* Istituto di Tecnologie Biomediche, Sezione di Bioinformatica e Genomica Comparata, Consiglio Nazionale delle Ricerche, Bari, Italy
Institute of Evolution, University of Haifa, Israel
Dipartimento di Biochimica e Biologia Molecolare, Università di Bari, Italy
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Abstract |
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Key Words: Spalax ehrenbergi mitochondrial DNA control region speciation adaptation molecular evolution
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Introduction |
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Comparative studies carried out in the control region of mammals have revealed that each of the three domains presents a distinct pattern of variation, with some regions evolving rapidly, such as the ETAS and CSB domains, and others maintaining a high degree of conservation across taxa, such as the central domain (Brown et al. 1986; Sbisà et al. 1997; Pesole et al. 1999). This rate heterogeneity allows the use of the control region for the study of the patterns of genetic variation at different levels of divergence within mammals. Thus, the most variable regions have been used for detailed studies of population and phylogeographic structure (Goldberg and Ruvolo 1997; Matsuhashi et al. 1999; Rosel et al. 1999; Koh, Lee, and Kocher 2000; Matson and Baker 2001), whereas conserved regions may prove to be useful for phylogenetic studies of more divergent taxa (Douzery and Randi 1997; Larizza et al. 2002). In spite of this, just a few studies have been carried out on the structure and patterns of variation in rodent species (Faulkes et al. 1997; Prager, Orrego, and Sage 1998; Matson and Baker 2001).
The subterranean mole rats of the Spalax ehrenbergi superspecies in Israel include four species that only very recently have been named formally: Spalax galili (2n = 52), S. golani (2n = 54), S. carmeli (2n = 58), and S. judaei (2n = 60) (Nevo, Ivanitskaya, and Beiles 2001). The oldest fossil record of this superspecies is dated 1.4 MYA from the "Ubeidiya Formation," an Early Pleistocene hominid site in the Jordan Valley (Tchernov 1987). Since then, a prolific diversification of the S. ehrenbergi superspecies has taken place, but the past 120,000 years of the Upper Pleistocene probably better reflect the active speciation process (Tchernov 1968). Thus, nowadays, these four species represent progressive stages of late speciation and constitute an excellent example of ecological speciation and adaptive radiation. The adaptive radiation of mole rats in Israel, from the Early Pleistocene to recent times, is closely associated with fossoriality, increasing aridity and progressive deforestation, and savannization, and hence with distinct climatic diversity in both the Mediterranean and steppic climatic regimes (Nevo 1991, 1999; Nevo, Ivanitskaya, and Beiles 2001). S. galili (2n = 52) is present in the cool and humid Upper Galilee Mountains; S. golani (2n = 54) ranges in the cool and semidry Golan Heights; S. carmeli (2n = 58) is distributed in the warm and humid Lower Galilee Mountains, Central Yizreel, and the coastal plain; and S. judaei (2n = 60) colonizes the warm and dry mountains of Samaria, Judea, the northern Negev, the southern part of the Jordan Valley, and the coastal plain (fig. 1). Several different studies, especially those based on DNA-DNA hybridization (Catzeflis et al. 1989; Nevo 1991), indicate that S. golani (2n = 54) is the oldest species, originating about 1.6 MYA. The split to S. galili (2n = 52) occurred next, about 0.35 MYA. S. carmeli (2n = 58) and S. judaei (2n = 60) are the most recent derivatives, with speciation event estimated at 0.2 MYA.
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Materials and Methods |
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The complete mtDNA control region (979 to 983 bp) was double-strand sequenced using primers 1FR and 4RV described above and four additional primers (fig. 2), 2FR (5'-TACCATCCTCCGTGAAACCA-3'), 2RV (5'-TGGTTTCACGGAGGATGGTA-3'), 3FR (5'-AGTCTAGCTGGACTTGTATG-3'), and 3RV (5'CATACAAGTCCAGCTAGACT-3'), designed on the basis of the complete mitochondrial genome of Spalax judaei (2n = 60) (unpublished data). Sequencing reactions were performed using the Thermo Sequenase Cy 5.5 Dye Terminator Cycle Sequencing Kit (Amersham Pharmacia Biotech) in 8 µl reaction volumes and following the manufacturer's instructions. After purification, DNA sequences were analyzed on a Seq4X4 automated sequencer (Amersham Pharmacia Biotech). Complete control region sequences have been deposited in EMBL under accession numbers AJ440417 to AJ440466.
