Penial differentiation in speciation of subterranean mole rats Spalax ehrenbergi in Israel

Geographic and ontogenetic variation was examined in the glans penes and bacula of 92 specimens representing four biological species with the karyotypes 2n=52, 54, 58 and 60 of the Spalax ehrenbergi superspecies in 13 populations from Israel and one disjunct population from northern Egypt. Five measured and three calculated penial characters were analysed and employed in multivariate statistics. Comparison was made between penial morphological characters and craniometric biochemical and chromosomal data. The baculum is strongly species-specific and its length bears no significant relationships with head and body length. The results of these analyses suggest that different bacular morphologies may provide a basis for species-specific prezygotic isolating mechanisms, thus, together with other factors, contributing to reproductive isolation and speciation.     

Speciation and adaptive radiation of subterranean mole rats, Spalax ehrenbergi superspecies, in Jordan

The major initial mechanism of speciation in subterranean blind mole rats, Spalacidae, is chromosomal, primarily through Robertsonian rearrangements. Here we highlight another scenario of chromosomal rearrangement leading to ecological speciation and adaptive radiation apparently initiated by pericentric inversions and genie divergence to different ecologies in mole rats in Jordan. We analysed karyotype, allozyme, size and ecological diversity across the range of mole rats in Jordan from mesic Irbid in the north to xeric Wadi Musa (Petra region) in the south, a transect of 250 km. We examined mole rats for chromosome ( N =71), size ( N =76), and allozyme ( N =67) diversities, encoded by 32 loci, in 12 populations of the Spalax ehrenbetgi superspecies in Jordan. By a combination of chromosome morphology, genetic distance, body size and ecogeography, we identified four new putative biological species. All species (except two animals in Madaba) share 2 n = 60 but vary in chromosome morphology, caused by pericentric inversions and/or centromeric shifts. The 'north Moav' species is karyotypically polymorphic for 2 n (2 n = 60; including locally also two animals with 2 n = 62). The distribution of the four species is associated with ecogeographical different domains and climatic diversity. Genetic diversity indices were low, but like chromosome arms (NFa) were positively correlated with aridity stress. Discriminant analysis correctly classified 91% of the individuals into the four species utilizing combinatorially chromosome, allozyme and size diversities. It is hypothesized that mole rat evolution underground is intimately associated with climatic diversity stress above ground.     

DNA sequence variation in the mitochondrial control region of subterranean mole rats,Spalax ehrenbergi superspecies,in Israel

The complete mitochondrial control region was sequenced for 60 individuals representing different populations for each of the four species of the subterranean mole rat Spalax ehrenbergi superspecies in Israel: Spalax galili (2n = 52), S. golani (2n = 54), S. carmeli (2n = 58), and S. judaei (2n = 60). The control region of all species and populations is very similar both in length (979 to 983 bp) and in base composition. As in agreement with previous surveys on mitochondrial control regions on mammals, the mole rat control region can be divided into a central domain and two flanking domains, ETAS (extended termination associated sequences) and CSB (conserved sequence blocks). Along with the common conserved blocks found in these domains (ETAS1, ETAS2, CSB1, CSB2, and CSB3), we have also detected in all individuals an ETAS1-like and a CSB1-like element, both in the ETAS domain. The most conserved region was the central domain, followed by the CSB and ETAS domains, showing important differences in the four species analyzed. Phylogenetic analysis supported the existence of two clades. One clade contained individuals belonging to Spalax galili (2n = 52) and S. golani (2n = 54), separated in two different branches depending on the species. The other clade contained individuals belonging to S. carmeli (2n = 58) and S. judaei (2n = 60) mixed together, suggesting a more recent event of speciation. Within species we have observed a southward trend of increasing variability. These results have been explained as a consequence of the adaptation of the species to ecological factors such as aridity and temperature stresses.     

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.     

