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     

Evolutionary diversification of class II P loci in the Mhc of the mole-rat Spalax ehrenbergi

The class II region of the major histocompatibility complex (Smh) in the mole rat, Spalax ehrenbergi, consists of only two gene families, P and Q, instead of the four families (P, O, Q, and R) found in all other mammals studied to date. The Spalax P family consists of at least four beta and three alpha genes or gene fragments. In DNA-hybridization experiments, two of the beta genes behave as bona fide P-family members in that they hybridize strongly with human DP beta probes and hybridize weakly with probes specific for other class II gene families. The other two beta genes, on the other hand, hybridize weakly with human DP beta probes and nearly as well with human DQ beta probes. To determine the evolutionary relationships among these P-like genes, we have sequenced one of them. The sequence reveals, on the basis of its organization, that the gene clearly belongs to the P family, yet, on the basis of its nucleotide sequence, it is only slightly more similar to human DP than to human DQ genes. These results indicate that in the Spalax the P family of genes split into two subfamilies, PA and PB. For unknown reasons, one of these subfamilies (PB) retained more similarity to the Q gene family than did the other (PA).     

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.     

Evolutionary expansion of Mhc class I loci in the mole-rat, Spalax ehrenbergi

A cosmid genomic library was prepared from a single individual of the rodent Spalax ehrenbergi, the mole rat, captured in Israel. The library was screened with a mouse probe hybridizing with all mouse class I major-histocompatibility-complex (Mhc) genes; the cross-hybridizing clones were isolated; and their restriction maps were prepared using five enzymes. A total of 93 class I-bearing clones could be identified in the library. Forty-five of these clones showed partial overlaps and could be arranged into 14 clusters. Eleven of these clusters could be shown to contain two class I genes each; the remaining clusters, as well as most of the non-overlapping clones, each contained one class I gene. After the elimination of clones with possible cloning artifacts and of clones that may carry allelic forms of a given gene in the heterozygous animal, the total number of class I loci identified in Spalax is approximately 65. The high number of loci probably arose from the duplication of either the entire class I set or the different class I families. The high number of gene copies might represent a means of selecting different functional genes from the family in different mammalian orders. Three of the approximately 65 Spalax class I genes cross-hybridize with a probe specific for the mouse K, D, and L genes; two of these genes are in the same cluster. These three elements might possibly be the functional class I genes of the mole rat.     

Adaptation of Pelage Color and Pigment Variations in Israeli Subterranean Blind Mole Rats,Spalax Ehrenbergi

Background

Concealing coloration in rodents is well established. However, only a few studies examined how soil color, pelage color, hair-melanin content, and genetics (i.e., the causal chain) synergize to configure it. This study investigates the causal chain of dorsal coloration in Israeli subterranean blind mole rats, Spalax ehrenbergi.

Methods

We examined pelage coloration of 128 adult animals from 11 populations belonging to four species of Spalax ehrenbergi superspecies (Spalax galili, Spalax golani, Spalax carmeli, and Spalax judaei) and the corresponding coloration of soil samples from the collection sites using a digital colorimeter. Additionally, we quantified hair-melanin contents of 67 animals using HPLC and sequenced the MC1R gene in 68 individuals from all four mole rat species.

Results

Due to high variability of soil colors, the correlation between soil and pelage color coordinates was weak and significant only between soil hue and pelage lightness. Multiple stepwise forward regression revealed that soil lightness was significantly associated with all pelage color variables. Pelage color lightness among the four species increased with the higher southward aridity in accordance to Gloger''s rule (darker in humid habitats and lighter in arid habitats). Darker and lighter pelage colors are associated with darker basalt and terra rossa, and lighter rendzina soils, respectively. Despite soil lightness varying significantly, pelage lightness and eumelanin converged among populations living in similar soil types. Partial sequencing of the MC1R gene identified three allelic variants, two of which were predominant in northern species (S. galili and S. golani), and the third was exclusive to southern species (S. carmeli and S. judaei), which might have caused the differences found in pheomelanin/eumelanin ratio.

