n = 52, 54, and 58) showed a prevalence of a positive homospecific mate preference. It is suggested that the three modal distribution is explicable even on the basis of one major gene with three genotypes. The evolution of ethological reproductive isolation proceeded presumably from a high polymorphism in the most recent derivative of speciation towards increasing monomorphism of positive assortative mating among ancestral species. If an assortative mating locus combines with sexual selection of the frequent male adapted optimally to the local environment, then speciation and adaptation will be tightly linked in the evolution of mole rats.     

Total body water and adaptive water turnover rate in four chromosomal species of subterranean mole rats of the Spalax ehrenbergi superspecies in Israel

Total body water (TBW) and water turnover rate (WTR) were measured in 24 subterranean mole rats comprising four populations, each belonging to a different chromosomal species of the Spalax ehrenbergi superspecies in Israel. The four species range in different climates: humid-cool (2n = 52); semiarid-cool (2n = 54); humid-warm (2n = 58) and arid-warm (2n = 60). TBW, as a percentage of body weight, measured by tritiated water (HTO), was 72.4%±4–7 in 2n = 52, significantly ( P < 005) higher than the similar estimates 61.7%± 7.2, and 59.4%± 5–3, for 2n = 60 and 58, respectively. A comparison of HTO space, as a percentage of TBW, closely approximated TBW, ranging from 97% to 108%. WTR was high, 218.1 and 230.9 ml/kg^0-75/day in the mesic populations of 2n = 58 and 52, respectively. By contrast, WTR estimates were significantly lower , ( P < 0.001), 150.2 and 148.9 ml/kg^0-75/day in the xeric populations of 2n = 54 and 60, respectively. The biological half-life time, T_1/2, was similar and faster, 32.7 and 27.9 hours in the mesic populations of 2n = 52 and 58, as compared with slower, 47.9 and 40.8 hours in the xeric populations of 2n = 54 and 60, respectively. Urine osmolarity in the most xeric northern Negev steppic population of 2n = 60 (737 ± 45 mmol/kg) was significantly ( P < 0.001) higher than in the other species.
We conclude that adaptive radiation in the Spalax ehrenbergi superspecies involves speciation in semiarid (2n = 54) and arid (2n = 60) climates by physiological adaptations of kidney water conservation, along with multiple morphological, physiological and behavioural syndromes of climatic adaptations to increasing aridity (Nevo, 1986).     

Centric fusion polymorphism in captive animals of family Bovidae

Chromosomes of 228 captive specimens of the family Bovidae have been investigated. The examined animals were classified into the subfamilies Aepycerotinae, Reduncinae, Antilopinae, Alcelaphinae, Hippotraginae and Bovinae. Polymorphism for one fusion was identified in the species: Aepyceros melampus, 2n = 59–60; Redunca fulvorufula, 2n = 56–57; Kobus e. ellipsiprymnus, 2n = 50–52; Kobus e. defassa, 2n = 52–54 and Syncerus c. nanus, 2n = 54–55. This is the first study to reveal fusion 7;29 in Kobus e. defassa and simultaneously the respective polymorphism. Variation in the diploid number of chromosomes is also known in species: Oryx g. dammah and Oryx g. leucoryx but in this study only fusion 1;25 was identified in both karyotyped species. Our study showed that 13% of investigated individuals were polymorphic for the centric fusion and demonstrated the important role of cytogenetic screening in captive animals at zoological gardens.     

Foraging strategy in a subterranean rodent,Spalax ehrenbergi: a test case for optimal foraging theory