Genetic polymorphism of urine deoxyribonuclease I isomerases of subterranean mole rats,Spalax ehrenbergi superspecies,in Israel: Ecogeographical patterns and correlates

Genetic polymorphism of urine deoxyribonuclease I (DNase I) of mole rats was analyzed by isoelectric focusing in a thin-layer polyacrylamide gel (IEF-PAGE). One hundred and three subterranean mole rats, comprising 13 populations belonging to the four chromosomal species (2n = 52, 54, 58, 60) of the actively speciating Spalax ehrenbergi superspecies in Israel, were tested. The following results were indicated. (i) Spalax DNase I consisted of 6-12 major isozymes. (ii) Four phenotypes (numbers in parentheses) were 1 (92), 1-2 (5), 1-3 (4), and 2 (1). The decreasing order of genetic diversity, He, in the four species was 0.37, 0.13, 0.10, and 0.0 for 2n = 58, 52, 54, and 60, respectively. (iii) Spearman rank correlations and multiple regression analyses indicated associations of allele frequencies and genetic diversity with climatic and vegetation factors. We concluded that (a) climatic selection, either directly or indirectly through plant (i.e., food resources) diversity, plays an important role in DNase genetic differentiation and (b) no gene flow and introgression occur between the recent derivative of speciation (2n = 60) and its ancestor (2n = 58), suggesting the operation of reproductive isolation between both species despite natural hybridization.     

Circadian genes in a blind subterranean mammal III: molecular cloning and circadian regulation of cryptochrome genes in the blind subterranean mole rat, Spalax ehrenbergi superspecies

The blind subterranean mole rat superspecies Spalax ehrenbergi is an extreme example of mammalian adaptation to life underground. Though this rodent is totally visually blind, harboring a drastically degenerated subcutaneous rudimentary eye, its daily activity rhythm is entrainable to LD cycles. This indicates that it confers light information to the clock, as has been previously shown by the authors in behavioral studies as well as by molecular analyses of its Clock/MOP3 and its three Per genes. The Cryptochrome (Cry) genes found in animals and plants act both as photoreceptors and as essential components of the negative feedback mechanism of the biological clock. To further understand the circadian system of this unique mammal, the authors cloned and characterized the open reading frame of Spalax Cry1 and Cry2. The Spalax CRY1 protein is significantly closer to the human homolog than to the mice one, in contrast to the evolutionary expectations. They have found two isoforms of Cry2 in Spalax, which differ in their 5' end of the open reading frame and defined their expression in Spalax populations. They found a large and significant excess of heterozygotes of sCry2 (sCry2L/S genotype). Both sCry1 and sCry2 mRNAs were found in the SCN, the eye, the harderian gland, as well as in a wide range of peripheral tissues. Their expression pattern under different LD conditions has also been analyzed. As was already shown for other circadian genes, despite being blind and living in darkness, the Cry genes of Spalax behave in a similar, though not identical, pattern as in sighted animals. Once again, the results indicate that the uniquely hypertrophied harderian gland of Spalax plays a key role in its circadian system.     

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).     

MtDNA polymorphisms: evolutionary significance in adaptation and speciation of subterranean mole rats

Patterns of mtDNA diversity in subterranean mole rats of the Spalax ehrenbergi superspecies (2n = 52, 54, 58 and 60) were previously studied in the main ranges of the four chromosomal species, and specifically in the 2n = 60 species and its peripheral steppe semi-isolates and desert isolates. In the present study we correlated mtDNA diversity indices, nucleon diversity, h , and nucleotide divergence, π, with physical (climatic), biotic (parasites) and biological (genetical, morphological, physiological and behavioural) factors, showing that mtDNA diversity is structured ecogeographically and biologically. The following significant correlations of mtDNA diversity were indicated with: (i) climatic heterogeneity and unpredictability; (ii) levels of ecto- and endoparasites; and (iii) biological diversities, primarily with physiological diversity associated with the energy budget. Small steppe semi-isolates and desert isolates harbour high levels of mtDNA haplotype diversity, some novel, which may be a prerequisite for future speciation events. We conclude that the ecogeographical and biological correlates, as well as the maintenance of mtDNA polymorphisms in small isolated populations, strongly suggest that mtDNA diversity is not neutral. Diversifying natural selection appears to be an important differentiating factor of mtDNA diversity in the twin evolutionary processes of adaptive radiation and active speciation. We suggest critical experiments to substantiate our conclusions and highlight the contribution of mtDNA diversity to fitness, i.e. to the biological function of mtDNA diversity in the evolutionary process.     

