Phylogeography of an Italian endemic salamander (genus Salamandrina): glacial refugia, postglacial expansions, and secondary contact

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1 Biological Journal of the Linnean Society, 2011, 104, With 4 figures Phylogeography of an Italian endemic salamander (genus Salamandrina): glacial refugia, postglacial expansions, and secondary contact MARCO MATTOCCIA*, SILVIO MARTA, ANTONIO ROMANO and VALERIO SBORDONI Department of Biology, University of Tor Vergata, Via Cracovia 1, Rome, Italy Received 3 June 2011; accepted for publication 17 June 2011bij_1747 The Italian endemic genus Salamandrina has been historically regarded as monotypic but, recently, studies based on both mitochondrial and nuclear markers have indicated the existence of two distinct species of spectacled salamanders: Salamandrina perspicillata, in central and northern Italy, and Salamandrina terdigitata, in southern Italy. We analyzed nucleotide variation at mitochondrial and nuclear genes [cytochrome b, 12S and 16S rrna, recombination activating gene (RAG 1)] in 223 individuals from 56 locations, aiming to investigate their genetic structure and recent evolutionary histories. Phylogenetic and phylogeographical analyses revealed the existence of three and two genetically distinct groups of populations in northern and southern salamander, respectively. Historical demographic analyses led to the inference of range expansion for both species in the late Pleistocene. During the last glacial stage, each salamander survived in a single refugium, namely the southern in Calabria and the northern in central Italy. At the end of this period, both lineages expanded northward and established secondary contact. Spatial distribution of RAG 1 haplotype variation revealed two differentiated population groups corresponding to the major mitochondrial (mt)dna clades. Nuclear pattern of introgressive hybridization was more extensive than the highly limited introgression of mtdna markers. From a conservation standpoint, southern Latium and Calabria proved to be the major genetic diversity reservoirs, thus deserving particular conservation efforts The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 104, ADDITIONAL KEYWORDS: Amphibians cytonuclar discordance hybridization mtdna nucdna. INTRODUCTION The phylogeographical pattern of a species is the result of the combined influence of contemporary and historical processes. Several studies have documented how the climatic oscillations of the Pleistocene have played a major role in determining the present range and genetic diversity of temperate species of animals and plants (Hewitt, 2000; Schmitt, 2007). The Iberian, Italian, and Balkan Peninsulas are assumed to have been the three main refugial areas in Europe because of their species richness and considerable level of endemism (Hewitt, 1996, 2000; Taberlet et al., 1998; Weiss & Ferrand, 2007). Allopatric fragmentation in isolated southern refugia *Corresponding author. marco.mattoccia@uniroma2.it brought about genetic differentiation among and within species. The number and origin of expanding populations, as well as the routes followed during the recolonization of northern regions, determined the phylogeographical pattern of the current species and the shaping of hybrid zones between distinct lineages or closely-related species (Remington, 1968; Hewitt, 2000). According to this scenario, the higher genetic diversity of southern populations of widespread species is explained in terms of their longer persistence in the refugial areas, whereas the homogeneity of northern populations is probably a result of the loss of genetic variation resulting from rapid recolonization. Nevertheless, a growing number of data have highlighted an even more composite picture of the different modes of both persistence and postglacial recolonization in a variety of European 903

2 904 M. MATTOCCIA ET AL. species. Additional refugial areas were identified in central and eastern Europe (Babik et al., 2004; Ursenbacher et al., 2006; Sotiropoulos et al., 2007; Provan & Bennett, 2008). The discovery of much cryptic diversity within several species or sibling species complexes revealed that the Iberian Peninsula consisted of multiple isolated glacial refugia ( refugia-withinrefugia ; Gómez & Lunt, 2007). It is now wellestablished that many taxa of Iberian flora and fauna consist of several genetically distinct lineages kept isolated in separate refugial areas through the Quaternary, which then expanded frequently until they formed secondary contact (Martínez-Solano et al., 2006; Gómez & Lunt, 2007). Amphibians are considered as a suitable model for studying historical processes associated with Pleistocene climatic oscillations. Indeed, they are poikilotherms and thus extremely sensitive to any drop in temperature. Furthermore, they are often phylogeographically deeply structured owing to their low dispersal capacities and strict habitat requirements (Sequeira et al., 2008; Gonçalves et al., 2009). The genus Salamandrina (Fitzinger, 1826) is an important Italian endemism, and it appears to be very divergent from the other salamandrid salamanders (Steinfartz et al., 2007; Zhang et al., 2008). The current range of the spectacled salamander is restricted to the Apennines, from Liguria to the tip of Calabria, although its distribution was more ample in the past. Fossil records ascribed to the genus Salamandrina and dating back to the Miocene and Pliocene, were found in Sardinia and Greece (Sanchíz & Młynarski, 1979), as well as Germany and Spain (Böhme & Ilg, 2003). Salamandrina has been historically considered a monotypic genus, although recent studies based on the analysis of both mitochondrial (mt)dna sequences (Mattoccia, Romano & Sbordoni, 2005; Nascetti, Zangari & Canestrelli, 2005) and allozymic loci (Canestrelli, Zangari & Nascetti, 2006b) have supported the existence of two genetically distinct lineages, which were considered as two cryptic species (Canestrelli et al., 2006b): the centralnorthern lineage was named as Salamandrina perspicillata (Savi, 1821; SPER), whereas the name Salamandrina terdigitata (Bonnaterre, 1789; STER) was retained for the southern Italian populations. The split between the two species has been dated to the upper Miocene, from 11 to 5.3 Mya, depending on the markers and molecular rates adopted (Mattoccia et al., 2005; Nascetti et al., 2005). These studies, however, were based on a relatively scanty sampling, and are certainly not sufficient to outline both the reciprocal distribution of the two species and their pattern of phylogeographical structuring. A thorough account on the distribution of both species has recently been provided by Romano et al along with the first description of morphological differences between the two taxa. SPER ranges from Liguria to northern Campania region, whereas STER inhabits the remainder of Campania, Basilicata, and Calabria region. Based on the analysis of mtdna markers, the distribution of the two lineages of spectacled salamanders proved to be clearly parapatric. However, a contact zone between them was confirmed by the sympatric occurrence of haplotypes of the two taxa in a restricted area in northern Campania (Romano et al., 2009). The present study analyzed spatial variation of mtdna sequences, aiming to investigate the genetic structure within each species, as well as attempting to trace the recent demographic and phylogeographical history of the genus Salamandrina in Italy. Moreover, we explored the pattern of variation at a fragment of the recombination activating gene (RAG 1) aiming to evaluate whether nuclear data are congruent with the genetic differentiation, geographical limits, and genetic structuring of the lineages as defined by mtdna sequences. A further goal, using the nuclear marker, was to verify the existence of hybridization within the contact zone. MATERIAL AND METHODS SAMPLE COLLECTION A total of 223 individuals belonging to Salamandrina were sampled from 56 locations covering the whole distribution range of the genus. Sampling locations and numbers of individuals collected are reported in Table 1. All tissue samples were obtained from live animals by removing approximately 2 mm 3 from the tail tip (Arntzen, Smithson & Oldham, 1999). Tissue samples were stored in 95% ethanol. Sampling was carried out under authorization of the Italian Ministero dell Ambiente e della Tutela del Territorio (permit DPN/2D/2004/17393). DNA EXTRACTION, POLYMERASE CHAIN REACTION (PCR) AMPLIFICATION, AND SEQUENCING For each specimen 12S, 16S, cytochrome b (cyt b), and RAG1 fragments (370, 552, 423, and 461 bp, respectively) were amplified. Conditions for DNA extractions, PCR amplifications and sequencing of mtdna genes were conducted sensu Mattoccia et al. (2005). Genbank accession numbers of mitochondrial sequences used are presented in Table A1 of the Appendix. We amplified RAG 1 with PCR using primers RAG1-C (Frippiat et al., 2001) and RAG1-D: 5 -GCA AGC CCC TGT GCC TCA TGC-3 (modified from RAG1F4; Evans et al., 2005). PCR products were sequenced in the same fashion as mtdna fragments.

