Sequence analySiS of mitochondrial dna in Salamandra infraimmaculata larvae from populationS in northern iSrael

Tali GoldberG1,2, oren Pearlson1,2, eviaTar nevo2, Gad deGani1,3

1 School of Science and technology, tel Hai academic college, upper Galilee 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, israelE‑mail: nevo@research.haifa.ac.il

3 author to whom all correspondence should be addressed. Prof. Gad degani; miGal‑Galilee technology center,P.O. Box 831, Kiryat Shmona 11016, israel; tel: +972‑4‑6953544; fax: +972‑4‑6944980. E‑mail: gad@migal.org.il

absTracT. the molecular dna 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 (GenBank accession nos. eu852723‑eu852738). a neighbor‑joining analysis had an optimal arrangement with a branch length sum of 0.00652338. 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 africa, and east asia, sur‑viving in various habitats and climates (reviewed by degani, 1996). Based on the morphological data, the species S. salamandra is subdivided into 16 subspe‑cies that can be found throughout europe, the middle east, and north africa (eiselt, 1958; fachbach, 1976; degani, 1986; Klewen, 1991).

the Salamandra subspecies, which can be differ‑entiated by plasma protein electrophoresis (Gasser, 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 dna sequences to study the evolution of salamanders be‑longing to the genus Plethodon (mizuno and mac‑Gregor , 1974; mizuno et al., 1976). Based on the sequence analysis of the mitochondrial d‑loop region and geological dates, Steinfartz et al. (2000) sug‑gested that five major monophyletic groups exist in europe (S. salamandra, S. infraimmaculata, S. cor‑sica, S. atra, and S. lanzai), one S. algira is located in africa (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 carm‑el, (2) Galilee (upper Galilee and Western Galilee),

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 breed‑ing sites are situated in three different regions in is‑rael (degani, 1986; degani, 1996). the relatively drier areas of mount carmel and the central Galilee have permanent (i.e., springs and streams) and sea‑sonal (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 dor‑sal 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 sig‑nificantly larger than those from moist habitats (de‑gani et al., 1999a). the molecular dna variation in salamanders from these two habitats has been stud‑ied using randomly amplified polymorphic dna polymerase chain reaction (rapd pcr). rapd pcr found genetic variation between populations in moist and semi‑arid habitats, but not between populations in semi‑arid habitats. rapd pcr data showed that salamanders from semi‑arid habitats

South american Journal of Herpetology, 4(3), 2009, 268‑274© 2009 Brazilian Society of herpetology

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 in‑habiting moist habitats.

herein, we examined the genetic variation of mito‑chondrial 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 vari‑ous moist breeding and semi‑arid habitats to assess their genetic variation.

MaTerials and MeThods

tissue samples were obtained from ten S. infraim‑maculata larvae, collected by dipnet from each breed‑ing site, as described previously by degani (1982); larvae from eight locations in Galilee, northern israel, are shown in figure 1. the ephemeral ponds (avail‑able between march and July) included manof pond – a winter pond in Gush 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: al 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 year‑round water at 16°c) (figure 2).

Genomic dna was extracted from clipped or whole tail of larvae with the qiaamp dna mini Kit that consists of proteinase K lysis and specific dna binding to the qiaamp 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 se‑quence 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 cy‑tochrome b (table 1).

pcr amplification was performed in a 50 μl so‑lution containing 10 mm tris‑hcl, 50 mm Kcl, 2.5 mm mgcl2, 0.5 mm of each dntp, 0.5 μm of each primer, 10‑500 ng genomic dna and 2.5 units of taq dna polymerase (promega, uSa). pcr was performed in a ptc‑150 minicycler (mJ research, uSa) as follow: 3 min denaturation at 94°c, fol‑lowed 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. amplified products were separated by electrophoresis on a 1.3% agarose gel, stained with ethidium bromide, and dna was extracted using Jet quick Gel extracting Spin Kit (Genomed, Germany); recovered dna was dissolved in deionized water. purified dna was sequenced in both directions with an aBi priSm 3100 Genetic analyzer (pe Biosystems, uSa).

the genetic similarity among samples was in‑ferred using the neighbor‑joining method (Saitou et al., 1987). Genetic distances were computed using the maximum composite likelihood method (tamura et al., 2004) and are presented as the number of base substitutions per site. arlequin analysis (Schneider et al., 2000) was carried out between and among eight sequences of different populations.

FiGure 1. Salamander sampling locations: a. al Balad Spring, B. manof pond, c. maalot pit, d. humema Spring, e. navuraya Spring, f. dovev pond, G. matityahu pond, h. tel dan Stream.

Table 1. primers used in this study.

Gene primer Sequence 5’‑3’ pcr product lengthd‑loop of the control region h‑12S1‑ml caaGGccaGGaccaaaccttta 807 bp

l‑pro‑ml GGcacccaaGGccaaaattctcytochrome b l14841 aaaaaGcttccatccaacatctcaGcatGatGaaa 361 bp

cyt b B2 ccctcaGaatGatatytGtcctca

269Goldberg, t. et al.

