Molecular Phylogenetics and Evolution 51 (2009) 399–404 Contents lists available at ScienceDirect Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev Short Communication Phylogenetic relationships of Sardinian cave salamanders, genus Hydromantes, based on mitochondrial and nuclear DNA sequence data Arie van der Meijden a,*, Ylenia Chiari b,1, Mauro Mucedda c, Salvador Carranza d, Claudia Corti e, Michael Veith a a Department of Biogeography, University of Trier, Am Wissenschaftspark 25-27, 54296 Trier, Germany b Department of Ecology and Evolutionary Biology, YIBS-Molecular Systematics and Conservation Genetics Lab, ESC 158, Yale University, 21 Sachem St., New Haven, CT 06520-8105, USA c Via Leopardi 1, Sassari, Gruppo Speleologico Sassarese, Italy d Institute of Evolutionary Biology (UPF-CSIC), Passeig Maritim de la Barceloneta 37-49, E-08003 Barcelona, Spain e Museo di Storia Naturale dell’Università di Firenze, Sezione di Zoologia ‘‘La Specola”, Via Romana, 17, 50125 Firenze, Italy article info Article history: Received 5 September 2008 Ó 2009 Elsevier Inc. All rights reserved. Revised 12 November 2008 Accepted 10 December 2008 Available online 24 December 2008 1. Introduction by five species, versus only three species in mainland Europe, and three in the western USA. Recently, Vieites et al. (2007) have The Tyrrhenian island of Sardinia is known for its high level of brought forward a compelling hypothesis for the highly disjunct endemism (Oosterbroek and Arntzen, 1992; Grill et al., 2007). distribution of the members of the genus Hydromantes between Beside the importance of this island as a refuge during the last gla- the western US and Europe by suggesting dispersal through the ciations, little is known about the origin and relationships of Sardi- Bering land bridge. The eight European species are distributed in nian species (reviewed in Grill et al., 2007). The origin of some southeastern France, north and central Italy and throughout Sardi- Sardinian lineages is attributed to the isolation of the Sardo-Cors- nia. Previous phylogenetic works based on immunological data and ican plate (e.g. Lanza, 1983; Caccone et al., 1994; Oliverio et al., allozymes (Wake et al., 1978), allozymes (Nascetti et al., 1996), 2000; Omodeo and Rota, 2008), others to the Messinian salinity morphology (Lanza and Caputo, 1995), morphology, caryology crisis (e.g. Marra, 2004) or to more recent events such as the and cytochrome b sequences (Jackman et al., 1997) and cyto- severe climate changes from the early Pliocene and throughout chrome b, 12S rRNA and 16S rRNA sequences (Carranza et al., the Pleistocene (Grill et al., 2007 and references therein). It still 2007) did not fully resolve the relationships among the European remains unclear which of these events are more relevant to explain species of Hydromantes. Some of these studies (e.g., Nascetti the current distribution of the European members of the genus et al., 1996; Nardi, 1991; but see also Carranza et al., 2007) support Hydromantes. Despite their common name, Hydromantes are not the hypothesis of H.(Atylodes) genei, which inhabits the southwest restricted to cave habitats, or even to limestone substrates, but of Sardinia, as the most basal European species. The eastern Sardi- are also found on exposed sites, even on granites and volcanic nian species (H.(Speleomantes) flavus, H.(S.) supramontis, H.(S.) rocks (Lanza et al., 2005). Their range is therefore not directly imperialis and H.(S.) sarrabusensis) are thought to be more closely restricted by these aspects of geology or even by plant cover. related to the mainland species (H.(S.) strinatii, H.(S.) ambrosii and Humid subterranean hiding places, such as the space between H.(S.) italicus). Several biogeographic hypotheses have been put the rocks of a rockslide, do seem to be correlated with their pres- forward to explain this pattern (reviewed in Lanza et al., 2005). ence (personal observation). The salamanders of the genus Hydro- All these biogeographic hypotheses hinge on the basal position of mantes have their highest diversity in Sardinia, being represented H.(A.) genei, which is generally considered to have split off from the ancestral stock well before the other species. The recent publication of Carranza et al. (2007) addresses the * Corresponding author. Fax: +49 (0)6512013851. phylogenetic relationships between the European species based E-mail addresses: [email protected] (A. van der Meijden), yle@ylenia- on three mitochondrial genes, but could not resolve H.(A.) genei chiari.it (Y. Chiari), [email protected] (M. Mucedda), [email protected] unambiguously as the most basal taxon, or the sister group rela- csic.es (S. Carranza), claudia.corti@unifi.it (C. Corti), [email protected] (M. Veith). 1 Present address: Institut des Sciences de l’Evolution, Université Montpellier 2, tionship between the mainland species and the eastern Sardinian Place Eugène Bataillon, Bat 22/RdC/CC064, 34095 Montpellier Cedex 5, France. species. Also the placement of H.