Conclusion/Significance

Darker dorsal pelage in darker basalt and terra rossa soils in the north and lighter pelage in rendzina and loess soils in the south reflect the combined results of crypsis and thermoregulatory function following Gloger''s rule.     

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.      

T-Specific DNA Polymorphisms among Wild Mice from Israel and Spain

Lehrach and his coworkers have isolated a series of DNA probes that specifically hybridize with different regions of mouse chromosome 17 within the t complex. The probes display restriction fragment length polymorphisms, RFLPs, which are specific for the t haplotypes in all laboratory mouse strains tested thus far. Some of these probes have been used to test wild mice populations for these t-associated DNA forms. It is demonstrated that populations from Germany, Switzerland, Italy, Greece, Yugoslavia, Australia, Costa Rica, and Venezuela contain chromosomes in which all the tested DNA loci display the t-specific polymorphisms. The frequency of mice carrying these chromosomes is as high as 31%. Wild mice from Israel and Spain, on the other hand, carry chromosomes displaying t-specific DNA forms only at one or two of the probed loci, while the other loci carry the wild-type (+) forms. These chromosomes thus resemble the partial t haplotypes known from the study of laboratory mice. One possible interpretation of these findings is that these DNA polymorphisms contributed to the assembly of the complete t haplotypes and that these haplotypes may have originated in the Middle East.     

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.     

The functional link between the immune suppression gene and Mhc class II molecules

The immune response to bovine insulin (BI) in the rat is controlled by the major histocompatibility complex (Mhc)-linked immune response gene (Ir-BI) and immune suppression gene (Is-BI). In the present study, we investigated the low responsiveness to BI in the WKAH rat (RT1 ^k ) and attempted to explore the functional link between Is-BI and Mhc class II molecules. Lymph node cells (LNC) from the low responder (WKAH) rats responded well to BI when a large amount of antigen was added to the culture in vitro or after OX8-bearing (OX8⁺) T cells were eliminated. These LNC, after the elimination of OX8⁺ cells, could show the RT1.D^k-restricted proliferative response upon in vitro challenge with BI, BI-B chain, or pork insulin. In addition, OX8⁺ T cells, which were activated with BI and antigen-presenting cells (APC) in vitro, suppressed the anti-BI response of W3/25-bearing proliferating T cells from BI-immunized rats. The results have demonstrated that proliferating T-cell repertoires do exist to BI, which recognize BI-B chain in the context of RT1.D^k molecules in the WKAH rat, and that the state of low responsiveness is mediated to a great extent by antigen-specific OX8⁺ suppressor T (Ts) cells. Furthermore, the elimination of APC or the addition to RT1.B^k-specific monoclonal antibody in the in vitro secondary activation culture of Ts cells diminished the suppressive activity of OX8⁺ Ts cells. In the induction phase of Ts cells it therefore seems to be necessary for these cells to recognize BI together with RT1.B^k molecules on APC.     

Individual Odours in Two Chromosomal Species of Blind,Subterranean Mole Rat (Spalax ehrenbergi): Conspecific and Cross-species Discrimination

Mole rats from two chromosomal species (2n = 58 and 2n = 60) of the Spalax ehrenbergi superspecies of Israel were tested to determine whether they were able to discriminate differences in the odour of urine from same-sex individuals of their own and of the other chromosomal species. An habituation-discrimination apparatus was designed for use with these solitary and blind subterranean rodents. Animals habituated to the odour of urine from one individual presented for 10 min at a centre sniffing area in the roof of a 50 cm long Perspex tunnel. The odour of urine from the original donor and from a second individual were presented at two other sniffing areas in the tunnel roof during a 5 min discrimination phase. Significant differences in the time spent investigating the two odours demonstrated successful discrimination between them. The results indicate that male and female mole rats of both species can discriminate between the individual-specific odour cues in urine from pairs of conspecifics and pairs of heterospecific mole rats.     

