mitochondrial dna polymorphisms in subterranean mole-rats of the spalax ekrenbergi superspecies in...

Mitochondrial DNA Polymorphisms in Subterranean Mole-Rats of the Spalax ekrenbergi Superspecies in Israel, and Its Peripheral Isolates’

Eviatar Nevo, * Rodney L. Honeycutt,Jf Hiromichi Yonekawa,$ p2 Kimberlyn Nelson,$ and Naoto Hanzawag y3 *Institute of Evolution, University of Haifa; TDepartment of Wildlife and Fisheries Sciences, Texas A&M University; *Department of Biochemistry, Saitama Cancer Center Research Institute; and §Biology Department, Pennsylvania State University

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.

Introduction

Mole-rats of the Spalax ehrenbergi superspecies in Israel provide an excellent evolutionary model of ecological speciation and adaptive radiation (Nevo 199 1) . The superspecies consists of four chromosomal species (2n = 52, 2n = 54, 2n = 58, and 2n = 60) that are distributed parapatrically in four climatic regimes in Israel. Behavioral and cytogenetic data suggest that these four species are reproductively isolated by pre- and postmating isolating mechanisms, with narrow hybrid zones along their parapatric contacts. The restricted gene flow between the chromosomal species has been docu- mented with numerous genetic markers (Nevo 199 1).

1. Key words: mtDNA diversity, mole-rats, speciation, gene flow, isolates, Spalax ehrenbergi.

2. Present address: Department of Laboratory Animal Science, Tokyo Metropolitan Institute of Medical Science, Honkomagome, Bunkyo-ku, Tokyo 113, Japan.

3. Present address: Department of Cell Genetics, National Institute of Genetics, Japan.

Address for correspondence and reprints: Eviatar Nevo, Institute of Evolution, University of Haifa, Mount Carmel, Haifa 3 1905, Israel.

Mol. Biol. Evol. 10(3):590-604. 1993. 0 1993 by The University of Chicago. All rights reserved. 0737-4038/93/1003-0007$02.00

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mtDNA of Mole-Rats in Israel 591

The patterns of polymorphism of S. ehrenbergi have been extensively tested (Nevo 199 1) . Genetic diversity at the allozyme level (Nevo and Shaw 1972; Nevo and Cleve 1978; E. Nevo, unpublished data) was low overall, with an increasing southward trend of heterozygosity toward regions of higher aridity and a more unpredictable climate. This increase resulted in the 2n = 60 species having the highest levels of genetic polymorphism and heterozygosity.

Across the S. ehrenbergi superspecies in Israel, populations are continuously distributed in their main ranges but become semi-isolated and isolated, particularly in the peripheral steppes and deserts surrounding the chromosomal species 2n = 60 (Nevo et al. 1982; Nevo 1989). The post-Wtirm relictual semi-isolates and isolates are separated from the main range by several to 10s of kilometers of harsh steppic and desert environments. They consist of low effective population sizes ( 10s to several 100s of individuals; - 100 in the main Sede Boqer isolate studied). We found that the semi-isolates and isolates harbor genetic polymorphisms [of allozymes, nuclear genes, mitochondrial DNA (mtDNA), and chromosomes] and phenotypic variances (in morphology, physiology, and behavior) similar in kind to (though lower in degree than) those of the continuous populations. We therefore devoted a separate mtDNA study to the 2n = 60 and its peripheral isolates (the latter are extensively described in Nevo 1989).

This paper is based on two somewhat overlapping yet independent studies and data sets of mtDNA variation in the S. ehrenbergi superspecies in Israel, addressing two different problems. The first data set was used to estimate mtDNA polymorphism, within and between the four chromosomal species of S. ehrenbergi. The second data set was focused on the 2n = 60 species and its peripheral semi-isolates and isolates. Here we show much mtDNA haplotype uniqueness and diversity in the main ranges of the S. ehrenbergi superspecies in Israel, even in small desert isolates.

Material and Methods Sampling

There are two data sets. The first data set consists of 64 mole-rats representing 12 populations from the main ranges of the four chromosomal species in Israel, an- alyzed for mtDNA patterns of diversity and divergence (fig. 1). In each species we tested three types of populations: (i) near the hybrid zones (NHZ); (ii) central (C); and (iii) ecologically marginal (M). The only population not in this data set is the central population of the 2n = 60 species (Lahav). The second data set consists of 69 mole-rats representing the 2n = 60 species of the Spalax ehrenbergi superspecies, collected from eight localities including main range, semi-isolated, and isolated populations (fig. 1).