3 PHYLOGEOGRAPHY OF SALAMANDRINA 905 Table 1. Details of the sampling sites, mitochondrial (mt)dna and nuclear haplotypes detected, mtdna population grouping, and sample sizes (N) in each locality for the genus Salamandrina Mithocondrial features Nuclear features Population codes Sampling site N Occurrence and code (in brackets) of haplotypes SAMOVA Population Grouping N Occurrence and code (in brackets) of haplotypes 1 LIG Liguria, GE, Genova 5 5(h1) NOR 5 4(a1), 3(a2), 3(a3) 2 LOR Liguria, GE, Lorsica 3 3(h1) NOR 3 2(a1), 2(a2), 2(a3) 3 BOL Emilia Romagna, BO, Casalecchio di R. 5 5(h1) NOR 5 4(a1), 4(a2), 2(a3) 4 BAG Emilia Romagna, FO, Bagno di Romagna 4 4(h1) NOR 4 3(a1), 2(a2), 3(a3) 5 PEN Tuscany, LU, Stazzema 4 3(h1), 1(h2) NOR 2 3(a1), 1(a2) 6 STZ Tuscany, LU, Stazzema 5 5(h1) NOR 5 7(a1), 1(a2), 2(a3) 7 VLN Marche, MC, Visso 2 2(h1) NOR 2 1(a2), 3(a3) 8 VIT Latium, VT, Canino 2 2(h1) NOR 2 1(a2), 3(a3) 9 PGA Umbria, PG, Piegaro 3 3(h1) NOR 2 1(a1), 3(a3) 10 UMB Umbria, TR, Terni 6 6(h4) CEN 5 6(a1), 1(a2), 3(a3) 11 TAN Latium, RI, Poggio Catino 5 4(h1), 1(h4) NOR 2 3(a1), 1(a3) 12 CES Latium, RM, Cesano 2 1(h1), 1(h4) CEN 2 2(a1), 1(a2), 1(a3) 13 SIM Latium, RM, Jenne 3 3(h3) CEN 2 3(a1), 1(a3) 14 FOS Abruzzi, CH, Fara San Martino 2 2(h3) CEN 2 1(a1), 1(a2), 2(a3) 15 PIZ Abruzzi, CH, Palena 2 2(h3) CEN 2 2(a1), 2(a3) 16 PRE Latium, RM, Ciciliano 4 4(h3) CEN 4 3(a1), 1(a2), 4(a3) 17 ROS Abruzzi, CH, Rosello 6 6(h3) CEN 6 9(a1), 1(a3), 2(a6) 18 CAS Molise, CB, Casacalenda 6 6(h3) CEN 6 6(a1), 5(a5), 1(a6) 19 INF Campania, CE, Castello del Matese 6 5(h3), 1(h6) CEN 6 10(a1), 2(a5), 2(a6) 20 VLT Apulia, FG, San Marco La Catola 7 7(h3) CEN 7 14(a1) 21 BAR Campania, BN, S. Bartolomeo in Galdo 8 7(h8), 1(h7) CEN 8 13(a1), 3(a3) 22 GUA Molise, CB, Guardiaregia 6 5(h3), 1(h7) CEN 6 8(a1), 2(a5), 2(a6) 23 FAG Molise, CB, Guardiaregia 1 1(h3) CEN 1 1(a5), 1(a6) 24 QUI Molise, CB, Guardiaregia 2 2(h3) CEN 2 4(a1) 25 ROC Latium, LT, Roccamassima 4 3(h3), 1(h1) CEN 4 2(a1), 2(a2), 4(a3) 26 TOR Latium, LT, Cisterna di Latina 7 7(h1) NOR 5 2(a1), 7(a2), 1(a3) 27 NRM Latium, LT, Norma 3 3(h3) CEN 2 3(a1), 1(a2) 28 GOR Latium, RM, Gorga 3 2(h12), 1(h3) VOL 3 2(a1), 4(a2) 29 LOM Latium, RM, Gorga 6 2(h12), 2(h14), 1(h13), 1(h3) VOL 7 5(a1), 3(a2), 6(a3) 30 SUP Latium, FR, Supino 4 4(h12) VOL 3 3(a1), 1(a2), 2(a3) 31 CAC Latium, LT, Patrica 3 3(h12) VOL 3 2(a1), 2(a2), 2(a3) 32 ERA Latium, LT, Roccagorga 2 2(h12) VOL 2 3(a1), 1(a3) 33 VPR Latium, LT, Maenza 3 3(h12) VOL 3 2(a1), 4(a2) 34 AUS Latium, LT, Monte San Biagio 3 2(h12), 1(h15) VOL 2 1(a1), 3(a3) 35 FIC Latium, LT, Monte San Biagio 2 2(h3) CEN 3 2(a1), 1(a2), 3(a3)