Summary The foraging behavior of the subterranean mole rat Spalax ehrenbergi (Rodentia, Spalacidae) was tested according to the framework of optimal foraging theory. We compared the frequencies of food species hoarded in storage chambers of mole rats with the frequencies of these species occurring in the vicinity of the mole rats' nest mounds during the winter and spring seasons. In addition, we examined the food composition of several summer nest mounds. Laboratory observations were conducted in order to test the foraging behavior of mole rats under simulated subterranean conditions. The mole rat is a generalist and collects a variety of food species. Out of 33 plant species that were hoarded by mole rats in the 21 studied nest mounds, 61% (n=20) were geophytes, 21% (n=7) perennial herbs, 15% (n=5) annual herbs and 3% (n=1) dwarf shrubs. The frequency of each collected species in the 16 winter and spring nest mounds is in general accordance with its frequency in the mole rat's territory. This implies that the mole rat randomly samples the food reserve of its territory without special preference or directed search for a particular species. The collection or avoidance of any food item is not dependent on the presence or absence of any other food item. We suggest that the foragin generalism of the mole rat is a product of the constraints of a subterranean niche — the necessity to hoard food as much as possible in a limited time period and the high energetic investment of tunneling to the food items.     

Chromosome Banding and 18S rDNA in situ Hybridization Analysis of Seven Species of the Family Achiridae (Teleostei: Pleuronectiformes)

Achiridae is an important family of the order Pleuronectiformes widely distributed in North, Central, and South America with freshwater and marine species. In the present study cytogenetic analyses comprising conventional and molecular techniques were carried out in seven species of this family. The following diploid numbers (2n) and fundamental numbers (FN) were obtained: Achirus declivis 2n = 34, FN = 52; Achirus lineatus 2n = 40, FN = 66; Catathyridium jenynsi 2n = 40 and FN = 50; Gymnachirus nudus 2n = 36 and FN = 50; Hypoclinemus mentalis 2n = 38 and FN = 54; Trinectes paulistanus 2n = 42 and FN = 52; and Trinectes sp. 2n = 38 and FN = 54. All species presented a single nucleolar organizer region (NOR) bearing chromosome pair and C-band positive segments mainly distributed at the pericentromeric position. The wide variation observed in chromosome number and FN suggests the occurrence of larger chromosome rearrangements in the family Achiridae if compared with other families of the same order.     

Heterochromatin study and geographical distribution of Microtus species (Rodentia,Arvicolinae) from Greece

In a sample of 108 underground voles from 23 Greek localities, the species Microtus felteni, M. guentheri, M. rossiaemeridionalis and M. subterraneus were identified, based on external body morphology and karyotype. Moreover, the implemented C-banding staining technique revealed the heterochromatin distribution in the chromosomes of the above species. All M. guentheri (2n=54, FN=54) and M. rossiaemeridionalis (2n=54, FN=56) specimens displayed the typical karyotypes of these species, respectively. The M. subterraneus specimens belonged to the chromosomal race with 2n=52, FN=60 of this species, apart from a single individual that demonstrated a medium-sized, subtelocentric autosome in heterozygous condition (2n=52, FN=61). Furthermore, M. felteni individuals, trapped again after many years, were karyologically studied (2n=54, FN=56) and the C-banding pattern for this species is hereby presented for the first time. Finally, the study of meiotic preparations in M. guentheri and M. rossiaemeridionalis males verified the asynaptic behaviour of their sex chromosomes. The karyotype of the four studied Microtus species does not seem to have diversified much from the putative ancestral arvicoline karyotype (2n=56, FN=56). On the other hand, the heterochromatin accumulation in the sex chromosomes, particularly prominent in M. felteni and M. rossiaemeridionalis, represents this common tendency in several Microtus species.     

Aotus vociferans ×Aotus nancymai: Sympatry without chromosomal hybridation

In a study relating geographic distribution ofAotus populations with karyotype and coat pattern,Hershkovitz (1983) suggested that this genus comprises nine species.A. vociferans (2n=46) is distributed along the left bank of the Maranon river, andA. nancymai (2n=54) along the right bank of the same river. In the present study we analyzed the karyotypes of 11A. vociferans and 11A. nancymai specimens using G- and C-banding and NOR-staining. Of these animals, fiveA. vociferans were sympatric with fiveA. nancymai in Isla Aramaza, Colombia; oneA. vociferans was sympatric with threeA. nancymai in Yahuma, Peru; oneA. vociferans was sympatric with threeA. nancymai in Terezinha I, Brazil. No signs of hybridation between the taxa was observed at any time, with allA. vociferans having 2n=46 and allA. nancymai 2n=54, as previously reported in the literature. These data suggest thatA. vociferans andA. nancymai should indeed be distinct species.     