Data Analyses
Complete mtDNA control regions were multialigned using the PILEUP program from GCG (1994). The alignment of the sequences was corrected by eye inspection, even though it was quite straightforward due to the high degree of similarity of the sequences and the low number of indels (table 2).
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The positions undergoing variation between sequences were evidenced and variability at these sites was calculated using the SiteVar program (Pesole and Saccone 2001). In order to determine the nature of the genetic change (i.e., the number and type of transitions and transversions), the data were analyzed using the PAUP program (Swofford 1998).
To investigate the origin, genetic structure, and relationships between and within the four species, several algorithms available in the ARLEQUIN package (Schneider, Roessli, and Excoffier 2000) were used. In particular, estimates of the nucleotide diversity and the analysis of molecular variance (AMOVA) that allows the partitioning of the observed variability in a hierarchical fashion were carried out with this package.
Phylogenetic relationships between individuals were established on the basis of a Neighbor-Joining tree applied to a distance matrix obtained using the Markov Stationary Method (also called the GTR method) implemented in the PAUP 4.0b10 package (Swofford 1998). Another Neighbor-Joining tree was obtained from a GTR distance matrix using a proportion of invariant sites (I) and a gamma distribution for variable sites (), being both parameters estimated from the data. Maximum Parsimony analysis was performed using PAUP 4.0b10 (Swofford 1998). Bootstrap values were based on 1,000 replicates.
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Results |
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In the ETAS domain, ranging from position 1 to 312 of the alignment, three conserved blocks have been found: ETAS1, ETAS2, and ETAS1-like. The ETAS1 is located between positions 81 and 139 of the multialignment and shows 15 variable positions out of a total of 59 (25.42%) for all species and four, four, five, and five variable positions in S. galili (2n = 52), S. golani (2n = 54), S. carmeli (2n = 58), and S. judaei (2n = 60), respectively (fig. 3). The consensus ETAS1 shows 18.05% dissimilarity with the consensus obtained from rodent species (data not shown). An ETAS1-like sequence is found to be present from position 1 to 59 of the multialignment (fig. 3). The consensus of the ETAS1-like sequence shows a 32.43% dissimilarity with the consensus ETAS1 of mole rats and a 36.07% dissimilarity with other ETAS1 from rodents (data not shown). The degree of variation of this ETAS1-like sequence is similar to that of ETAS1, with 14 variable positions out of 59 (23.72%) for all species and four, four, seven and seven variable positions in 2n = 52, 54, 58, and 60 species, respectively (fig. 3). ETAS2 is located between positions 141 and 197 of the multialignment and shows a higher degree of variability than the previous elements. Indeed, 23 of 57 (40.35%) nucleotide positions were variable for the whole data set and nine, 10, nine, and 11 variable positions in S. galili (2n = 52), S. golani (2n = 54), S. carmeli (2n = 58) and S. judaei (2n = 60), respectively (fig. 3). In agreement with the higher degree of variation, the dissimilarity of the consensus ETAS2 with that of rodent species is 22.41% (data not shown).
