Sequence analysis of mitochondrial DNA in Salamandra infraimmaculata larvae from populations in northern Israel

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1 South merican Journal of Herpetology, 4(3), 2009, Brazilian Society of Herpetology Sequence analysis of mitochondrial DN in Salamandra infraimmaculata larvae from populations in northern Israel Tali oldberg 1,2, Oren Pearlson 1,2, Eviatar Nevo 2, ad Degani 1,3 1 School of Science and Technology, Tel Hai cademic College, Upper alilee 12210, Israel. E mail: talig@migal.org.il; orenp@migal.org.il 2 Institute of Evolution and Department of Evolutionary and Environmental Biology, University of Haifa, Israel E mail: nevo@research.haifa.ac.il 3 uthor to whom all correspondence should be addressed. Prof. ad Degani; MIL-alilee Technology Center, P.O. Box 831, Kiryat Shmona 11016, Israel; Tel: ; Fax: E mail: gad@migal.org.il bstract. The molecular DN variation among Salamandra infraimmaculata larvae populations, representing eight breeding sites in Israel, was studied. Samples from larvae were analyzed by sequence analysis of the mitochondrial cytochrome b fragment and D loop regions (enbank accession nos. EU EU852738). neighbor-joining analysis had an optimal arrangement with a branch length sum of The genetic distances, which were computed by the maximum composite likelihood method and which are presented as the number of base substitutions per site, demonstrated that there are two sub-populations. One consists of larvae from unpredictable breeding sites, of which most are winter ponds, and the other one includes populations from perennial water sources, mostly streams and springs. Keywords. genetics; mitochondria; xeric habitat; Salamandra infraimmaculata; adaptation; breeding sites; neighbor joining method. Introduction Salamanders of the genus Salamandra are present throughout Europe, North frica, and East sia, surviving in various habitats and climates (reviewed by Degani, 1996). Based on the morphological data, the species S. salamandra is subdivided into 16 subspecies that can be found throughout Europe, the Middle East, and North frica (Eiselt, 1958; Fachbach, 1976; Degani, 1986; Klewen, 1991). The Salamandra subspecies, which can be differentiated by plasma protein electrophoresis (asser, 1978; Degani, 1986; Joger et al., 1994; Joger et al., 1995) and allozyme data (Veith, 1994), do not show clear morphological differentiation. Previous studies used chromosomes and DN sequences to study the evolution of salamanders belonging to the genus Plethodon (Mizuno and Mac regor, 1974; Mizuno et al., 1976). Based on the sequence analysis of the mitochondrial D loop region and geological dates, Steinfartz et al. (2000) suggested that five major monophyletic groups exist in Europe (S. salamandra, S. infraimmaculata, S. corsica, S. atra, and S. lanzai), one S. algira is located in frica (Escoriza et al., 2006), and the salamanders in Israel belong to S. infraimmaculata. The populations of S. infraimmaculata found in northern Israel are located near breeding sites in three different and isolated areas: (1) Mount Carmel, (2) alilee (Upper alilee and Western alilee), and (3) Hermon Mountain (Veith et al., 1992). There are physiological (Degani, 1981a; Degani, 1981b), morphological and biological differences among these isolated populations of salamanders, which are affected by habitat conditions (Degani et al., 1978; Degani, 1996). Various types of breeding sites are situated in three different regions in Israel (Degani, 1986; Degani, 1996). The relatively drier areas of Mount Carmel and the central alilee have permanent (i.e., springs and streams) and seasonal (i.e., temporal ponds and rain pools) breeding sites (Warburg, 1992; Warburg, 1997; Degani et al., 1999b). In the xeric areas, there are many types of breeding sites, including streams, springs, rock pools, rain pools, and large ponds, where water is available during different time periods and under various conditions. No variation has been found among the populations of these areas regarding dorsal color patterns, serum proteins (Degani, 1986), or in the ten enzyme system with 14 loci (Veith et al., 1992). Salamanders from semi-arid habitats are significantly larger than those from moist habitats (Degani et al., 1999a). The molecular DN variation in salamanders from these two habitats has been studied using randomly amplified polymorphic DN polymerase chain reaction (RPD PCR). RPD PCR found genetic variation between populations in moist and semi-arid habitats, but not between populations in semi-arid habitats. RPD PCR data showed that salamanders from semi-arid habitats