Experimental Procedure

The first data set was obtained in Dr. Honeycutt’s laboratory. mtDNA was isolated from frozen tissues by using CsCl-propidium iodide gradient centrifugation (Brown 1980; Densmore et al. 1985). The mtDNAs were digested with 13 six-cutter restriction endonucleases-AccI, BamHI, BclI, BglII, BstII, ClaI, EcoRI, EcoRV, HindIII, PvuII, SaZI, XbaI, and XhoII-and one five-cutter, HincII. The mtDNA fragments were end- labeled with 32P-dNTP by using large ( IUenow ) fragment DNA polymerase I (Brown 1980). Blunt-end fragments were labeled with T4DNA polymerase prior to standard end-labeling. The labeled fragments were separated by gel electrophoresis using 1.2% agarose and 3.5% polyacrylamide vertical gels. HindIII-digested lambda DNA and



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LEBANON ., t Qiryal Shemono.q

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J I Wadi Fara l 12 . . . . . . . . . . . . . . . . . . . . . . . . . ~.~.~.~.~.~.~.~.~.~.~.~.~.~.~.~.~.~.~.~.~.~.~.~.~.~ ~.~.~.~.~.~.~.~.~.~.~.~.~.~.~.~.~.~.~.~.~.~.~.~.~. / 19 / . . , . . . . . . . . . . . . . . . . . . . . . . . . ~:::_~::. Jiftlik ‘a3 1

Jerusalem n

0 60

Lahav l 15

Beer Sheva n

Ramai Chovav . 16

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A Dimona l 17

/

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FIG. 1 .-Geographic distribution of the four chromosomal species belonging to the Spalax ehrenbergi superspecies in Israel, separated by narrow hybrid zones. The 18 populations tested for mtDNA polymor-



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HinfI-digested SV40 DNA were used as molecular-weight standards on each gel. The end-labeled fragments were made visible by autoradiography. Restriction sites were mapped for each individual restriction endonuclease and were used to confirm site maps produced by the above procedure (table 1). The sites in both data sets, those of Honeycutt and those of Yonekawa (described later), were not mapped relative to the D-loop but, rather, were mapped relative to each other. Thus, the site maps that we have are maps for each restriction endonuclease separately. Since AccI was not completely mapped for all haplotypes of Honeycutt’s data set, it was eliminated from the calculations of all genetic indexes of these data. All haplotypes produced by a given enzyme were assigned a letter, and a composite haplotype was constructed for each individual. Complete site maps for each restriction endonuclease, as well as the alphabetical classification of haplotypes, are available on request.

The second data set was obtained in Dr. Yonekawa’s laboratory, involving eight populations of the 2n = 60 species. mtDNA was purified from liver tissue by using differential centrifugation as described by Bustamante et al. ( 1977) and modified by Yonekawa et al. ( 1980, 198 1) for the enhancement of mtDNA retrieval from frozen tissues. The mtDNA was digested with the following 10 restriction endonucleases: Hind11 ( = HincII, considered as 5-base restriction enzyme in the calculations), AccI, &I, HpaI, AvaI, HaeII, BarnHI, BqlI, and HindIII. After digestion, the restriction fragments were electrophoresed on 1% agarose gels, and the fragment patterns were made visible under UV light by staining with ethidium bromide (Yonekawa et al. 1978). Restriction fragments ~500 bp were detected by gel electrophoresis on 4% polyacrylamide gels stained with ethidium bromide. If mtDNA fragment variation could not be visualized with ethidium bromide staining, Southern blot hybridization was performed using a purified mtDNA molecule as a probe (Yonekawa et al. 1978). The mtDNA probe was labeled by the random priming method of Feinberg and Vogelstein ( 1984). Lambda DNA and mouse mtDNA digested with Hind111 were used as molecular-weight standards for each gel. All haplotypes produced by a given enzyme were assigned a letter, and a composite haplotype was constructed for each individual. Each haplotype was designated by a roman numeral. The haplotypes of the second data set, generated at Dr. Yonekawa’s lab, were designated by a roman numeral accompanied by an asterisk.