4 906 M. MATTOCCIA ET AL. Table 1. Continued Mithocondrial features Nuclear features Population codes Sampling site N Occurrence and code (in brackets) of haplotypes SAMOVA Population Grouping N Occurrence and code (in brackets) of haplotypes 36 AUR Latium, LT, Itri 5 3(h5), 2(h12) VOL 4 1(a1), 1(a2), 6(a3) 37 ESP Latium, FR, Esperia 1 1(h12) CEN 1 1(a2), 1(a3) 38 CHI Latium, FR, S. Biagio Saracinisco 2 2(h3) CEN 2 2(a1), 2(a4) 39 PET Campania, CE, Mondragone 7 4(h9), 2(h3), 1(h10) CEN 7 2(a1), 4(a2), 8(a3) 40 POZ Campania, CE, Caserta 5 4(h3), 1(h11) CEN 5 7(a3), 1(a5), 2(a6) 41 LAV Campania, BN, Cusano Mutri 5 3(h3), 2(h20) 5 4(a1), 3(a3), 1(a5), 2(a6) 42 MOR Campania, BN, Morcone 6 4(h20), 2(h3) 8 11(a1), 1(a3), 2(a5), 2(a6) 43 BUO Campania, BN, Buonalbergo 4 4(h20) BAC 6 8(a1), 2(a3), 2(a6) 44 PAR Campania, AV, S. Martino Valle Caudina 4 4(h20) BAC 4 2(a5), 6(a6) 45 PIC Campania, SA, Acerno 1 1(h20) BAC 1 2(a6) 46 SAL Campania, SA, Cava de tirreni 4 4(h20) BAC 6 1(a1), 2(a5), 9(a6) 47 TRF Basilicata, PZ, Rionero in Vulture 3 3(h20) BAC 5 4(a5), 6(a6) 48 RIN Basilicata, PZ, Rionero in Vulture 2 2(h20) BAC 1 2(a6) 49 SCA Basilicata, MT, Accettura 2 2(h20) BAC 2 4(a6) 50 CIR Basilicata, MT, Cirigliano 1 1(h20) BAC 1 1(a5), 1(a6) 51 TLT Basilicata, PZ, Calvello 2 2(h20) BAC 2 4(a6) 52 POL Basilicata, PZ, San Severino Lucano 5 5(h20) BAC 4 1(a5), 7(a6) 53 SOS Calabria, CS, San Sosti 4 3(h16), 1(h17) CAL 4 4(a5), 3(a6), 1(a7) 54 SER Calabria, VV, Serra San Bruno 3 1(h16), 1(h18), 1(h19) CAL 2 1(a5), 3(a6) 55 LUC Calabria, RC, San Luca 5 5(h16) CAL 4 5(a5), 3(a6) 56 ASP Calabria, RC, Samo 3 3(h16) CAL 3 6(a5), 2(a6) Total N SAMOVA, Spatial analysis of molecular variance.

5 PHYLOGEOGRAPHY OF SALAMANDRINA 907 MITOCHONDRIAL DATA Genetic diversity, phylogeographical, and demographic analyses Estimates of haplotype (h) and nucleotide diversity (p) (Nei, 1987) were calculated within each lineage using DNASP, version (Librado & Rozas, 2009). Intraspecific gene genealogies were inferred using TCS, version 1.21 (Clement, Posada & Crandall, 2000), which implements the statistical parsimony method of Templeton, Crandall & Sing (1992). Connections among haplotypes within the network are justified by 95% parsimony criterion. Nested clade analysis (NCA; Templeton, Routman & Phillips, 1995) was conducted to explore relationships between geographical and genetic structuring and to infer the population history of spectacled salamanders. Nesting design was constructed by hand in accordance with the nesting procedures of Templeton, Boerwinkle & Sing (1987). The statistical analyses of the associations between geographical and genetic variation were conducted using GEODIS, version 2.6 (Posada, Crandall & Templeton, 2000) with permutations. Significant values obtained from GEODIS were then interpreted using Templeton s inference key ( pdf). Several recent studies have debated about the reliability of phylogeographical interpretations from NCA (Knowles & Maddison, 2002; Panchal & Beaumont, 2007; Garrick et al., 2008; Petit, 2008; Templeton, 2008). However, NCA still proves to be a useful tool for relating population structure to population history. Nevertheless, taking into account the criticisms of NCA, we employed several other methods to minimize the possibility of incorrect inferences. Population genetic structure was assessed by performing the spatial analysis of molecular variance (SAMOVA, version 1.0; Dupanloup, Schneider & Excoffier, 2002). Populations were partitioned into three, four, five, and six groups to maximize the among-groups variation (F ct). The statistical significance of the variance components was computed by permutations. Moreover, to exclude the effect of a scanty size of some samples on the performances of the SAMOVA, we first clustered localities closer than 10 km into single samples, which were spatially defined by their centroids. Subsequently, we excluded from the dataset all of the resulting localities with a sample size smaller than four individuals and reran the analysis. DNASP was used to calculate the nearestneighbour statistic, Snn (Hudson, 2000) with permutations. This statistic measures how often the nearest neighbours in sequence space are from the same location in geographical space. Tajima s D (Tajima, 1989) and Fu s F S (Fu, 1997) tests were calculated to infer the historical demography of each lineage using ARLEQUIN, version 3.1 (Schneider, Roessli & Excoffier, 2000). In the absence of selection, negative values of these tests can be interpreted as signatures of expanding populations (Rogers, 1995). Fu s and Li s D* and F* (Fu & Li, 1993) were computed using DNASP with permutations to determine whether significant departures of Fu s F S test from 0 were caused by selection or population expansion (Fu, 1997). The observed distributions of pairwise differences between mtdna haplotypes were calculated and tested against the demographic expansion model of Rogers & Harpending (1992) with DNASP. Long-term stable populations are expected to have multimodal mismatch distributions, whereas those expanded in the past are predicted to have unimodal distributions (Rogers & Harpending, 1992). The sum of square deviations between the observed and expected mismatch (Schneider & Excoffier, 1999) and the raggedness index (r; Harpending, 1994) were computed with ARLEQUIN to assess the significance of the distribution s fit to that of an expanding population. The population expansion timing was determined using the equation t=2ut (u = the mutation rate per generation over the fragment assayed, t = time in generations; Rogers & Harpending, 1992). Parameter t and confidence intervals were calculated by parametric bootstrap ( replicates) with ARLEQUIN. Because no calibration of the molecular clock specific for Salamandrina is available, we utilized two different mutation rates, as used previously in Mattoccia et al. (2005). These derived from calibrations made for other salamandrids, based on the split of Euproctus montanus from Euproctus platycephalus (Caccone et al., 1997) and on the split of Pleurodeles poireti and Pleurodeles waltl (Carranza & Arnold, 2004), respectively. Genetic distances between the congeneric species of Euproctus and Pleurodeles were calculated from the sequences available in GenBank homologous to the gene regions used in the present study, and corrected according to the Tamura Nei substitution model (Tamura & Nei, 1993) chosen by MODELTEST. The recalculated evolutionary rates were in the range mutations/site/myr for 12S and 16S and in the range mutations/site/myr for cyt b. The demographic history of each lineage was also inferred by estimating the effective population size through time using the Bayesian Skyline Plot (BSP; Drummond et al., 2005), which is a Bayesian coalescent approach as implemented in BEAST, version (Drummond & Rambaut, 2006). A posterior distribution of the effective population size through time was estimated using the Markov chain Monte Carlo

