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.     

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.     

Microsatellite diversity in populations of blind subterranean mole rats (Spalax ehrenbergi superspecies) in Israel: speciation and adaptation

The genetic diversity, structure, and relationships of 12 populations of blind subterranean mole rats belonging to four species of the Spalax ehrenbergi superspecies were examined. Diversity levels and divergence of 19 nuclear microsatellite markers were analysed. Results indicate that the 12 populations fall into two major clusters representing the species pairs Spalax galili (2 n  = 52) and S. golani (2 n  = 54) vs. S. carmeli (2 n  = 58) and S. judaei (2 n  = 60). There is very little gene flow between the species pairs, except for one population (Afiq) of S. carmeli (2 n  = 58), which is located near the hybrid zone between S. golani (2 n  = 54) and S. carmeli (2 n  = 58). Higher values of the mean for alleles per locus, gene diversity, and number of unique alleles were observed for the southern species pair, S. carmeli and S. judaei, than for the northern species pair, S. galili and S. golani . Microsatellite diversity increases southward toward the Negev Desert and is positively correlated with aridity stress. Climatic factors of water availability coupled with temperature account for 76.7% of microsatellite variance. Natural selection appears to adaptively determine microsatellite evolution in Spalax regionally across Israel.  © 2004 The Linnean Society of London , Biological Journal of the Linnean Society , 2004, 83 , 229–241.     

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).     

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.      

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.     

Genetic variation in a subterranean mammal,Spalax ehrenbergi

The mole rat, Spalax ehrenbergi, occurs in Israel and vicinity as four clinally distributed chromosome forms which are probably sibling species. By the method of starch gel electrophoresis of enzymes, genetic variation of 13 proteins controlled by 17 loci was studied in a total of 383 animals from the four chromosome forms. There was little genetic difference among the four groups, suggesting that speciation can occur without changes in many of the genes. Average degree of polymorphism per chromosome form was 19.1%, ranging from 5.9 to 29.4% for different chromosome forms. Individuals were, on the average, heterozygous at 3.7% of their loci, ranging from 1.8 to 5.6% in the four different chromosome forms. These relatively low values of genetic variation may possibly be associated in part with the ecologically relatively monotonous subterranean niche of Spalax.This work was supported in part by research grant GM-15597 from the National Institutes of Health, Bethesda, Maryland.     

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.     

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.     

Genetic polymorphism of human urine deoxyribonuclease I

Summary A genetic polymorphism of human urine deoxyribonuclease I (DNase I) has been detected by the technique of polyacrylamide gel isoelectric focusing (IEF-PAGE) followed by immunoblotting with anti-DNase I antibody. Family studies showed that the three common phenotypes —DNASE1 1, 1–2, and 2 — and the other four rare phenotypes — DNASE1 1–3, 2–3, 2–4, and 3–4 — represent homozygosity or heterozygosity for four autosomal codominant alleles, DNASE1 ^* 1, ^* 2, ^* 3, and ^* 4. The frequencies of the DNASE1 ^* 1, DNASE1 ^* 2, DNASE1 ^* 3, and DNASE1 ^* 4 alleles in a studied Japanese population were 0.5453, 0.4396, 0.0117, and 0.0034, respectively.     

Aggression patterns in three chromosome forms of the mole rat, Spalax ehrenbergi.

Three chromosome forms of the mole rat, Spalax ehrenbergi, 2n = 52, 2n = 58 and 2n = 60 were studied in an especially designed apparatus which simulated a natural territorial situation by permitting the animal free movement into and out of its home cage. Observed were variables representing agonistic, conflict and territorial behaviour. The data were analysed by means of a Smallest Space Analysis, SSA-II, which gave a graphic portrayal of the interrelations of behaviour categories where differences between means of single traits had been of no significance. 2n = 52 animals were found to be 'losers' in encounters. 'Lose', 'win' and 'draw' could be characterized by specific behaviour patterns: each outcome of encounter was surrounded by a cluster of behaviour categories consisting of agonistic, conflict and territorial activities that characterized its region of the SSA space.     