Data Analysis

We measured mtDNA diversity of S. ehrenbergi by four indices: total and unique number of haplotypes, nucleon (haplotype) diversity (h) between haplotypes, and nucleotide sequence divergence (K) between mtDNA haplotypes. h was derived from equations given by Nei and Tajima ( 198 1). Overall n; was estimated from the site data (Nei and Li 1979; Nei 1987, p. 256) by using Tajima’s computer program, provided by M. Nei. Calculations of averages, frequencies, and h were conducted by

phisms, their abbreviations (for figs. 2 and 3), and their ecogeographic nature (in parenthesis) are as follows: 2n = 52-l = Maalot, Ma (NHZ); 2 = Kerem Ben Zimra, KBZ (C); and 3 = Qiryat Shemona, QS (M); 2n = 54-4 = Mt. Hermon, He (M); 5 = Quneitra, Qu (C); and 6 = El-Al, EA (NHZ); 2n = 58-7 = Kabri, Ka (NHZ); 8 = Zippori, Zi (C); 9 = Mt. Carmel, Ca (C); and 10 = Afiq, Af (M; NHZ); and 2n = 60-l 1 = Anza, An (NHZ); 12 = Wadi Fara, WF (SI); 13 = Jiftlik, Jif (SI); 14 = Jerusalem, Jer (M); 15 = Lahav, Lah (C); 16 = Ramat Chovav, RC (SI); 17 = Dimona, Dim (I); and 18 = Sede Boqer, SB (I).



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the SpSSx (1986, pp. 101-l 16, 148-161, 248-255, 372-383) package. Phylogenetic trees were constructed by the PHYLIP package, version 3.4 (Felsenstein 199 1)) using the Do110 and Wagner parsimony methods (DOLPENNY, DOLBOOT, MIX, and BOOT programs).

Results mtDNA Patterns Between and Within Species (First Data Set)

Restriction-endonuclease digestions used 13 enzymes (Accl site variation was not mapped for this data set). There were 28 mtDNA haplotypes revealed, involving 68 restriction sites ( 23 unvaried and 45 varied; table 1) . There is considerable geographic structure and population subdivision both within and between the chromosomal species of the Spalax ehrenbergi superspecies. Few mtDNA haplotypes are shared between chromosomal species. Only one haplotype is shared between the 2n = 52 and 2n = 54 species, and only one is shared between 2n = 58 and 2n = 60. Even within a chromosomal species there is much inter-population subdivision (table 1 and fig. 2 ) . Nearly all enzymes involving polymorphic sites distinguished between the two major species pairs, the northern (2n = 52 and 2n = 54) and the southern (2n = 58 and 2n = 60) (table 1). Some enzymes also differentiate between 2n = 52 and 2n = 54.

The mtDNA summary statistics of all 12 populations and four species (table 2 ) were calculated from the restriction-site data presented in table 1. h increases southward with increasingly warmer, drier, and more unpredictable climates. Likewise, n: ( =average of 6’s in Nei and Li 1979 ) values displayed a similar pattern (table 2).

Phylogenetic Analyses

Interspecific and interpopulational distance data of nucleotide divergence estimates ( dA; Nei 1987, p. 276) were calculated (and are obtainable from E.N.). Two types of phylogenetic analyses were performed on the mtDNA data. First, a phylogenetic tree for the four species was constructed by using the Do110 parsimony method (DOLPENNY and DOLBOOT programs). The tree was derived from the restriction- site data of table 1 and separated the northern from the southern species pairs. Boot- strapping, based on 1,500 replications, indicated that the tree was significant (P > 0.998). Second, a phylogenetic tree for all haplotypes was constructed by following the Wagner parsimony method, with programs MIX and BOOT, analyzing the restriction-site data presented in table 1 (fig. 2).