In the CSB domain, ranging from position 627 to position 983 of the multialignment, three conserved blocks, CSB1, CSB2, and CSB3, have been found. CSB1 is located between positions 665 and 688 of the alignment and shows only two variable positions out of 24 (8.33%), in S. carmeli (2n = 58) and S. judaei (2n = 60), exclusively (fig. 3). The high degree of conservation of this block is also stressed by the fact that the consensus sequence for all species shows just a 12.00% dissimilarity with the consensus obtained for all rodent species (data not shown). The other two conserved blocks, CSB2 and CSB3, placed at positions 754 to 770 and 805 to 822 of the multialignment, respectively, displayed no variable positions in mole rats (fig. 3). The dissimilarity with the consensus sequence for rodent species was 11.76% and 0.00% for CSB2 and CSB3, respectively (data not shown). In addition to the previously documented CSB elements, we have also identified a CSB1-like element. This element, however, is not located in the CSB domain, but in the ETAS domain, between positions 215 and 239. This block shows a relatively low degree of dissimilarity with CSB1 both from mole rats (28%) and from the consensus of other rodent species (20%). However, it is less conserved than the CSB1, and nine of 25 (36%) positions were variable (fig. 3).
Control Region Length and Base Composition
The control region of all species and populations is very similar both in length and base composition. The length of the control region of most sequences is 981 bp. It is noteworthy that all individuals from S. galili (2n = 52) present a 1-bp insertion in position 60 of the alignment, resulting in control regions of 982 bp. Only three other individuals show length variation: one from Zippori, which presented a 2-bp insertion in position 59 of the alignment (983 bp), and two from Kabri, which showed a 2-bp deletion in position 61 of the alignment (979 bp).
Regarding base composition, only small differences are detected between populations and species (table 2). All taxa showed similar biased base composition in the three domains of the control region: A>T>C>G in the ETAS domain (38.50%, 33.95%, 18.78%, and 8.77%, respectively), T>C>A>G in the central domain (30.68%, 26.65%, 23.60%, and 19.07%, respectively), and A>T>C>G in the CSB domain (36.46%, 31.39%, 22.48%, and 9.6%, respectively). As observed, G is strongly avoided in the two peripheral domains, but it is surprisingly high in the central domain, which is in agreement with previous observations made in other rodent species and in mammals in general.
Site Variability Heterogeneity Between Populations
The number of variable sites differed significantly across the control region, the central domain being the most conserved, followed by the CSB and the ETAS domains (figs. 3 and 4). This general pattern of variability distribution was observed in all four species, but important differences between species were detected (fig. 4). S. galili (2n = 52) represents the species with the lowest site variability, regarding both the number of variable sites and the relative variability per site. S. golani (2n = 54) shows a slight higher degree of variability in the ETAS domain, mainly due to higher values of relative variability rather than to a higher number of variable sites (fig. 4 and table 2). In the case of S. carmeli (2n = 58) and S. judaei (2n = 60), we detected a higher number of variable sites in all three domains with respect to the other two species (fig. 4 and table 2).
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The pattern of transition and transversion accumulation between populations and species was further investigated by plotting the number of TS against the number of TV (fig. 5). Linear regression and correlation between TS and TV is detected (y = 2.32x + 6.44; r = 0.88, P < 0.001) for comparisons involving individuals of the same population (open circles), populations of the same species (filled circles), and species with similar chromosomal number (open triangles), that is, S. galili (2n = 52) against S. golani (2n = 54) and S. carmeli (2n = 58) against S. judaei (2n = 60). However, for comparisons involving populations with different chromosomal number (filled triangles), that is, S. galili (2n = 52) and S. golani (2n = 54) against S. carmeli (2n = 58) and S. judaei (2n = 60), the slope of the curve decreases (y = 1.22x + 12.28; r = 0.84, P < 0.0001), most likely reflecting the beginning of the saturation of TS relative to TV. The curve that better explains all points is a logarithmic type, y = 20.34logx + 7.74 (r = 0.89, P < 0.001), which clearly shows the beginning of saturation transitions relative to transversions.