Burrow patterns of the mole rat Spalax ehrenbergi in two soil types (terra-rossa and rendzina) in Mount Carmel, Israel

Complete burrow systems of the mole rat Spalax ehrenbergi (Rodentia; Spalacidae), from two sites on Mount Carmel in Israel, with different soil types (terra-rossa and rendzina), were excavated and described here for the first time.
A comparison was made of burrow structure patterns in the two soils and of the sexes, with special attention to the features of the feeding tunnels.
The pattern in the rendzina revealed longer burrows with a longer main tunnel and fewer branches per metre of the main tunnel, while the pattern in the terra-rossa revealed shorter burrows with a shorter main tunnel and a relatively higher number of branches. These differences can primarily be related to the different levels of food availability, which is higher in the terra-rossa. It is suggested that each of the patterns reflects the mole rat's ability to optimize foraging efficiency in accordance with its given food availability.
The average total length of the males' burrows was much greater than those of the females' burrows in the rendzina soil. It appears that food requirements determine different burrow features of the sexes rather than reproduction requirements.
Other tunnel features (e.g. structural complexity, depth and width, segment length and spatial arrangement) and the factors which may affect them, as well as burrow structure of young mole rats and evidence of the underground dispersion of young mole rats, were presented and discussed.
Many similarities were found in a comparison of S. ehrenbergi burrow features with those of other solitary subterranean rodents.     

Variations of molar morphology in the Spalax ehrenbergi superspecies: adaptive and phylogenetic significance

This study compares and contrasts variations of enamel fold pattern on the crowns of the molars of subterranean mole rats ( Spalax ) belonging to two superspecies, S. ehrenbergi and S. leucodon , and involving about 20 chromosomal species, distributed parapatrically and ranging on different soil types. The sample studied involved 397 skulls, subdivided as follows: 280 of S. ehrenbergi from Israel, representing the four chromosomal species 2n = 52, 54, 58 and 60; 59 of S. ehrenbergi from Egypt, Lebanon, Syria, Jordan and Turkey; 58 of S. leucodon from Turkey.
In making the comparisons it was necessary to take into account the marked changes of molar pattern that take place as a result of wear.
There is a reasonably good agreement between differences of molar pattern and genetic and immunological distances. The species in Israel and Egypt form a compact group, clearly differentiated from Turkish leucodon; ehrenbergi from Turkey and Syria are intermediate, in accordance with their geographical distribution.
In Israel, differences associated with soil type are at about the same level as differences between species. Local populations show a higher level of diversity, and in a number of cases neighbouring populations differed significantly, sometimes over very short distances. Population differences within species are generally associated with differences of soil and vegetation, indicating the influence of natural selection. There are also differences across species boundaries, implying a phylogenetic (heritage) effect. The nature of the adaptive link between molar pattern and soil is unknown: possible factors are contamination of the food by soil, and toughness of the food which necessitates changes of enamel fold length to improve grinding efficiency.     

Adaptive hypoxic tolerance in the subterranean mole rat Spalax ehrenbergi: the role of vascular endothelial growth factor.

Spalax ehrenbergi has evolved adaptations that allow it to survive and carry out normal activities in a highly hypoxic environment. A key component of this adaptation is a higher capillary density in some Spalax tissues resulting in a shorter diffusion distance for oxygen. Vascular endothelial growth factor (VEGF) is an angiogenic factor that is critical for angiogenesis during development and in response to tissue ischemia. We demonstrate here that VEGF expression is markedly increased in those Spalax tissues with a higher capillary density relative to the normal laboratory rat Rattus norvegicus. Upregulation of VEGF thus appears to be an additional mechanism by which Spalax has adapted to its hypoxic environment.     

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.     

Synaptonemal complex analysis of mole rats (Spalax ehrenbergi): unusual polymorphisms of chromosome 1

Two unusual structural polymorphisms in the largest chromosomal pair of the Israeli mole rat, Spalax ehrenbergi, were analyzed from surface-spread and silver-stained preparations of synaptonemal complexes. A C-band negative polymorphism for the length of the 1p arm was visible as axial length differences during late zygonema and early pachynema. This region underwent synaptic adjustment resulting in a fully paired, mid-pachytene synaptonemal complex with equalized axial lengths. The somatically variable and nonargentophilic secondary constriction in the 1q arm was evident as a distinct silver-stained thickening along the synaptonemal complex. Presence of this structure on the synaptonemal complex varied both among individuals and among cells within individuals. The intraindividual variation of this region is hypothesized to represent differential biochemical activity with its cellular visualization being regulated in a manner similar to that of nucleolus organizer regions.