6 908 M. MATTOCCIA ET AL. analysis, with parameters sampled every 1000 iterations over a total of 60 million generations. The first eight million steps were discarded as burn-in. TRACER, version 1.3 (Rambaut & Drummond, 2004) was used to check adequate sampling and convergence to the stationary distribution and to create BSP. To infer the expansion time and the age of the most recent common ancestor (tmrca) the aforesaid mutation rates were employed. The BSPs for each species and for single clades was performed under a HKY model of nucleotide substitution (Hasegawa, Kishino & Yano, 1985), which was selected using MODELTEST (Posada & Crandall, 1998) according to the Akaike information criterion. The age of tmrca of the two species was estimated under a GTR+G model chosen by MODELTEST for the data set including both salamanders. The results obtained with the two different substitution rates were combined. NUCLEAR DATA Diploid amplified sequences of RAG 1 fragments were separated into two haplotypes (GenBank accession nos. JN to JN695274). Haplotypes were determined both through haplotype subtraction (Clark, 1990) and using the haplotype reconstruction algorithms provided by PHASE (STEPHENS,SMITH &DON- NELLY, 2001), as implemented in DNASP. The latter software was also used to estimate the minimum number of recombination events (R m; Hudson & Kaplan, 1985) in the history of the samples. A minimum spanning network was performed with TCS (95% parsimony criterion). RESULTS MITOCHONDRIAL DATA Genetic diversity Of the 20 mtdna haplotypes identified, 15 were found in S. perspicillata (SPER) and five in S. terdigitata (STER). Within SPER, ten unique haplotypes were found at single locations, whereas five haplotypes were shared among different localities. Within STER, four haplotypes were exclusive to Calabrian populations (Ca) and only one widespread haplotype was found in the remaining part of the range (Table 1). The average estimates of haplotype and nucleotide diversity were almost twice as large in SPER (h = , p = ) as in STER (h = , p = ). Overall, the average numbers of nucleotide differences per site were extremely low in both lineages. Most populations had a single haplotype, whereas the population with the highest gene diversity (h = ) occurs in southern Latium (LOM). Phylogeographical analysis Geographical distribution of haplotypes is reported in Figure 1. The statistical parsimony analysis sorted haplotypes from the two species into independent networks, shown in Figure 2. Seventy-nine mutational steps are required to link SPER to STER). The network of SPER displayed a star-like pattern of relationship with a central haplotype, at higher frequency, surrounded by several other, less frequent, haplotypes that were only one or two mutational steps away. The haplotypes h3 and h16 had the highest outgroup weights (0.421 and 0.726) and are indicated as ancestral for SPER and STER, respectively. According to the reduced number of haplotypes and, primarily, the differences among haplotypes, the nesting design structure of NCA (Templeton et al., 1995) was kept very simple (Fig. 2). NCA detected only two nesting levels in SPER and a single clade in STER. Within the SPER species, three clades can be distinguished: 1 1 (N), including haplotypes h1 h2 from the northern and central range of SPER (Liguria, Emilia Romagna, Tuscany, Marche, Umbria, northern Latium); 1 2 (Ce), embracing all haplotypes (h3 h11) from the southern portion of the lineage range (southern Umbria, Latium, Molise, Abruzzi, northern Campania, Apulia); and 1 3, which clusters haplotypes h12 h15 (V) from a small group of populations all localized in the Volsci chain (south-western Latium; Lepini Mts, Ausoni Mts, Aurunci Mts). The most common haplotypes in the different clades were: h1, fixed in large part of the centralnorthern Italy; the ancestral h3, amply widespread in central Italy; and h12, confined in the inland area of Volsci chain. Furthermore, in the clade 1 2, the haplotype h4 was found only in northern Latium (TAN, CES) and in a close population from Umbria (UMB), whereas h8 and h9 were distinctive of the southeastern (BAR) and southwestern (PET) boundaries of the lineage range, respectively (Table 1). The haplotypes of STER were all connected in a single group (clade 1 4); the ancestral haplotype h16 was the most common in Calabria, whereas h20 was the sole haplotype discovered in Campania and Basilicata. In the SPER species, the NCA showed a significant association between haplotype nesting and geographical location at two clade levels: clade 1 2 and the entire cladogram (P < 0.001). Some tip haplotypes were more widespread geographically than the ancestral haplotypes (Table 2), as expected for a population that has undergone a range expansion. Using Templeton s inference key, contiguous range expansion was inferred for the entire cladogram, whereas past gradual range expansion followed by fragmentation was the most likely explanation for the pattern observed in clade 1 2. For STER (clade 1 4), the geographical distribution of haplotypes was