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Ecogeographic Pattern of Diversity
Levels of genetic diversity have been found to vary between and within species of S. ehrenbergi. The clearest difference is the one observed between northern, S. galili (2n = 52) and S. golani (2n = 54), and southern, S. carmeli (2n = 58) and S. judaei (2n = 60), populations. As mentioned above, the number of variable sites and the relative variability per site are higher in southern than in northern populations (fig. 4). Furthermore, the levels of nucleotide diversity (table 2) are also on the average higher in southern populations. This trend is detected even within each species. For example, in the case of S. golani (2n = 54), nucleotide diversity from northern to southern Golan populations are 0.0061 in Hermon, 0.0099 in Quneitra, and 0.0106 in El Al (table 2). Two particular cases are Afiq, which, due to the low sample size, presents only one haplotype, and Lahav, which, compared with previous estimates, displays a lower level of diversity than expected, most likely due to sampling variations. Thus, regarding the general trend, population nucleotide diversity is highly and significantly correlated with latitude (y = -45.62x + 33.46; r = -0.84, P = 0.0025) when Afiq and Lahav populations are not considered in the analysis (table 1 and 2).
Climatic conditions in Israel are highly determined by the latitude, and thus, the lower the latitude, the lower the mean annual rainfall and the number of rainy days, and the higher the temperature and the day-night temperature differential. Therefore, when we searched for correlations between climatic parameters and levels of genetic variability (table 1 and 2), we found a significant correlation between nucleotide diversity and mean number of rainy days (y = -664.84x + 63.54; r = -0.73, P = 0.0164) and moderate correlations, even though not significant, with both day-night and season temperature differences (r = -0.41, P = 0.2341 and r = 0.41, P = 0.2336), always with the exclusion of Afiq and Lahav populations.
Furthermore, observed nucleotide diversity in the mitochondrial control region is significantly correlated with estimates of cytochrome b variability (y = 335.62x - 2.46; r = 0.83, P = 0.003) (Nevo, Beiles, and Spradling 1999), all four nuclear allozyme diversity measures, A, P, H, and He (r = 0.67 to 0.82, P = 0.033 to 0.0033) (Nevo, Filippucci, and Beiles 1994), and RAPD number of polymorphic loci (y = 143.53x + 3.75; r = 0.66, P = 0.0371) (Ben-Shlomo, Fahima, and Nevo 1996), with the exclusion of Afiq and Lahav populations.
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Discussion |
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Important differences in base composition have been detected in the three different domains. However, they follow the general trend described for mammals (Sbisà et al. 1997) and the same base content within statistical fluctuations in Clethrionomys species (Matson and Baker 2001). It is noteworthy to mention the relatively high G content of the central domain (19.07%), which is higher than that observed in both the other two domains and the whole mitochondrial genome of mammals (Reyes et al. 1998). Moreover, the presence of the dinucleotide and trinucleotide GG and GGG (14 and four, respectively) is higher than expected at random (11 and two, respectively), as also described for other rodent species (Larizza et al. 2002). The presence of such motifs suggests that this region could be involved in the interaction between mitochondria and cytoskeletal elements (Jackson, Barlett, and Cook 1996).
In addition to the differences in base composition, the three described domains show important differences in site variability (figs. 3 and 4). The central domain is the most conserved region (only 7.7% variable sites), which is in agreement with previous studies (Sbisà et al. 1997; Larizza et al. 2002), and its level of conservation has been shown to be similar to that of mitochondrial 12S and 16S rRNAs, tRNAs, and nonsynonymous sites of protein-coding genes (Pesole et al. 1999). This high degree of conservation contrasts with the apparent lack of function of this region, even though its possible involvement in the interaction with cytoskeletal elements (Jackson, Barlett, and Cook 1996) could partially explain such conservation. The two peripheral domains, ETAS and CSB, are less conserved (28.53% and 13.17% variable sites, respectively), the former being more variable than the latter (fig. 3 and 4) and almost reaching the level of variability found in the synonymous positions of protein-coding genes (Pesole et al. 1999). However, within these regions, highly conserved blocks are found: ETAS1, ETAS2, ETAS1-like, CSB1, CSB2, CSB3, and CBS1-like (figs. 2 and 3). Some of these elements have been reported to be conserved across different mammalian orders (Brown et al. 1986; Saccone, Attimonelli, and Sbisà 1987; Sbisà et al. 1997), and different functions have also been proposed based on experimental evidence (Doda, Wright, and Clayton 1981; Chang and Clayton 1987; Ghivizzani et al. 1993; Tullo et al. 1995). ETAS1 has been proposed as the region containing multiple processing and/or termination signals functioning bidirectionally, whereas ETAS2 could contain the binding sites for termination factors (Sbisà et al. 1997). Nevertheless, a recent survey carried out in rodents has showed that only ETAS1 is present in all species and that ETAS2 could not be present or be much less conserved (Larizza et al. 2002). In the case of Spalax species, ETAS1 is much more conserved than ETAS2 (25.42% and 40.35% variable sites, respectively), as also found in closely related species of Clethrionomys (Matson and Baker 2001), giving further support to the idea that ETAS2 could be functionally less important or shows a species-specific evolutionary pattern. A similar pattern is observed in the case of CSB blocks, where CSB1 is the least conserved block even though it has been suggested to be the most important from the point of view of functionality, since it is the only one present in all species of mammals examined so far (Sbisà et al. 1997; Matson and Baker 2001; Larizza et al. 2002).