All 2 10 replicates of bootstrapping result in the same grouping ( 2n = 52-54 and 2n = 58-60), obtained by Do110 parsimony. Little discordance occurs between chromosomal species and mtDNA markers. Paraphyly is evident in both the 2n = 52-54 and 2n = 58-60 groups. In 2n = 58 two haplotypes from Afiq (XIX and XX) had diverged from both 2n = 58 and 2n = 60 (fig. 2). In 2n = 60 one haplotype from Anza (XXVIII) appears also in Zippori and groups with 2n = 58. One haplotype of 2n = 54 (XII) also appears in the 2n = 52 population of Qiryat Shemona, whereas the remaining 2n = 52 and 2n = 54 haplotypes group separately. The 2n = 60 Anza haplotypes represent two divergent groups (XXI-XXIII and XXIV-XXV), suggesting population substructuring at this locality. This phenomenon was also observed in the second data set ( fig. 3 ) .

For populations 2n = 52 and 2n = 54 the interpopulation differences within each of the two species are of the same magnitude as the intrapopulation divergence (data not given). Thus, the net divergence ( dA) between the populations within these two species is zero or nearly so. The haplotypes of 2n = 52 and 2n = 54 are closely related.



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52 c I Ma KBZ QS 52 II KBZ 52 III KBZ 52 IV KBZ 52 V KBZ

EA QS 58 XVII Zi 58 XVI Zi 58 XV Zi 58 XIII Ka

# $8 XIV Ka Zi 58 XVIII Ca 58 60 XXVIII Zi An ~a-58 XIX Af

60 XXIV An 13L60 XXV An

CO XXI An

er FIG. 2.-Unrooted consensus phylogenetic tree of haplotype divergence of the 28 haplotypes of the

four chromosomal species of the Spalax ehrenbergi superspecies. The tree was constructed from the restriction- site data (table I), according to the Wagner parsimony method, by the MIX program of the PHYLIP package. The program generated 162 parsimonous trees, each of which consists of 66 steps, from which the consensus tree was derived by the CONSENSE program. The haplotype (in roman numerals) appears between the chromosomal species (2n’ = 52, 54, 58, and 60), on its left-hand side, and the abbreviated population name (given in the legend to fig. 1)) on its right-hand side. Only branch lengths of two and more steps are shown. The bootstrapping is based on 210 replications, and the results are shown in circles.



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Table 2 Summary of mtDNA Indexes of First Data Set of Spalax ehrenbergi Superspecies in Israel

Populations? 2n = 52:

1. Maalot ............ 2. Kerem Ben Zimra ... 3. Qiryat Shemona .....

Subtotal ............ Mean ..............

2n = 54: 4. Mt. Hermon ........ 5. Quneitra ........... 6. El-Al ..............

Subtotal ............ Mean ..............

2n = 58: 7. Kabri ............ 8. Zippori ........... 9 Mt. Carmel ........

10. Afiq .............. Subtotal ............ Mean ..............

2n = 60: 11. Anza ............. 14. Jerusalem .........

Subtotal ............ Mean ..............

Species 1. 2n=52. ............. 2. 2n = 54 .............. 3. 2n=58.. ............ 4. 2n=60.. ............

Overall .........

No.

No. OF HAPLOTYPES

Total Unique

DIVERSITY INDEX

hb xc + Standard Error

1 0 0.0 0.0 + 0.0 5 4 0.905 0.00263 + 0.00033 2 0 0.400 0.00865 + 0.002 11

0.435 0.00376

3 1 0.700 0.00116 + 0.00039 5 3 0.857 0.00338 + 0.00044 2 1 0.667 0.0040 1 + 0.00 160

0.741 0.00285

2 1 5 3 1 1 2 2

0.500 0.00125 + 0.00054 0.786 0.00190 + 0.00023 . . . . . .

0.667 0.00433 + 0.00069

0.65 1 0.00249

6 5 0.889 0.00665 + 0.0009 1 2 2 0.400 0.00 166 + 0.00070

0.645 0.00416

6 5 7 6 9 8 8 7

38

0.588 0.00365 + 0.00041 0.586 0.003 18 + 0.00027 0.912 0.01213 + 0.00067 0.895 0.00727 + 0.00045

IV . . . 0.953

5 7 5

ii

5 7

3 15

4 8 1 4

ii

10 5

Is

17 15 17 15 -

64 0.03863 + 0.00057

a Numbered as in fig. 1. b The probability of sampling two different haplotypes simultaneously. ’ The average no. of nucleotide differences per site between two haplotype sequences.