7 PHYLOGEOGRAPHY OF SALAMANDRINA 909 N Ce S. perspicillata V Syntopic population 1 Li La 14 Ab Em 23 6 Mo Tu La 43 9 Um Ma Ap 7 Ab h20 Ca 46 Mo S. terdigitata 47 Ap Cp 50 Ba Cl Cp Km Figure 1. Map of Italy showing the geographical distribution of the different mitochondrial DNA haplogroups, as detected by phylogenetic and phylogeographical analyses for the genus Salamandrina. N, Ce, and V represent haplotype groups found in central-northern Italy, central Italy, and Volsci chain populations, respectively; Ca represents the haplotype group found in Calabria. The geographical distribution of the sole haplotype h20 is shown in green. Numbers identify the populations, as listed in Table 1. Li, Liguria; Em, Emilia Romagna; Tu, Tuscany; Um, Umbria; Ma, Marche; La, Latium; Ab, Abruzzi; Mo, Molise; Cp, Campania; Ap, Abulia; Ba, Basilicata; Cl, Calabria.

8 910 M. MATTOCCIA ET AL. h2 Clade 1-1 h17 h18 h16 Clade 1-4 h19 h1 Clade 1-2 h7 h8 h20 h6 h5 h4 h13 h15 h12 h11 h14 h3 Clade 1-3 S. perspicillata h9 h10 Clade 2-1 S. terdigitata Figure 2. Nested design constructed on maximum parsimony haplotype networks of STER and SPER. Each line represents a single nucleotide substitution, whereas each circle corresponds to one observed haplotype. Haplotypes are identified by the same codes as in Table 1 and sizes of circles are proportional to the haplotype frequency. not significantly different (P = 0.073) from random expectations. The genetic structuring of populations was further supported by SAMOVA (Dupanloup et al., 2002). Because the spatial analysis of molecular variance lead to similar F ct values, considering both clustered and unclustered localities (SPER, three groups, unclustered: F ct = 0.664; SPER, three groups, clustered: F ct = 0.598; STER, two groups, unclustered: F ct = 0.893; STER, two groups, clustered: F ct = 0.945), we report uniquely the results obtained when analyzing the complete dataset. By means of SAMOVA, we tested different hierarchical arrangements of SPER populations (Table 3). When populations were partitioned into three groups (NOR, CEN, VOL), these corresponded to the three haplogroups (N, Ce, V) as in the NCA analysis. By contrast, UMB plus CES, BAR, and PET were kept separate from CEN in the other analytical schemes adopted (four, five, and six groups, respectively). UMB, CES, BAR, and PET have unique haplotypes and are located at the edges of distribution of the central group (CEN). The among-groups variation was maximized (F ct = 0.773; P < 0.001) with six groups of populations. Only 1.05% of variance was a result of differences among populations within these groups. Whatever the partitioning pattern, estimates of genetic diversity revealed marked differences among the three main population groups (Table 4). The northern populations were clearly the least variable, whereas those inhabiting the Volsci chain were the most heterogeneous. This is plain especially considering the six-group pooling scheme, when UMB, CES, BAR, and PET were excluded from the central population group (CEN*). The diversity of VOL was a noteworthy finding, when considering that this group is confined to a small area of southern Latium. SAMOVA revealed significant genetic subdivision also across STER populations (F ct = 0.893, F st = 0.885, P < 0.001) detecting two groups: the first included Calabrian populations (CAL), with four haplotypes, whereas the other (BAC), monomorphic for a unique haplotype, included all of the remaining sampling locations (Tables 3, 4). The results for the Snn (Hudson, 2000) also confirmed the significant association between the mtdna sequence divergence and the geographical location of the different population groups of SPER (Snn = 0.942, P < for the three-group pooling scheme and Snn = 0.901, P < for six groups) and of STER (Snn = 1, P < 0.001). Demographic analysis The observed mismatch distributions for both species were unimodal, as expected in the case of a population expansion (curves not shown; results summarized in Table 5). Similar results were

9 PHYLOGEOGRAPHY OF SALAMANDRINA 911 Table 2. Results of nested clade analysis of mitochondrial DNA haplotypes within Salamandrina Haplotypes One-step clades Two-step clades Clade Dc Dn Clade Dc Dn Clade h h L I-T h S S h S L h h h S S S h h S h h S I-T L S h h h S S h I-T I-T S S h S L h h h h S S I-T L The nested design is given in Fig. 2, as are the haplotype and clade designations. For any nested clade, Dc (clade distances) and Dn (nested clade distances) are reported. Within each nested group, the differences for Dc and Dn between interior and tip clades are indicated by I-T. Bold values with a superscript L and S indicate distance measures significantly larger or smaller than those expected when haplotype distribution is random. Table 3. Spatial analysis of molecular variance of the mitochondrial haplotypes found in Salamandrina populations Percentage of variation Among groups Among populations within groups Fixation indices Within populations F sc F st F ct Salamandrina perspicillata NOR-VOL-CEN NOR-VOL-CEN*-UMB, CES NOR-VOL-CEN*-UMB, CES-BAR NOR-VOL-CEN*-UMB, CES-BAR-PET Salamandrina terdigitata CAL-BAC The genetic structure of S. perspicillata was examined clustering the samples in three, four, five, and six geographical groupings. P-values calculated using simulations were all significant at the 99% level.