An ETAS1-like and a CSB1-like element have been identified in the ETAS domain (fig. 2). The presence of ETAS1-like elements has been described in different species of mammals and even within rodent species (Sbisà et al. 1997; Larizza et al. 2002). The relatively low degree of dissimilarity with ETAS1 (36.06% in the case of Spalax species) suggests that they could be derived from this element by a slippage event. All four species of Spalax present this ETAS1-like element, and it is quite conserved in all of them, supporting the idea of a single slippage event before the speciation. It is important to note that the position of the ETAS1-like element corresponds with the position where indels have been found. Regarding the CSB1-like element, it shows a low degree of dissimilarity with CSB1 (28.0%) but is located in the ETAS domain. CSB1-like elements have been described in different mammals, always in the CSB domain (Sbisà et al. 1997); only in the case of Clethrionomys species have they been found in the ETAS domain (Matson and Baker 2001). While the most likely hypothesis for their origin is slippage event, the underlying mechanism that led to the location of this CSB1-like element in the ETAS domain remains unclear.
Phylogenetic Content and Pattern of Diversity of the Control Region
Transitions were shown to be at the beginning of saturation with respect to transversions when distantly related populations are involved in the comparison (fig. 5), as previously observed for the control regions of other mammals (e.g., Wills 1995; Douzery and Randi 1997). At the species level, a bias toward transitions over transversions was observed, but important differences were detected in the four Spalax ehrenbergi species, with ratio values ranging from 18 to 1.63 (table 2). This bias has also been observed in other rodent species such as Rattus (Brown et al. 1986) and Clethrionomys (Matson and Baker 2001). Moreover, within transition, we have detected a significant bias toward pyrymidine over purine transitions in most populations that reach a ratio of 6.5 to 1, as in the case of Quneitra (table 2). These results are in agreement with those found in Clethrionomys (Matson and Baker 2001), where a significant bias toward pyrymidine over purine transitions are also observed. However, they differ from the results obtained in Rattus, where no pyrimidine transitional bias is detected (Brown et al. 1986). This different behavior of murid rodents, when compared with other rodent species, has also been observed at the level of change of nucleotide composition in both whole mitochondrial genome (Karlin and Mrazek 1997) and nuclear genes (e.g., Cortopassi and Wang 1996; Gissi et al. 2000; Michaux, Reyes, and Catzeflis 2001).
Despite these differences in the mutational pattern in the different species of mole rats, the analysis of the control region has proven to be suitable for establishing phylogenetic relationships among individuals, populations, and species (fig. 6). According to these figures, populations can be divided into two groups: northern populations belonging to Spalax galili (2n = 52) and S. golani (2n = 54) showing a clear separation of these two species, and southern populations from S. carmeli (2n = 58) and S. judaei (2n = 60) where the degree of speciation is less evident. These results are in agreement with the postulated speciation process for these species, that is, a progressive southward trend of late speciation (Nevo 1991, 1999; Nevo, Ivanitskaya, and Beiles 2001). According to this, northern species represent the oldest stages of speciation, whereas southern species represent more recent stages of speciation. Indeed, we can see that northern populations are nonoverlappingly clustered according to the species they belong to, whereas southern populations are mixed, suggesting a more recent speciation event (fig. 6). These results are also confirmed on the basis of other molecular markers (Catzeflis et al. 1989; Nevo et al. 1993; Nevo, Filippucci, and Beiles 1994; Ben-Shlomo, Fahima, and Nevo 1996; Nevo, Beiles, and Spradling 1999).