(S.) sarrabusensis could not be 1055-7903/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2008.12.011 400 A. van der Meijden et al. / Molecular Phylogenetics and Evolution 51 (2009) 399–404 unambiguously resolved. Increased taxon sampling and enlarge- default settings (best accuracy). The resulting alignments were ment of the dataset have been shown to increase phylogenetic res- checked by eye, but were not found to require additional editing. olution (Pollock et al., 2002; Brinkmann and Philippe, 2008). The final aligned dataset was deposited in the TreeBase database Therefore, in an effort to resolve these relationships, in the current (Study accession number S2274). study we extended the existing dataset considerably by adding two Missing genes were coded as ‘‘missing” (?) in the concatenated nuclear genes and additional mitochondrial sequence data, as well dataset (see Table 1). In Bayesian Inference (BI) and Maximum Par- as greatly extend the number of Sardinian populations represented simony (MP) analyses of large multi-gene data sets of hylid frogs, in the dataset from eight to 16 populations. Our data confirm the no relationship was found between completeness of the sequence previous findings by Carranza et al. (2007), but despite the larger data of a taxon, and the support values the taxon receives (Wiens dataset, several relationships among the European members of et al., 2005), suggesting that the limited amount of missing data the genus Hydromantes still remain poorly resolved. in our concatenated alignment is unlikely to distort the phyloge- netic results. Furthermore, the placement of taxa with missing data 2. Materials and methods in our analysis was congruent for well supported branches (boot- strap support >80%) between the single gene analyses in which 2.1. Specimen selection the taxon was represented by a complete sequence, and the com- bined dataset, suggesting that omission of one or more genes from A total of 66 European Hydromantes specimens were used in the combined set did not influence the placement of particular taxa this study. Of these 33 samples are the same as used in the study (data not shown). by Carranza et al. (2007), while the other 33 samples were col- A homogeneity partition test (Farris et al., 1994) as imple- lected in Sardinia in April 2007 (Table 1). We included a total of mented in PAUPÃ version 4.0b10 (Swofford, 2002) did not reject 27 populations. Sardinian species were represented by samples homogeneity of the five different markers (P = 0.17). Besides an from across the range of each species from a total of 16 populations analysis of the combined data set we also performed independent (Fig. 1). A maximum of three specimens from every sampling of analyses for each gene and for the combined mitochondrial and the each population were randomly selected to be included in this combined nuclear genes in the dataset. Phylogeny reconstruction study (Table 1). Samples taken from sites at very close proximity based on the separate and combined datasets was performed using were counted as a single population. Maximum Likelihood (ML) and BI methods. The best fitting models of sequence evolution were determined by the AIC criterion in 2.2. DNA extraction, PCR and sequencing Modeltest 3.7 (Posada and Crandall, 1998). ML tree searches were performed using PhyML, version 2.4.4 (Guindon and Gascuel, DNA was extracted from tail tips preserved in 99% ethanol using a 2003). Bootstrap branch support values were calculated with 500 DNeasyÒ blood and tissue kit (Quiagen). Primers for one fragment of replicates for the combined datasets, and 200 replicates in the sin- the 12S rRNA gene and one fragment of the 16S rRNA gene were gle gene analyses. The BI analyses of the combined and separate 12SA-L and 12SB-H and 16SA-L and 16SB-H, used as in Palumbi datasets were conducted with MrBayes 3.1.2 (Huelsenbeck and et al. (1991). Primers for cytochrome b were designed based on an Ronquist, 2001), using models estimated with Modeltest under alignment of Hydromantes sequences available in GenBank. These the AIC criterion, with 2,500,000 generations, sampling trees every were Cyt-BYf1 (50-CAARTCCTYACCGGRYTATTT-30) as the forward 100th generation (and calculating a consensus tree after omitting primer and Cyt-BYr1 (50-TTCGRTKTTTTGADGTRTTTA-30) as the the first 6250 trees). Log likelihood scores for the remaining trees reverse primer. This fragment was amplified using an initial dena- were graphed in Tracer 1.4 (http://beast.bio.ed.ac.uk/Tracer) and turation step of 2 min at 94 °C, followed by 35 cycles of denaturation checked for appropriateness of the burnin-period. The topologies at 94 °C for 20 s, annealing at 52 °C for 30 s, and extension at 72 °C for resulting from the ML and BI analyses of the combined dataset 90 s. Two nested pairs of primers to amplify a single 1400 bp frag- were compared using a Shimodaira–Hasegawa (SH) test as imple- * ment of Rag-1 were designed based on an alignment of available sal- mented in PAUP (Swofford, 2002), with RELL optimization and amander Rag-1 sequences from GenBank.