2 oldberg, T. et al. 269 Table 1. Primers used in this study. ene Primer Sequence 5 3 PCR product length D loop of the control region H-12S1-ML CCCCCCCTTT 807 bp L-Pro-ML CCCCCCTTCT cytochrome b L14841 CTTCCTCCCTCTCCTT 361 bp cyt b B2 CCCTCTTTYTTCCTC shared 94% of their bands, whereas comparing populations from semi-arid and those from moist habitat only shared 85 86% (Degani et al., 1999a). However no comparisons have been made to assess the level of genetic variation among populations inhabiting moist habitats. Herein, we examined the genetic variation of mitochondrial cytochrome b and control regions (D loop) segments in salamanders from eight populations that reside in streams, springs, and rain ponds in northern Israel. Sequences were used to study genetic variation among populations of S. infraimmaculata from various moist breeding and semi-arid habitats to assess their genetic variation. Materials and Methods Tissue samples were obtained from ten S. infraimmaculata larvae, collected by dipnet from each breeding site, as described previously by Degani (1982); larvae from eight locations in alilee, northern Israel, are shown in Figure 1. The ephemeral ponds (available between March and July) included Manof Pond a winter pond in ush Segev (elevation 340 m), Dovev Pond (740 m), Matityahu Pond (682 m), and the Maalot Pit (596 m a two-meter deep hole on a hill) (Figure 2). The permanent water bodies are: l Balad Spring (a year-round spring on Mount Carmel), Navuraya Spring (663 m), Humema Spring (900 m, a layer spring in a cave located on Mount Meron), and the permanent Tel Dan Stream (150 m, with yearround water at 16 C) (Figure 2). enomic DN was extracted from clipped or whole tail of larvae with the QIamp DN Mini Kit that consists of proteinase K lysis and specific DN binding to the QIamp silica-gel membrane through which contaminants pass. The primer sets consisted of: L Pro ML and H 12S1 ML (Steinfartz et al., 2000) to amplify (807 base pairs segment) and sequence the D loop of the control region and L14841 and a modified primer cyt b B2 (Kocher et al., 1989) to amplify (361 base pairs segment) and sequence cytochrome b (Table 1). PCR amplification was performed in a 50 μl solution containing 10 mm Tris HCl, 50 mm KCl, 2.5 mm MgCl 2, 0.5 mm of each dntp, 0.5 μm of each primer, ng genomic DN and 2.5 units of Taq DN polymerase (Promega, US). PCR was performed in a PTC 150 MiniCycler (MJ Research, US) as follow: 3 min denaturation at 94 C, followed by 32 cycles of 1 min at 94 C, annealing for 1 min at 52 C, elongation at 72 C for 1 min, and a final 10 min elongation period at 72 C. mplified products were separated by electrophoresis on a 1.3% agarose gel, stained with ethidium bromide, and DN was extracted using Jet Quick el Extracting Spin Kit (enomed, ermany); recovered DN was dissolved in deionized water. Purified DN was sequenced in both directions with an BI PRISM 3100 enetic nalyzer (PE Biosystems, US). The genetic similarity among samples was inferred using the neighbor-joining method (Saitou et al., 1987). enetic distances were computed using the maximum composite likelihood method (Tamura et al., 2004) and are presented as the number of base substitutions per site. rlequin analysis (Schneider et al., 2000) was carried out between and among eight sequences of different populations. Figure 1. Salamander sampling locations:. l Balad Spring, B. Manof Pond, C. Maalot Pit, D. Humema Spring, E. Navuraya Spring, F. Dovev Pond,. Matityahu Pond, H. Tel Dan Stream.

3 270 Figure 2. Salamander sampling sites. Mt-DN in Salamandra infraimmaculata larvae

4 oldberg, T. et al. 271 ll positions containing gaps and missing data were eliminated from the dataset (complete deletion option), with a total of 156 positions in the final dataset Results DN sequences were analyzed from a 361 bp fragment of cytochrome b and an 807 bp fragment of the control region (enbank accession nos. EU EU852738). The cytochrome b fragment varied at position 77, in which the Dovev, Maalot, Tel Dan, and Humema populations had an whereas all other populations had a (Table 2). Three nucleotide sites in the control region fragment, i.e., 24, 459, and 520, varied among locations. The l Balad, Maalot, and Manof populations all had C, C, and T for those positions respectively, the Navuraya, Dovev, Humema, and Tel Dan populations had T, T and C, and the Matityahu population was polymorphic, with C/T in all positions (Table 2; enbank acc. nos. EU EU852738). The D loop sequence of Salamandra infraimmaculata differed from S. corsica by 6.5 7%. The lowest level of genetic differentiation in the control region (0%) was found among individuals inhabiting springs and streams (Humema Spring, Tel Dan Stream, and Navuraya Spring) located in the northern region of the studied area (Table 3). similar situation was found when comparing the variation of cytochrome b among S. infraimmaculata populations. The analysis, using rlequin v. 3.1 (Schneider et al., 2000), of eight sequences revealed that a gene diversity of 1.00 (± ), the mean number of pairwise differences was (± 0.569), and the average gene diversity over loci was (± 0.651). Figure 3. Calculated standard parameters of nucleotide variation among populations. The evolutionary history was inferred using the neighbor-joining method (Saitou et al., 1987). The evolutionary distances were computed using the maximum composite likelihood method (Tamura et al. 2004) and are presented as the number of base substitutions per site. Table 2. Nucleotide differences among populations. Ten larvae were examined from each population. Nucleotide Differences Cytochrome b Nucleotide position 77 l Balad Spring Manof Pond Navuraya Spring Dovev Pond Matityahu Pond Tel Dan Stream Maalot Pit Humema Spring Control region Nucleotide position l Balad Spring C C T Manof Pond C C T Navuraya Spring T T C Dovev Pond T T C Matityahu Pond C/T C/T C/T Tel Dan Stream T T C Maalot Pit C C T Humema Spring T T C In this analysis, there are two sub-clustering of populations, one the Humemas, Navuraya, Dovev, and Tel Dan cluster and a second one consisting of l Balad, Maalot, Manof, and Matityahu populations (Figure 3). Discussion The biological and ecological variation among Salamandra infraimmaculata larvae from different populations in Israel have been described previously, but they are less than the variation found between S. infraimmaculata and European salamanders (Degani, 1996; Steinfartz et al., 2000; Escoriza et al., 2006; Weisrock et al., 2006). enetic variation in S. salamandra has been studied intensively (reviewed by Steinfartz et al., 2000); earlier studies focused on geographical and taxonomical aspects, examining differences between populations (described as subspecies, Degani, 1986). The parameters by which differences among S. salamandra populations are determined, include morphology (Eiselt, 1958; Thorn, 1968; Degani, 1986; Degani, 1994; Veith, 1994; Degani et al., 1995), plasma proteins (Degani, 1986; Joger et al., 1994), and isozyme studies (Nicklas, 1992; Veith et al., 1992; lcobendas et al., 1994; Nicklas et al., 1994). However, these parameters have low