Seismic communication in the blind subterranean mole-rat: patterns of head thumping and of their detection in the Spalax ehrenbergi superspecies in Israel

Patterns of seismic communication and somatosensory responses were studied in four chromosomal species (2n= 52, 54, 58, 60) of blind subterranean mole-rats, belonging to the Spalax ehrenbergi superspecies in Israel. The patterns involved head thumping signals and surface-recorded cerebral potentials evoked by vibrational signals. The patterns correspond to the differences in territory size and population density which characterize the four species. It is suggested that this correspondence is related to the adaptive significance of the seismic communication.     

Activity of free‐living subterranean blind mole rats Spalax galili (Rodentia: Spalacidae) in an area of supposed sympatric speciation

Subterranean rodents forage underground, which is energetically costly. Therefore, they can be expected to economize burrowing activity in response to food supply and soil characteristics. We analyzed the activity of radio‐tracked blind mole rats, Spalax galili, on a locality sharply subdivided into harder but relatively food‐rich, basaltic soil and softer, relatively food‐poor rendzina. It was recently proposed that the mole rats in this locality are undergoing sympatric ecological speciation. We predicted that mole rats from basaltic soil would be less active than those from rendzina as a result of the reduced need for burrowing to reach food. By contrast to our predictions, mole rats from basaltic soil were more frequently located outside the nest and observed pushing soil above ground. We suggest that this is a result of territorial behaviour due to high population density. All mole rats exhibited a unimodal daily activity pattern likely related to temperature. Large males had large but gradually decreasing home‐ranges, likely indicating the end of the mating season. We conclude that the ecological differences between the habitats cause behavioural differences in the mole rats, which indicates different selection pressures. The genetic divergence previously found between the populations might have arisen via density‐dependent selection.     

Mitochondrial DNA polymorphisms in subterranean mole-rats of the Spalax ehrenbergi superspecies in Israel, and its peripheral isolates

Patterns of mitochondrial DNA (mtDNA) variation were examined in 133 mole-rats constituting all four chromosomal species (2n = 52, 2n = 54, 2n = 58, and 2n = 60) of the Spalax ehrenbergi superspecies in Israel, as well as the peripheral isolates of 2n = 60. In the main range of the complex, a total of 28 mtDNA haplotypes were found in 64 mole-rats, with most haplotypes being unique to either a single chromosomal species or population. mtDNA divergence increased from low to high diploid number in a north-to-south direction in Israel. Overall levels of mtDNA diversity were unexpectedly the highest in the 2n = 60, the youngest species of the complex. The mtDNA haplotypes can be separated into two major groups, 2n = 52-54 and 2n = 58-60, and a phylogenetic analysis for each group revealed evidence of a few haplotypes not sorted by diploid number. The overall patterns of mtDNA divergence seen within and among the four chromosomal species are consistent with the parapatric mode of speciation as suggested from previous studies of allozyme and DNA hybridization. In a separate data set the patterns of mtDNA variation were examined across the main geographic range and across peripheral semi-isolates and isolates of the 2n = 60 chromosomal species. Fifteen haplotypes were found in 69 mole-rats. High levels of mtDNA diversity characterized the main range, semi-isolated, and even some desert isolated populations. The peripheral isolates contain much mtDNA diversity, including novel haplotypes.      

Odour–genes covariance within a natural population of subterranean Spalax galili blind mole rats

Previous research, using habituation techniques with multiple rodent species as subjects, has demonstrated (from kin to across species) the greater perceptual similarity in the qualities of individual odours of more closely genetically related individuals ('odour–genes covariance', abbreviated 'OGC'). This predictable relationship between individual genotypes and individual odours has only been assessed in rodents in which the genetic similarities and dissimilarities are known either through laboratory breeding or by selecting odour donors from different populations or species. To study OGC within a natural population of rodents, we genotyped Spalax galili blind mole rats using ten microsatellite markers, and conducted pairwise genetic distance comparisons to estimate genetic similarity and to calculate the relatedness coefficient between pairs of individuals. We then used habituation-generalization techniques to assess whether perceived odour similarities covaried with the genetic similarities we had identified. Indeed, animals treated odours from genetically closer donors as more similar than odours from less genetically similar donors. The results suggest an accurate and subtle ability to resolve genetically determined odour distinctions among familially unrelated animals within a population. Graded differential responses to conspecific odours based on degrees of similarity between other individuals' odours and one's own have been demonstrated in kinship and species discriminations and preferences. The evidence presented here provides the basis for hypothesizing a similar process that could promote optimal outbreeding and inclusive fitness within populations of conspecifics. The evolutionary implications of these findings are discussed.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 96 , 483–490.     