In contrast, the population pattern of 2n = 58 and 2n = 60 displays a larger net interpopulation divergence ( figs. 2 and 3 ) . Three populations of 2n = 5 8 (Mt. Carmel, Zippori, and Kabri) are located on one branch, whereas the fourth population ( Afiq) is located on the branch of the 2n = 60 populations ( Anza and Jerusalem). Divergence of the Afiq 2n = 58 population represents a faster mtDNA evolutionary rate than do all other populations of the superspecies in Israel.

Patterns of mtDNA Variation in 2n = 60 and Its Isolates (Second Data Set)

Fifteen mtDNA haplotypes were found for 69 mole-rats of the S. ehrenbergi 2n = 60 species and its peripheral steppic semi-isolates and desert isolates (table 3 ) . The Hind11 ( = HincII) and AccI enzymes yielded the largest number of phenotypes (five;



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I V* WF

t

VI* WF Jif

III* An WF

LI* An

VII* Jer

21X* Jer

X* Jer Lah

Jer

-XIII* Lah RC Dim

XI* Lah

-XII* Lah

SB

FIG. 3.-Unrooted consensus phylogenetic tree of 15 mtDNA haplotype divergence from eight populations of the 2n = 60 species. The tree was constructed from the restriction-site data (table 3 ), according to the Wagner parsimony method, by the MIX program of the PHYLIP package. The program generated 30 parsimonous trees, each of which consists of 27 steps, from which the consensus tree was derived by the CONSENSE program. Cluster B contains haplotypes II*, IV*, V *, and VIII*, whereas others belong to



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derived from table 3) and, hence, the highest level of polymorphism. The three enzymes BarnHI, BgZI, and Hind111 showed no polymorphisms. Nevertheless, the BamHI and BgZI enzymes proved diagnostic for the 2n = 60 species relative to the northern species, 2n = 52 and 2n = 54. A large proportion of these haplotypes were rare and mostly confined to a particular locality, but four of the six more common haplotypes were shared between two or three localities (table 3).

High Intrapopulation mtDNA Diversity (Second Data Set)

High (unexpected) intrapopulation mtDNA haplotype diversity characterizes the 2n = 60 species and its peripheral isolates (tables 3 and 4). Ranges of haplotype sequence divergences within populations were d = 0.35%-2.17% in Anza, 0.2 l%- 2.33% in Jerusalem, 0.20%-0.88% in Lahav, 0.20%-1.94% in Wadi Fara, 0.20% in Sede Boqer, and 1.02% in Dimona. Likewise, it appears evident that all populations with more than two sampled animals consisted of plural matriarchies. Two haplotypes were even found in each of the two small desert isolates of Sede Boqer and Dimona, consisting of only - 100 and - 25 individuals, respectively.

The mtDNA summary statistics of all eight populations (table 4) were calculated from the restriction-site data presented in table 3. Both mtDNA estimates-h and n-were high in all populations, though both were lower in the Sede Boqer isolate compared with the other isolates. Nevertheless, distinctly high levels of h and n: were found in the semi-isolates (Wadi Fara) and isolates (Dimona). Unique haplotypes characterized the main populations (Jerusalem) and even occurred in the desert isolates (e.g., Sede Boqer) . When samples consisted of one animal, it was impossible to calculate the estimates. Therefore, we also calculated estimates for the pooled isolates, where high uniqueness is indicated in the eastern semi-isolates (Wadi Fara and Jiftlik) and in the southern isolates ( Ramat Chovav, Dimona, and Sede Boqer ) (table 4).

Sequence Divergence of Haplotypes (Second Data Set)

Sequence divergence among pairs of haplotypes in the second data set was calculated by pair-wise comparison using d estimates. The estimates were varied: the smallest estimate of sequence divergence was between haplotypes II* and VIII* (0.18% ) , while the largest was between haplotypes II * and XII * (2.9 1% ) . The smallest and largest estimates of branching times between haplotypes, according to Wilson et al’s ( 1985) suggestion (2%-4% divergence/ 1 Myr), are 0.05-0.09 Myr and 0.75- 1.45 Myr, respectively.