10 912 M. MATTOCCIA ET AL. Table 4. Mitochondrial DNA diversity statistics for the population groups of Salamandrina Salamandrina perspicillata Salamandrina terdigitata NOR VOL CEN CEN* BAC CAL Number of haplotypes (h) Haplotype diversity (H D) Nucleotide diversity (p) CEN* corresponds to the CEN group excluding UMB with CES, BAR, and PET as detected in the six population groupings by spatial analysis of molecular variance. Table 5. Results of neutrality tests and mismatch analysis for population groups of Salamandrina Salamandrina perspicillata CEN VOL CAL obtained when the analysis was run using separated groups. Because the northern populations of SPER (NOR) and STER populations from Campania and Basilicata (BAC) were poorly variable, they were not considered. Results of neutrality tests are also reported in Table 5. CEN and CAL exhibited significant negative D (Tajima, 1989) and F S (Fu, 1997) values, whereas, for VOL, only the F S value was significant. F* and D* (Fu & Li, 1993) values were not statistically significant, indicating that the departure of the F s values from 0 was a result of population expansion rather than selection (Fu, 1997). Salamandrina terdigitata Tajima s D P Fu s F S P Fu and Li s F* P 0.10 > P > 0.05 P > > P > 0.05 Fu and Li s D* P 0.10 > P > 0.05 P > > P > 0.05 t 0.6 ( ) 1.1 ( ) ( ) SSD P SSD r P r T A ( ) ( ) ( ) T B ( ) ( ) ( ) t [95% confidence interval (CI)] is the time of population expansion estimated in mutational units; SSD is the sum of square deviations between the observed and expected mismatch; r is the raggedness index; P SSD and P r measure the probability of deviation from a hypothesis of sudden expansion according to the mismatch distribution and raggedness index, respectively. T A and T B (95% CI) are the times of population expansion estimated in years, assuming two different divergence rates: the first (A) based on the split of Euproctus montanus from Euproctus platycephalus (Caccone et al., 1997), the second (B) on the split between Pleurodeles poireti and Pleurodeles waltl (Carranza & Arnold, 2004) The expansion timing for each population group is reported in Table 5. Confidence intervals of these datings were rather large. The BSPs for the single population groups (CEN, VOL, and CAL) showed a moderate demographic expansion (data not shown). The expansion time estimate of the CEN group was very similar to that inferred by mismatch analysis. The BSP relative to the whole SPER species (Fig. 4) estimated a constant population size until approximately / years BP (depending on the mutation rate considered), when the population began to increase, at first slowly and then (by / years BP)

11 PHYLOGEOGRAPHY OF SALAMANDRINA 913 rapidly. The historical demographic reconstruction for STER species (Fig. 4) showed a trend similar to that of SPER, although the expansion was more recent: / years BP for the start and / years BP for the rapid growth. NUCLEAR DATA Genetic diversity and phylogenetic analysis Sequencing of a 461 bp RAG 1 fragment returned only seven haplotypes in the overall Salamandrina sampling. The four-gamete test suggested that only one recombination event took place within the RAG1 region (R m = 1), producing the haplotype a1. Haplotype genealogy and geographical distribution are shown in Figure 3. All RAG 1 haplotypes were connected in the same network. Haplotype a1 occurs at highest frequency in the sample and shows a reticulated pattern. Haplotypes a5 and a6 occur almost entirely in samples of STER as defined by mtdna sequences, and appear to be more closely-related to some alleles of SPER than to one another. The haplotypes a1, a2, a3, and a4 mostly occur in SPER. Indeed, they were present only in populations containing SPER mtdna, except for the sites where SPER and STER mtdnas occurred syntopically (41 and 42) and in two other nearby localities (43, 46). By contrast, haplotypes a5, a6, and a7 were found in STER mtdna populations. However, they were shared between the two forms in several localities (17, 18, 19, 22, 23, 40, 41, and 42). In the mixed populations, heterospecific heterozygotes (involving more than two haplotypes, in particular: a1, a3 and a5, a6) were found. These heterozygotes appear to be more frequent in samples 41 and 42 (sample 17 = 33%, 18 = 33%, 19 = 33%, 22 = 33%, 40 = 20%, 41 = 60%, 42 = 50%, 43 = 33%, 46 = 17%). Moreover, in four individuals of these two sites, STER mtdna was linked with typical nuclear SPER genotypes (a1/a1 and a1/a3). The reduced size of most samples and the small number of RAG1 haplotypes did not allow the possible existence of genetic structure, within each species, to be addressed. DISCUSSION GEOGRAPHICAL DISTRIBUTION AND HYBRIDIZATION BETWEEN THE TWO SPECIES In the present study, we conducted a significantly increased sampling in terms both of localities and individuals compared to previously published studies (Mattoccia et al., 2005; Nascetti et al., 2005; Canestrelli et al., 2006b), aiming to investigate genetic diversity and population structuring within the two Salamandrina species. Moreover, we supplemented the mitochondrial genetic analysis, screening the samples for variation at RAG 1 gene to verify the congruence between mtdna and nuclear data. The use of nuclear markers was decisive with respect to checking for the occurrence and extent of hybridization processes. The distribution of the two salamanders, as defined by mtdna analysis, is clearly parapatric and the occurrence of a contact zone between them has been confirmed by the sympatric occurrence of haplotypes of both species in two geographically nearby sites. The area where SPER and STER overlap appears to be very narrow and limited to a piedmont zone of the Matese Massif (northern Campania; Fig. 1). The detection of this contact zone raised the question of whether the two species hybridize or not. The nuclear marker used in the present study failed to reveal two phylogenetically distinct haplogroups, which instead was indicated by the mtdna analysis. Such a discordance could reflect incomplete lineage sorting in RAG 1 sequences, which is compatible with the relatively shallow topology of the nuclear (nuc)dna network. However, it is also possible that conflicting genealogy of nuclear and mitochondrial markers may be the results of an old event of introgressive hybridation. Indeed, the recombination test offered support to such a scenario. Moreover, preliminary data from another nuclear marker, a pseudogene of the elongation factor-1a (EF-1 a), would indicate that STER and SPER are detectable as two monophyletic groups of haplotypes, with the pattern of spatial variation for this gene being similar to that of RAG 1, and hybrids between the two forms occurring in the mixed samples (M. Mattoccia, unpubl. data). Despite reciprocal monophyly not being observed, the pattern of spatial variation of RAG 1 is not in contradiction with the mtdna data, which in turn define two genetically distinct population groups corresponding to the SPER and STER species (with the exception of a few localized populations). This result is congruent with the high genetic differentiation at 29 allozymic loci (average D Nei = 0.47) reported by Canestrelli et al. (2006b) between SPER and STER. Although admixture between the two species was not found in their study, it was in the present study. Most likely, this is because the previous study did not include crucial populations. Admixture was detected only in some populations, which, at various degrees, was supported by both mtdna and nucdna. The pattern of geographical variation of RAG 1 gene is consistent with a hypothesis of introgressive hybridization in the contact zone between the two lineages. Admixture of the two RAG 1 haplotype groups appears to be limited to the northern Campania and Molise, which represent the transition area between mtdna lineage ranges.