Allozyme variations in Anatolian populations and cytotypes of the blind mole rats (Nannospalax)

Enzymatic proteins encoded by 28 putative loci in 326 samples of 12 mol rat cytotypes collected from 97 localities in Anatolia were investigated by standard horizontal starch-gel electrophoresis. A total of 61 alleles were determined for 28 isozyme loci and 23 of the 28 were polymorphic. Eight of the 23 polymorphic loci were agreeable to the Hardy–Weinberg equilibrium. It was determined that deviations from the Hardy–Weinberg equilibrium in the examined populations were due to a heterozygote deficiency. It was revealed by allozyme analyses that mole rat populations in Anatolia have formed 4 cytotypes groups, represented by 4 species (Nannospalax xanthodon, Nannospalax ehrenbergi, N. cilicicus, and N. nehringi). Cytotypes in western Anatolia (2n = 36, 2n = 38, 2n = 40, 2n = 52) showed private alleles in different enzyme systems; therefore, these cytotypes were genetically different, both from each other and other cytotypes. Although cytotypes in central Anatolia (2n = 52S, 2n = 56, 2n = 58, and 2n = 60) contained a different diploid chromosome number, they showed identical patterns in terms of their allele content in the 28 enzymatic loci.     

New Karyotypes and Some Considerations about the Chromosomal Diversification of Ctenomys minutus (Rodentia: Ctenomyidae) on the Coastal Plain of the Brazilian State of Rio Grande do Sul

The dominant mammals occupying the subterranean niche in South America are rodents of the genus Ctenomys, which form a large group of 56 species with chromosome numbers ranging from 2n= 10 to 70. In southern Brazil, Ctenomys minutus is the species with the widest geographic distribution, inhabiting sandy fields and dunes extending from Jaguaruna beach in the state of Santa Catarina to the town of São José do Norte in the state of Rio Grande do Sul. Eleven karyotypes (2n= 42; 2n= 46a; 2n= 46b; 2n= 47a; 2n= 47b; 2n= 48a; 2n= 48b; 2n= 49a; 2n= 49b; 2n= 50a and 2n= 50b) were described for this species and zones of hybridization are also known. A sample of 51 C. minutus specimens was collected from five sampling sites about 20 km apart along the coastal plain of Rio Grande do Sul between the municipalities of Tavares (31°23′S 51°09′W) and São José do Norte (31°52′S 51°54′W). We were able to extend the known geographic distribution of C. minutus by 90 km, from Tavares southwards to São José do Norte. During our study we found five karyotypes (2n= 46b, 47b, 48b, 49b and 50b), four of which (2n= 47b, 48b, 49b and 50b) have not previously been described for this species.     

Comparative gene mapping in cattle,Indian muntjac,and Chinese muntjac by fluorescence in situ hybridization

The Indian muntjac (Muntiacus muntjak vaginalis) has a karyotype of 2n = 6 in the female and 2n = 7 in the male. The karyotypic evolution of Indian muntjac via extensive tandem fusions and several centric fusions are well documented by molecular cytogenetic studies mainly utilizing chromosome paints. To achieve higher resolution mapping, a set of 42 different genomic clones coding for 37 genes and the nucleolar organizer region were used to examine homologies between the cattle (2n = 60), human (2n = 46), Indian muntjac (2n = 6/7) and Chinese muntjac (2n = 46) karyotypes. These genomic clones were mapped by fluorescence in situ hybridization (FISH). Localization of genes on all three pairs of M. m. vaginalis chromosomes and on the acrocentric chromosomes of M. reevesi allowed not only the analysis of the evolution of syntenic regions within the muntjac genus but also allowed a broader comparison of synteny with more distantly related species, such as cattle and human, to shed more light onto the evolving genome organization. For convenience and to avoid confusion we added for each species a three letter abbreviation prior to the chromosomal band location discussed in this paper: BTA, Cattle chromosome; HSA, Human chromosome; MMV, M. m. vaginalis chromosome; MRE, M. reevesi chromosome.