FiGure 2. Salamander sampling sites.

270 mt‑dna in Salamandra infraimmaculata larvae

all positions containing gaps and missing data were eliminated from the dataset (complete dele‑tion option), with a total of 156 positions in the final dataset

resulTs

dna sequences were analyzed from a 361 bp fragment of cytochrome b and an 807 bp fragment of the control region (GenBank accession nos. eu852723‑eu852738). the cytochrome b fragment varied at position 77, in which the dovev, maalot, tel dan, and humema populations had an a whereas all other populations had a G (table 2). three nucleotide sites in the control region fragment, i.e., 24, 459, and 520, varied among locations. the al Balad, maalot, and manof populations all had c, c, and t for those positions respectively, the navuraya, dovev, hu‑mema, and tel dan populations had t, t and c, and the matityahu population was polymorphic, with c/t in all positions (table 2; GenBank acc. nos. eu852723‑eu852738).

the d‑loop sequence of Salamandra infraimmac‑ulata 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).

a similar situation was found when comparing the variation of cytochrome b among S. infraimmaculata populations. the analysis, using arlequin v. 3.1 (Sch‑neider et al., 2000), of eight sequences revealed that a gene diversity of 1.00 (± 0.0625), the mean number of pairwise differences was 0.678 (± 0.569), and the average gene diversity over loci was 0.678 (± 0.651).

in this analysis, there are two sub‑clustering of popu‑lations, one the humemas, navuraya, dovev, and tel dan cluster and a second one consisting of al 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 (de‑gani, 1996; Steinfartz et al., 2000; escoriza et al., 2006; Weisrock et al., 2006). Genetic 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 sub‑species, degani, 1986). the parameters by which differences among S. salamandra populations are de‑termined, include morphology (eiselt, 1958; thorn, 1968; degani, 1986; degani, 1994; veith, 1994; de‑gani et al., 1995), plasma proteins (degani, 1986; Jo‑ger et al., 1994), and isozyme studies (nicklas, 1992; veith et al., 1992; alcobendas et al., 1994; nicklas et al., 1994). however, these parameters have low

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 evolu‑tionary 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 differencescytochrome b

nucleotide position 77al Balad Spring Gmanof pond Gnavuraya Spring Gdovev pond amatityahu pond Gtel dan Stream amaalot pit ahumema Spring a

control regionnucleotide position 24 459 520al Balad Spring c c tmanof pond c c tnavuraya Spring t t cdovev pond t t cmatityahu pond c/t c/t c/ttel dan Stream t t cmaalot pit c c thumema Spring t t c

271Goldberg, t. et al.

sensitivity in elucidating genetic variation among populations belonging to the same species, in com‑parison to rapds (degani et al., 1999a).

using rapds 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 vari‑ation among populations. however, an arlequin anal‑ysis (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 lim‑ited 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 bio‑logical variability is the result of ecological adapta‑tion (degani, 1994; degani, 1996). our study shows differences among S. infraimmaculata within the same geographic region. these results contrast de‑gani (1986) and veith et al. (1992) who found that in‑dividual populations were completely monomorphic, but agrees with degani et al. (1999a) who reported genetic variation among populations. Geographic distances among the examined sites seem to corre‑late 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 (de‑gani, 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 molecu‑lar polymorphisms in mitochondrial dna 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 dna. a sequencia do dna mitocondrial foi analisada nas regioes do citocromo e d‑loop (numero de acesso

Table 3. percent of identity of the control region (a) and cytochrome b (b).

a al Balad dovev humema maalot manof matityahu navurayatel dan 99.6 100 100 99.6 99.6 99.6 100al Balad * 99.6 99.6 100 100 99.6 99.6dovev * * 100 99.6 99.6 99.6 100humema * * * 99.6 99.6 99.6 100maalot * * * * 100 99.6 99.6manof * * * * * 99.6 99.6matityahu * * * * * * 99.6navuraya * * * * * * *B al Balad dovev humema maalot manof matityahu navuraya1 tel dan 99.7 100 100 99.7 99.7 99.7 1002 al Balad * 99.7 99.7 100 100 100 99.73 dovev * * 100 99.7 99.7 99.7 1004 humema * * * 99.7 99.7 99.7 1005 maalot * * * * 100 100 99.76 manof * * * * * 100 99.77 matityahu * * * * * * 99.78 navuraya * * * * * * *

272 mt‑dna in Salamandra infraimmaculata larvae

GenBank eu852723‑eu852738). a neighbor‑joining analysis had an optimal arrangement with a branch length sum of 0.00652338. a analise demonstrou a existencia de duas sub‑populacoes, a primeria encontrada nas pocas de inverno e a segunda encontrada nas pocas de primavera.

acKnowledGMenTs

We would like to acknowledge dr. dani Bercovich from the human molecular Genetics & pharmacogenetics dept. of miGal – Galilee 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 mtdna.

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Submitted 26 february 2009 accepted 09 September 2009

274 mt‑dna in Salamandra infraimmaculata larvae