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.     

Microsatellite diversity associated with ecological factors in Hordeum spontaneum populations in Israel

Microsatellite diversity at 18 loci was analysed in 94 individual plants of 10 wild barley, Hordeum spontaneum (C. Koch) Thell., populations sampled from Israel across a southward transect of increasing aridity. Allelic distribution in populations was not distributed randomly. Estimates of mean gene diversity were highest in stressful arid-hot environments. Sixty-four per cent of the genetic variation was partitioned within populations and 36% between populations. Associations between ecogeographical variables and gene diversity, H(e), were established in nine microsatellite loci. By employing principle component analysis we reduced the number of ecogeographical variables to three principal components including water factors, temperature and geography. At three loci, stepwise multiple regression analysis explained significantly the gene diversity by a single principal component (water factors). Based on these observations it is suggested that simple sequence repeats are not necessarily biologically neutral.     

Stress,adaptation, and speciation in the evolution of the blind mole rat,Spalax, in Israel

Environmental stress played a major role in the evolution of the blind mole rat superspecies Spalax ehrenbergi, affecting its adaptive evolution and ecological speciation underground. Spalax is safeguarded all of its life underground from aboveground climatic fluctuations and predators. However, it encounters multiple stresses in its underground burrows including darkness, energetics, hypoxia, hypercapnia, food scarcity, and pathogenicity. Consequently, it evolved adaptive genomic, proteomic, and phenomic complexes to cope with those stresses. Here I describe some of these adaptive complexes, and their theoretical and applied perspectives. Spalax mosaic molecular and organismal evolution involves reductions or regressions coupled with expansions or progressions caused by evolutionary tinkering and natural genetic engineering. Speciation of Spalax in Israel occurred in the Pleistocene, during the last 2.00–2.35 Mya, generating four species associated intimately with four climatic regimes with increasing aridity stress southwards and eastwards representing an ecological speciational adaptive trend: (Spalax golani, 2n = 54 → S. galili, 2n = 52 → S. carmeli, 2n = 58 → S. judaei, 2n = 60). Darwinian ecological speciation occurred gradually with relatively little genetic change by Robertsonian chromosomal and genic mutations. Spalax genome sequencing has just been completed. It involves multiple adaptive complexes to life underground and is an evolutionary model to a few hundred underground mammals. It involves great promise in the future for medicine, space flight, and deep-sea diving.     

Aggression patterns in adaptation and speciation of subterranean mole rats

The patterns and correlates of aggressionwithin andbetween actively speciating subterranean mole rats of theSpalax ehrenbergi superspecies in Israel were tested in an attempt to approach an evolutionary theory of aggression related to both adaptation and speciation. Laboratory experiments were conducted on 314 adult mole rats (188 males and 126 females) caught in nature, representing 12 populations and comprising four chromosomal species (2n = 52, 54, 58, 60). The present analysis concentrates on “total aggression” comprising four aggressive variables: attacks, bitings, head-ons and sniffing with open mouth, out of 23 recorded variables. The results indicate that (a) aggression is distributed as a multipeak, discontinuous phenotypic parameter, displaying polymorphism within all 12 populations and 4 species tested; (b) the level of aggression was higher in males than in females, in the breeding than in the nonbreeding season, and displayed clinal geographic variation in both sexes across the superspecies range, where “militancy” increased northwards; (c) aggression was significantly correlated with ecological, physiological, genetical and ethological factors. These results support an evolutionary theory of aggression related to the intertwined processes of adaptation and speciation.     