According to the neutral theory, older species are expected to show higher levels of genetic variability than younger ones. However, we have found higher nucleotide diversity values, number of haplotypes, and relative variability (table 2, fig. 4) in southern young species (S. carmeli [2n = 58] and S. judaei [2n = 60]) than in northern old species (Spalax galili [2n = 52] and S. golani [2n = 54]). These results are also supported by allozyme, DNA-DNA hybridization, mtDNA RFLPs, DNA minisatellite fingerprinting, RAPD, and cytochrome b sequence data (Catzeflis et al. 1989; Nevo et al. 1993; Nevo, Filippucci, and Beiles 1994; Ben-Shlomo, Fahima, and Nevo 1996; Nevo, Beiles, and Spradling 1999). Therefore, we can say that the observed pattern of distribution of gene variability is nonrandom, and it has been explained as a consequence of the adaptation of the species to ecological factors (Nevo 1991, 1999; Nevo, Ivanitskaya, and Beiles 2001). Aridity and temperature show a clinal distribution in Israel, with higher values in the south than in the north (table 1). Adaptation of Spalax species and populations to these ecologically different environments from the Pleistocene up to the present might have resulted in a north to south trend of variability. Thus, a significant correlation between nucleotide diversity in the control region of Spalax populations and latitude has been found and subsequently with the mean number of rainy days and to some extent with seasonal temperature differences.
This adaptation process has also involved many other variables regarding morphology (skull and body variables) (Nevo, Tchernov, and Beiles 1988; Nevo 1991) and physiology (basic metabolic rates, nonshivering thermogenesis, thermoregulation, respiration, heartbeat and breathing frequency, and hematocrite and hemoglobin concentration) (Nevo and Shkolnik 1974; Arieli et al. 1984; Arieli et al. 1986a, 1986b; Haim et al. 1984; Haim, Heth, and Nevo 1985). All these adaptations would be directed to the optimization of the energetic balance across the geographic range of Spalax species (Nevo 1991, 1999; Nevo, Ivanitskaya, and Beiles 2001). On one hand, adaptation is directed to the reduction of water expenditure and overheating in xeric environments and in environments that are hot during the daytime and the summer. On the other hand, thermoregulation enables better resistance to the cold environment during the winter and the night. Behavioral adaptations, including activity, exploratory, habitat selection, and aggression patterns also contribute to the optimization of the energy. Thus, for example, species living in a cooler climate, S. galili (2n = 52) and S. golani (2n = 54), display less rest periods than those living in warmer climates, that is, S. carmeli (2n = 58) and S. judaei (2n = 60) (Nevo et al. 1982). Aggression intensity also varies geographically and, at least partly, in accordance with aridity and temperature. Aggressive phenotype frequency decreases southward: S. galili (2n = 52) > S. golani (2n = 54) > S. carmeli (2n = 58) > S. judaei (2n = 60) (Nevo 1986; Nevo, Heth, and Beiles 1986).
The importance of adaptation to the clinal distribution of ecological factors found in Israel is also highlighted by the fact that many unrelated species of plants, invertebrates, and vertebrates show the same north to south trend of variability (Nevo 1988). In these cases, southern populations display higher levels of genetic variability. The fact that diverse species sharing similar ecological distribution show a genetic parallelism gives further support to the idea that selection is involved in the genetic differentiation of populations in Israel, including the control region of Spalax mitochondrial genome.
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Acknowledgements |
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Footnotes |
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