12 914 M. MATTOCCIA ET AL. S. perspicillata S. terdigitata S. terdigitata (a5) a2 1 Li La 14 Ab Em 6 Mo Tu La 43 9 Um Ma Ap 7 Ab a4 46 Mo a3 a6 a7 47 Ap Cp 50 Ba Cl a1 a5 B A Cp Km Figure 3. A, map of Italy showing the geographical distribution of the different RAG 1 haplogroups in the genus Salamandrina. Numbers identify the populations, as listed in Table 1. Li, Liguria; Em, Emilia Romagna; Tu, Tuscany; Um, Umbria; Ma, Marche; La, Latium; Ab, Abruzzi; Mo, Molise; Cp, Campania; Ap, Abulia; Ba, Basilicata; Cl, Calabria. B, haplotype tree of Salamandrina. Each line represents a single nucleotide substitution. Each circle corresponds to one observed haplotype and sizes of circles are proportional to the haplotype frequency. Open circles indicate missing intermediate haplotypes.

13 PHYLOGEOGRAPHY OF SALAMANDRINA 915 Figure 4. Bayesian skyline plot showing the effective population size fluctuations through time of the two major lineages within Salamandrina. The black lines correspond to median estimates of population size; grey lines indicate the 95% highest posterior probability intervals. Time estimates along the x-axis were obtained assuming two different divergence rates, the first (A) based on the split of Euproctus montanus from Euproctus platycephalus (Caccone et al., 1997), the second (B) on the split between Pleurodeles poireti and Pleurodeles waltl (Carranza & Arnold, 2004). The extent of the hybrid zone appears to be very different, as inferred by mtdna and nuclear variation. The known range of mtdna introgression appeared to be very restricted, whereas the analysis of RAG 1 sequences suggests that introgression has occurred over a broader area of central Italy (approximately 130 km). Several factors might be responsible for this cytonuclear discordance, such as cytonuclear incompatibility, selection acting on the nuclear or mtdna genes, Haldane s rule or sex differences in dispersal (Scribner & Avise, 1994; Abe, Spence & Sperling, 2005; Di Candia & Routman, 2007). To

14 916 M. MATTOCCIA ET AL. discriminate among them, we need to analyse more samples in the hybrid zone at several independent nuclear markers. Such work should help us to understand whether isolating mechanisms maintain the hybrid zone or whether a neutral mixing of haplotypes occurs in this area. The lack of correspondence between patterns of population differentiation at different markers within the contact zone suggests that the two species are not randomly interbreeding. In the contact zone of the Salamandrina species, introgression detected at mtdna appeared to be very restricted compared to that at nucdna. Similar results was reported also for other endemic taxa of Iberian and Italian Peninsulas (Pinho, Harris & Ferrand, 2007; Godinho, Crespo & Ferrand, 2008; Sequeira et al., 2008; Gonçalves et al., 2009). An interesting case study is that of Lacerta schreiberi (Godinho et al., 2008), an Iberian endemic lizard, where the observed phylogeographical pattern was explained, assuming that the two lineages, divergent beginning from the Pliocene, have undergone events of introgression, expansion, and contraction during the Pleistocene. Evidence was provided from mtdna and different nuclear markers, including the detection of a likely nuclear recombinant between haplotypes of the two lineages. POPULATION STRUCTURING, DEMOGRAPHIC HISTORIES, AND POTENTIAL REFUGIA Although two of the three mitochondrial markers used in the present study (12S and 16S) represent relatively slow-evolving genes, our data retain adequate information to reconstruct the phylogeographical history of both Salamandrina species. Phylogeographical analysis revealed three distinct haplogroups within SPER, corresponding to genetically homogeneous groups of population (NOR = central-northern Italy populations; CEN = central Italy populations; VOL = Volsci Mountains populations). The genetic distinctiveness of these groups was further supported by the results for Snn and SAMOVA. Within STER, the Calabrian populations (CAL) were distinguishable from all the others (BAC) on the basis of their unique haplotypes. This geographical structuring apparently is not supported by the RAG 1 spatial variation, although it is consistent with the pattern of allozymic variation reported for the same clades by Canestrelli et al. (2006b). Those data, although based on a limited number of populations, appear to support the same groupings found with mtdna markers. The groupings identified by phylogeographical analyses also differed in their level of genetic variability (Table 4). The survey of the genetic diversity highlighted the notable homogeneity of NOR and BAC within SPER and STER, respectively. Among the remaining groups, VOL stood out for its relatively high genetic diversity, in particular considering the restricted range of these populations. Only few of these differences were found between the samples employed in the nuclear genes analysis (Canestrelli et al., 2006b), although the reduced number of samples, the low values of heterozygosity, and the overlap of the standard errors of the estimates could explain the incomplete agreement. A proper sample size and the use of variable markers as microsatellite DNA loci would be better suited to test for concordant patterns in genetic diversity of mtdna and nuclear data. Multiple independent analyses were employed to reconstruct the evolutionary processes that gave origin to the current genetic structure of the two Salamandrina species. All the inferences from NCA, mismatch analysis, neutrality tests, and BSP agree in suggesting a population expansion for both SPER and STER. Furthermore, the haplotype star-like phylogeny of SPER and low nucleotide diversity associated with a relatively high haplotype diversity in both species provide further strong evidence for a recent and rapid population expansion (Rogers, 1995). Average dates of the expansion, as suggested by the mismatch analysis and Bayesian Skyline Plot, must be considered with caution because of the large confidence interval. However, these were substantially identical for SPER. For STER, the time inferred by BSP (30 000/ BP) was lower than that obtained with the mismatch analysis (41500/ BP). In any case, the beginning of these demographic events overlapped with the Würm glaciation, even if the BSPs dated the crucial moment of exponential growth in both lineages (30 000/ years BP for SPER and / for STER) at the end of that glacial period. These time intervals do not rely on a Salamandrina mtdna explicit mutation rate. Because the mutation rates used in the present study were calibrated over deep phylogenies, our estimates could suffer a time-dependency effect (Ho et al., 2005, 2007; Gratton, Konopiński & Sbordoni, 2008; Gentile et al., 2010) and be overestimations. Thus, for Salamandrina species, the occurrence of a population expansion after the last glacial period would appear to be a more realistic scenario, as expected for most of temperate species. Bayesian coalescent analysis estimated the most recent common ancestor (tmrca) of SPER at / years BP and at / years BP for STER. Although the confidence intervals are large, these results suggest that Pleistocene climatic changes had a deep impact on the evolutionary history of both species. Spectacled salamander is a temperate genus associated with mesophilous forestal habitats. It is