The structure of mast cell secretory granules in the blind mole rat (Spalax ehrenbergi)

Eosinophils, basophils, and mast cells produce and secrete active substances whose role is to attack invading parasites and protect the host. In this study we use morphometric methods to study mast cells in the blind mole rat (Spalax ehrenbergi). The subterranean and solitary way of life of this species has led to the evolutionary development of special anatomical, morphological, behavioral, and physiological adaptations. Because of its particular lifestyle, the mole rat is less exposed to parasites than other rodents. This could provide a unique model for research into the pathobiology of mast cells. The paracrystalline structure of the mast cell granule content is composed of parallel plates. Diffraction analysis of electron micrographs of thin sections of araldite-embedded tissues indicated that each crystal line plate is a periodic array of parallelograms. The crystal unit cell volume is approximately 930 nm(3), suggesting that each unit cell is composed of one heparin molecule and one to three additional adsorbed proteins. Morphometric data show that characteristics of the secretory granules of mast cells of the blind mole rat resemble those of other rodents. The mast cell unit granule volume in the present study was calculated to be 0.055 microm(3), similar to that of rat peritoneal mast cells.     

Evolution of crystallins: expression of lens-specific proteins in the blind mammals mole (Talpa europaea) and mole rat (Spalax ehrenbergi)

The mole (Talpa europaea; Insectivora) and the mole rat (Spalax ehrenbergi; Rodentia) both have degenerated eyes as a convergent adaptation to subterranean life. The rudimentary eye lenses of these blind mammals no longer function in a visual process. The crystallin genes, which display a lens-specific expression pattern, were studied in these blind mammals and in related species with normal eyes by hybridizing their genomic DNAs with probes obtained from cDNA clones for alpha A-, alpha B-, and beta Bp-crystallins from calf and gamma 3- crystallin from the rat. For all crystallin genes examined, the hybridization signals of mole and mole rat genomic DNA were comparable, respectively, with those of shrew and of rat and mouse, normal-vision representatives of the orders Insectivora and Rodentia. The expression of the crystallins at the protein level was tested by using antiserum specific for alpha-crystallin in immunofluorescence reactions on lens sections of mole and mole rat eyes and by using antisera against the beta- and gamma-crystallins on sections of the mole eye. All antisera gave positive fluorescence reactions exclusively with lens tissue of these blind mammals, indicating that the crystallins are still normally expressed despite the fact that these lenses have had no function in a visual process in these mammals for at least many million years. These findings apparently imply that some unknown selective advantage has conserved the crystallin genes and their expression after the loss of normal function of the lenses.      

Karyotype and genetic evolution in speciation of subterranean mole rats of the genus Spalax in Turkey

Karyotype (2fl) and allozymc diversity at 37 gene loci were determined in 69 subterranean mole rats in Turkey belonging to the two superspecies: the ancestor Spalax leucodon ( n = 55; 20 populations) and the descendant S. ehrenbergi ( n = 14; lour populations). We identified remarkable variation of diploid chromosome numbers in the S. leucodon superspecies: 2 n = 38. 40. 50, 54, 60 and 62; and in the S. ehrenbergi superspecies: 2 n = 52, 56 and 58. Genetic diversity indices were low on average in both S. leucodon and S. ehrenbergi superspecies: Allele diversity, A = 1.081 and 1.074; polymorphism, P-5%= 0.077 and 0.068; heterozygosity, H = 0.038 and 0.027; and gene diversity, H, = 0.038 and 0.034, respectively. H ranged from 0 in mesic or semimesic regions to 0.088 in arid Anatolia. We consider the populations with different diploid chromosome numbers, 2 n , as good biological species. Karyotypic diversity may mark extensive ecological speciation. Nei's genetic distances, D (average 0.174, range 0.002 0.422) and ecogeographical criteria suggest that almost each population may represent a different biological species, but critical future testing is necessary to support this claim. Karyotypes and allozymes are nonrandomly distributed across Turkey, displaying remarkable correlations with climatic and biolir factors. Both In and H are significantly correlated with aridity stress (2 n /rainfall, r , =–0.74; P < 0.001), and in our region also with climatic unpredictability. These results support the niche-width genetic variation hypothesis in space and time. Climatic selection in Turkey appears to be a major architect of karyotype and genetic (allozymc) diversity and divergence in mole rat evolution, in both speciation and adaptation.