5 272 Mt-DN in Salamandra infraimmaculata larvae Table 3. Percent of identity of the control region (a) and cytochrome b (b). l Balad Dovev Humema Maalot Manof Matityahu Navuraya Tel Dan l Balad * Dovev * * Humema * * * Maalot * * * * Manof * * * * * Matityahu * * * * * * 99.6 Navuraya * * * * * * * B l Balad Dovev Humema Maalot Manof Matityahu Navuraya 1 Tel Dan l Balad * Dovev * * Humema * * * Maalot * * * * Manof * * * * * Matityahu * * * * * * Navuraya * * * * * * * sensitivity in elucidating genetic variation among populations belonging to the same species, in comparison to RPDs (Degani et al., 1999a). Using RPDs genetic differences were found among salamander populations in Israel (Degani et al., 1999a). These results are not in agreement with those of previous studies, in which no variation was found in coloration and allozymes (Veith et al., 1992) among populations from different habitats in Israel. In this study, mitochondrial genes exhibited low variation among populations. However, an rlequin analysis (Schneider et al., 2000) detected high similarity among populations inhabiting permanent breeding sites. Moreover, the population (Dovev Pond) at the highest altitude with conditions comparable to the permanent breeding sites was genetically similar to populations of springs and streams. Patterns of plasma proteins and allozymes are limited to protein variation, so it is difficult to use them to determine genetic variations within populations or between populations of the same species. However, it seems that at least some of the physiological and biological variability is the result of ecological adaptation (Degani, 1994; Degani, 1996). Our study shows differences among S. infraimmaculata within the same geographic region. These results contrast Degani (1986) and Veith et al. (1992) who found that individual populations were completely monomorphic, but agrees with Degani et al. (1999a) who reported genetic variation among populations. eographic distances among the examined sites seem to correlate less with the genetic variation than the ecological conditions in the habitats (Degani, 1996; Degani et al., 1999a). There are many dissimilarities among salamanders from different areas, e.g., body size (Degani, 1986), number of larvae born per parturition (Degani et al., 1995), survival under dry conditions (Degani, 1981a), and reproductive cycle (Sharon et al., 1996; Degani et al., 1997). The eight habitats studied are geographically near to each other, with minor differences in rainfall, photoperiod, or other meteorological conditions. However, the semi-arid conditions in four of them, where the water source dries up during the summer, differ greatly from the moist conditions in the other four sites, where water is available year-round. This difference could account for the biological, morphological, physiological, and possible responsible for the genetic variations we found. We have shown here that although the molecular polymorphisms in mitochondrial DN are small, they reflect a sharp ecological separation between seasonal breeding sites and permanent water sources. These are presumably adaptive changes caused by natural selection. Resumo Nesse estudo a variabilidade genetica entre oito populacoes da Salamandra infraimaculata foi caracterizada atraves do sequenciamnto de DN. sequencia do DN mitocondrial foi analisada nas regioes do citocromo e D loop (numero de acesso

6 oldberg, T. et al. 273 enbank EU EU852738). neighborjoining analysis had an optimal arrangement with a branch length sum of analise demonstrou a existencia de duas sub-populacoes, a primeria encontrada nas pocas de inverno e a segunda encontrada nas pocas de primavera. cknowledgments We would like to acknowledge Dr. Dani Bercovich from the Human Molecular enetics & Pharmacogenetics Dept. of MIL alilee Technology Center and Dr. Liran Shlush from the Laboratory of Molecular Medicine of the Faculty of Medicine in the Technion for their assistance in the analyses of the sequences of the mtdn. Literature Cited lcobendas, M. M., H. Dopazo, and P. lberch enetic structure and differentiation in Salamandra salamandra populations from the northern Iberian peninsula. Mertensiella, 4:7 23. Degani,. 1981a. The adaptation of Salamandra salamandra (L.) from different habitats to terrestrial life. British Journal of Herpetology, 6: Degani,. 1981b. 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