Ancestral Polymorphism of Mhc Class II Genes in Mice: Implications for Balancing Selection and the Mammalian Molecular Clock

To investigate the evolutionary dynamics at Mhc class II DR genes of mice (genus Mus), we sequenced the peptide binding regions (PBRs) of 41 DRB (=Eβ) genes and eight DRA (=Eα) genes from 15 strains representing eight species. As expected trees of these PBR sequences imply extensive maintenance of ancestral DRB alleles across species. We use a coalescent simulation model to show that the number of interspecific coalescent events (c) observed on these trees was higher than the number expected for neutral genealogies and similar sample sizes and is more consistent with balancing selection than with neutrality. Patterns of ancestral polymorphism in mouse DRB alleles were also used to examine the tempo of synonymous substitution in the PBR of mouse class II genes. Both absolute and relative rate tests on DRA and DRB genes imply increased substitution rates at two- and fourfold degnerate sites of mice and rats relative to primates, and decreased rates for the DRB genes of primates relative to ungulate and carnivore relatives. Thus rates of synonymous substitution at Mhc DR genes in mammals appear to be subject to generation time effects in ways similar to those found at other mammalian genes.     

Chromosomal Polymorphisms of Constitutive Heterochromatin and Inversions in Drosophila

A simple technique for preparing mitotic metaphases from a larval ganglion of Drosophila is described. Parallel examination of polytene and metaphase chromosome groups shows that inversion polymorphism in chromosome 3 of D. recticilia from East Maui (Hawaii) manifests a one-to-one correlation with a metaphase karyotype polymorphism due to the presence of an extra heterochromatic portion. These observations are consistent with the previous findings on other species of Hawaiian Drosophila. They strongly support the hypothesis that when one breakpoint of a long inverted segment of a chromosome element occurs in the vicinity of the constitutive heterochromatin, it may exert an effect in eliciting the production of heterochromatic material in the same chromosome.     

Chromosomal malsegregation and micronucleus induction in vitro by the DNA topoisomerase II inhibitor fisetin

The plant flavonol fisetin is a common dietary component that has a variety of established biological effects, one of which is the inhibition of the enzyme DNA topoisomerase II (topo II). Compounds that inhibit topo II can exert genotoxic effects such as DNA double strand breaks, which can lead to the induction of kinetochore- or CREST-negative micronuclei. Despite reports that fisetin is an effective topoisomerase II inhibitor, its genotoxic effects have not yet been well characterized. Genotoxicity testing of fisetin was conducted in TK6 and HL60 cell lines and the cells were analyzed for malsegregating chromosomes as well as for the induction of micronuclei. Using the cytokinesis-blocked CREST micronucleus assay to discriminate between micronuclei formed from chromosomal breakage (CREST-negative) and chromosomal loss (CREST-positive), a statistically significant increase in CREST-positive micronuclei was seen for all doses tested in both cell lines. CREST-negative micronuclei, however, were significantly increased at the higher test concentrations in the TK6 cell line. These data indicate that at low concentrations fisetin is primarily exerting its genotoxic effects through chromosomal loss and that the induction of DNA breaks is a secondary effect occurring at higher doses. To confirm these results, the ability of fisetin to inhibit human topoisomerase II-alpha was verified in an isolated enzyme system as was its ability to interfere with chromosome segregation during the anaphase and telophase periods of the cell cycle. Fisetin was confirmed to be an effective topo II inhibitor. In addition, significant increases in the number of mis-segregating chromosomes were observed in fisetin-treated cells from both cell lines. We conclude that fisetin is an aneugen at low concentrations capable of interfering with proper chromosomal segregation and that it is also an effective topo II inhibitor, which exerts clastogenic effects at higher concentrations.     

Microgeographic Differentiation of Chromosomal and Enzyme Polymorphisms in DROSOPHILA PERSIMILIS

We studied microgeographic and temporal genetic differentiation in natural populations of Drosophila persimilis with respect to chromosome inversion and enzyme polymorphisms. Both inversion frequencies and allozyme frequencies varied significantly over short distances. Neither differed significantly between morning and evening collections. Because several studies of the dispersal behavior of this species have been performed, we attempt to fit the observed data to mathematical models which relate dispersion to random genetic drift and to spatially varying selection coefficients. We conclude that the observations are due at least partly to behavioral differences among genotypes. i.e., habitat preferences. These results have implications for genetic load theory and models of selection in heterogeneous environments.