15 PHYLOGEOGRAPHY OF SALAMANDRINA 917 likely that, during the Pleistocene glacial periods, both SPER and STER experienced repeated contractions of their habitat as a result of the cold and arid climate. Therefore, the two species probably became extinct over a large part of their range, whereas some populations persisted in restricted refugial areas where suitable habitats were still available. The reciprocal monophyly as well as the recent coalescence time of SPER and STER supports the hypothesis of a single refugium for each lineage during the last glacial stage. Recognizing ancestral haplotypes and populations displaying genetic diversity higher than the average could help to pinpoint refugial areas. Based on these assumptions, the putative glacial refugium for STER should be located in Calabria, whereas, for SPER, it could be identified in central Italy. More precisely, the refugium of the northern lineage should coincide with the Tyrrhenian coast between southern Latium and northern Campania. All the main haplotypes have been found in this area, including the putative ancestral one (h3). Furthermore, LOM and PET are by far the most variable populations. This scenario is also compatible with the climate and landscape history of the identified area. Paleobotanical and palynological studies have generally confirmed that Latium maintained residual forests important for the survival of thermophilous and mesophilous trees during the whole last glacial period (Follieri & Magri, 1997). In particular, relicts of temperate forests were located in the Pontine marshes along the coast of southern Latium (Tongiorgi, 1936). A few glacial refugia in Italy were located certainly outside the southernmost part of the Peninsula. However, the possible concordance between the location of the refugial area of SPER and that of other uncorrelated taxa (Fratini et al., 2005; Canestrelli, Cimmaruta & Nascetti, 2007; Barbanera et al., 2009) highlights the potential role of central Italy, particularly the Tyrrhenian side, as a supplementary source of geographical divergence of refugial populations. For STER, the Calabrian populations hold the ancestral haplotype (h16) and encompass the highest levels of diversity, suggesting that this species survived in this area during the last glacial period. The location of the putative refugium of STER coincides with what inferred for a variety of taxa (Steinfartz, Veith & Tautz, 2000; Grivet & Petit, 2003; Magri et al., 2006; Canestrelli et al., 2007; Grill et al., 2009), and led to the identification of Calabria as the main refugium for the Italian fauna and flora (Petit et al., 2003; Schmitt, 2007). However, in this region, some species consist of several genetically distinct lineages (Santucci, Nascetti & Bullini, 1996; Canestrelli, Cimmaruta & Nascetti, 2008) that, in a few cases, probably originated via allopatric isolation in multiple refugia even at the scale of the sole Calabria (Podnar, Mayer & Tvrtković, 2005; Canestrelli et al., 2006a). We found a single mitochondrial lineage of Salamandrina in that area, although we cannot exclude that further sampling in new localities might change the overall observations. As the climate conditions became more favourable, both species of Salamandrina expanded their range northward, recolonizing the newly suitable habitat. The high genetic homogeneity of population groups located towards the northern edges of each range (NOR and BAC) suggests that recolonization was a rapid event in both species. This pattern of genetic diversity meets the expectations of the leading-edge expansion model that foresees a decrease of genetic heterogeneity across wide areas, as a by-product of repeated founder events (Ibrahim, Nichols & Hewitt, 1996). The more recent expansion of STER could explain the lower genetic diversity of this clade (i.e. compared to SPER), although a more severe bottleneck, following the contraction of the range, cannot be excluded. The first hypothesis takes into account the most recent coalescence of sequences of STER in comparison with SPER. For the southern species, the Pollino Massif could have represented a strong barrier to the dispersal outside of Calabria. Indeed, at present, these mountains appear to maintain the two main population groups of STER separated from each other. Furthermore, the Pollino mountains maintained some glaciers until the Younger Dryas, years BP (Palmentola, Acquafredda & Fiore, 1990). Thus, the beginning of the exponential phase of demographic growth, as detected by BSP, probably coincided with the crossing of Pollino Massif. Although the expansion of STER gave rise to a single genetically homogeneous population group distributed across Campania and Basilicata (BAC), the geographical structuring of SPER populations would suggest that this species could have expanded northwards and eastwards, from its original refugial area, as could be tentatively inferred from the current distribution of haplotypes. It is also possible that, during the recolonization, UMB and BAR populations (within CEN group) became temporarily isolated. Indeed, these populations became fixed for private or rare haplotypes and, for the CEN group, the NCA indicated both range expansion and population fragmentation. Also the group including samples of the Volsci chain (VOL) showed signals of recent demographic expansion. The VOL group most probably evolved in this mountain range along the southern coast of Latium, although it did not expand beyond its borders. Demographic fluctuations in these mountains could have occurred during climatic changes as a result of variations in the altitudinal limit of the available habitat for the species.