We constructed from the site data of table 3 a phylogenetic tree of the haplotypes, following the Wagner parsimony method by the MIX and BOOT programs (fig. 3). The tree showed two major clusters (A and B). Haplotypes belonging to cluster A (haplotypes I *, III*, VI*, VII*, and IX*-XV * ) were commonly found in all eight localities, including the semi-isolated, and in very small populations of Ramat Chovav and Jiftlik, each represented by only one animal. These haplotypes were also common

cluster A. Note the separated and significant ( 196/200) branch including the three haplotypes (XI*, XII*, and XIII* ) which appears in the lower part of the figure. The abbreviated names of the populations are given in fig. 1 and appear on the right-hand side of the haplotypes, which are designated by roman numerals with asterisks. Only branch lengths of two or more steps are shown. The bootstrapping is based on 200 replications, and the results are shown in circles.



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Table 4 Summary of mtDNA Indexes (Second Data Set) of the 2n = 60 Species of Spalux ehrenbergi Superspecies in Israel

POPULATION No.

No. OF

HAPLOTYPES

Total Unique

DIVERSITY INDEX

h” TP + Standard Error

11. Anza 12. Wadi Fara 13. Jiftlik 14. Jerusalem 15. Lahav . 16. Ramat Chovav 17. Dimona 18. Sede Boqer

Overall

Pooled isolates: a. Easternb . b. Southernc . .

18 4 6 4

14 14

3 12 -

69

7 4 2 0.8 1 0.0074 + 0.003 19 16 3 2 0.34 0.0024 + 0.00028

4 4

2 2

15 . . . 0.89 0.0112 + 0.00017

0.7 1 0.87 . . .

0.50 0.74 . . .

0.67 0.17

0.0079 + 0.00054 0.0075 + 0.00127

. . . 0.0048 + 0.00057 0.0025 + 0.00024

0.0059 + b.00183 0.0003 + 0.00001

a Defined as in table 2. b Includes Wadi Fara and Jiftlik. ’ Includes Ramat Chovav, Dimona, and Sede Boqer.

across populations: of 69 animals, 44 (64%) consisted of these haplotypes. On the other hand, haplotypes belonging to cluster B (haplotypes II *, IV*, V*, and VIII * ) were found only in a few populations but displayed a southward gradient: Anza (44%), Wadi Fara ( 33% ), and Jerusalem ( 7%) (derived from data in table 3). As can be seen in figure 3, cluster A in the southern populations (Lahav, Dimona, and Ramat Chovav) evolved rapidly.

Discussion mtDNA Divergence Estimates and the Speciation Model in the Spalax ehrenbergi Superspecies

The 7c of the entire S. ehrenbergi superspecies was 0.0386 (table 2). This value is considerably higher than that seen in comparisons of different species (Ferris et al. 198 1; Avise and Lansman 1983; Kessler and Avise 1984), including intraspecific levels of divergence in the endemic African subterranean mole-rats, the Bathyergidae, Heterocephalus glaber (0.029)) Cryptomys hottentotus (0.0 18)) and C. natalensis (0.017) (Honeycutt et al. 1987, 199 1). The only exception is Georychus capensis, which had a higher x value, 0.07 1. Within-species (in our sample) variation for all chromosomal species of the 5’. ehrenbergi superspecies was low, with the 2n = 52, 2n = 54,2n = 58, and 2n = 60 species having n; values of 0.004,0.003,0.012, and 0.007, respectively. Low mtDNA variation within highly subdivided populations is a rather common observation for most species examined (Avise and Lansman 1983 ) .

The interspecific sequence divergence ( dA) (data not shown) and the evolutionary tree of figure 2 accord with the speciational trend from lower diploid numbers (2n = 52 and 2n = 54) to the higher diploid numbers (2n = 58 and 2n = 60). The geographic trend of this speciational model was derived from other lines of evidence-



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602 Nevo et al.

including allozymes, scnDNA hybridization, natural hybridization, mate preference polymorphisms, and morphogenetics (overviewed in Nevo 199 1 )-and not from the mtDNA trees. Our calculations are based on the assumption of a mean rate of divergence of -2%-4% /Myr for mammalian mtDNA (Wilson et al. 1985). The relative divergence (calculated from the average dA values) between the northern (2n = 52 and 2n = 54) and southern (2n = 58 and 2n = 60) pairs of species is 1.2-2.4 Mya. The divergence time of the major group 2n = 52-54 is 0.44-0.88 Mya, and that of 2n = 60 is 0.07-o. 13 Mya. While this major speciational trend is also substantiated by analysis of hybrid meiosis and synaptonemal complexes (E. Nevo, unpublished data), there are discrepancies in divergence times derived from mtDNA, allozyme (Nevo and Cleve 1978), and scnDNA (Catzeflis et al. 1989) methodologies. The oldest fossil Spalax known from the Lower Pleistocene of Israel is 1.4 Myr (Tchernov 1987), basically supporting the molecular time estimate of the superspecies origin.

Consequently, the S. ehrenbergi superspecies is young and has evolved by several speciation events from the early Pleistocene to recent times. The differences between some of the estimates derive from the lack of reliable calibration and from rate het- erogeneity.

mtDNA and Levels of Gene Flow between Species

The mtDNA evidence indicates that no gene flow appears to cross the hybrid zones separating the species 2n = 52-58 or 2n = 58-54 and even that of the youngest one, 2n = 58-60. Thus, interspecific introgression cannot be invoked for mtDNA differentiation. Even within the species, mtDNA uniqueness in populations is high, reflecting indirectly that local selection and/or random fixation may have played an important role in this extranuclear haploid maternal DNA, similar to the evolutionary forces operating on nuclear DNA. Gene flow (i.e., dispersal) appears to be relatively low even within species, particularly in the species 2n = 58 and 2n = 60.

mtDNA Divergence and Evolutionary History of the 2n = 60 Species

The higher levels of mtDNA diversity of 2n = 60 are seen in both data sets. This is reflected in all mtDNA diversity measures (table 2), as well as in their ranking in the two populations held in common ( Anza > Jerusalem). The expanded second data set of 2n = 60 indicates that the high levels of mtDNA diversity hold not only in the main range but also in the peripheral steppic semi-isolates and desert isolates. The separation of 2n = 60, the recent derivative of speciation in the complex, was dated to 0.07-o. 13 Mya both by allozymes and mtDNA (Nevo and Cleve 1978 and the present study, respectively) and to 0.18-0.75 Mya by DNA-DNA hybridization (Catz- eflis et al. 1989).

The general evolutionary trend southward from mesic to xeric open new environments in Spalax, and particularly in the S. ehrenbergi species 2n = 60 (Nevo 199 1 ), is substantiated by mtDNA haplotype evolution. The latter is decipherable through a colonization process that starts from the relatively mesic Anza population in the north, through increasingly xeric Jerusalem, Lahav, and Dimona to the desert isolate of Sede Boqer. The four haplotypes (VII * --) X * -XIV * -XV * ) differ among themselves by successive single mutational changes, and VII* is only two mutational changes from III *. The above-mentioned trends within cluster A can be observed in figure 3.

The high levels of intraspecific mtDNA diversity in 2n = 60, especially between cluster A and B haplotypes (fig. 3 and dA values in Results), may suggest that 2n



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= 60 is an old species. However, the first data set and additional lines of evidence (allozymes, scnDNA, and others; see Nevo 199 1) suggest the recency of this species. This discrepancy may be resolved by several models, including (i) introgression, (ii) natural selection, and (iii) prespeciation polymorphisms. Elsewhere we explain the discrepancy by the model of natural selection.

Acknowledgments

We are grateful to both Dr. Osamu Gotoh of the Saitama Cancer Center Research Institute and Dr. Avigdor Beiles of the Institute of Evolution for statistical analysis. We thank both Dr. Yusaku Tagashira of the Saitama Cancer Center Research Institute and Dr. Avigdor Beiles for their helpful discussions and encouragements. We are grateful to our reviewers and to Dr. Walter Fitch for their constructive comments, which improved the manuscript. This study was supported by grant 84-00343 from the United States-Israel Binational Science Foundation (BSF), Jerusalem, Israel, to E.N. and R.L.H.; by National Science Foundation grant BSR 85084479 to R.L.H.; by financial support from the Israel Discount Bank Chair of Evolutionary Biology, and by the Ancell-Teicher Research Foundation for Genetics and Molecular Evolution, established by Florence and Theodore Baumritter of New York.

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MASATOSHI NEI, reviewing editor

Received September 9, 199 1; revision received January 15, 1993

Accepted January 15, 1993



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