Molecular Phylogenetics and Evolution 38 (2006) 266–273 www.elsevier.com/locate/ympev Short communication Reexamination of the Iberian and North African (: ) phylogeny based on increased mitochondrial DNA sequencing

Catarina Pinho ¤, Nuno Ferrand, D. James Harris

CIBIO, Centro de Investigação em Biodiversidade e Recursos Genéticos, Campus Agrário de Vairão, 4485-661 Vairão, Departamento de Zoologia e Antropologia, Faculdade de Ciências da Universidade do Porto, 4099-002 Porto, Portugal

Received 16 March 2005; revised 9 June 2005; accepted 9 June 2005 Available online 2 August 2005

1. Introduction rian and North African form a clade, with the exception of P. muralis, which exists nowadays in North- In recent years, molecular studies have revealed com- ern Iberia and is widely distributed across Europe, most plex patterns of diVerentiation within many European likely after an expansion from the Italian Peninsula herpetological taxa. Wall lizards (Podarcis spp.) were one (Harris and Arnold, 1999; Oliverio et al., 2000). of the most studied groups, perhaps due to the complex- Recently, Iberian forms have been characterized using ity of deWning taxonomical entities within this genus by morphology (Geniez, 2001; Sá-Sousa et al., 2002), morphological analysis only, due to an extreme intraspe- sequencing of partial cytochrome b and 12S rRNA mito- ciWc variability coupled with low variation between spe- chondrial genes (Harris et al., 2002b; Harris and Sá- cies. Regarding Iberian forms, this diYculty is illustrated Sousa, 2001, 2002) and allozyme data (Pinho et al., 2003, by the variety of diVerent proposals for the classiWcation 2004). All these studies clearly showed that the actual of these lizards (Arnold and Burton, 1978; Geniez, 2001; variation within Iberian lizards is much more complex Klemmer, 1959; Mertens and Müller, 1940; Sá-Sousa, than that concealed behind ’ names, with multiple 2001). Despite the controversy, the most common view forms being present both in the Iberian Peninsula and (Arnold and Burton, 1978) accepted three species within North . The Wrst outcome of these studies was the the Iberian Peninsula: P. muralis Laurenti 1760, P. hispa- elevation of P. carbonelli Pérez-Mellado, 1981 to species nica (Steindachner 1870), and P. bocagei (Seoane 1884). status after morphological and molecular data corrobo- According to some authors (Barbadillo et al., 1999), P. rated its distinctiveness from P. bocagei, in which this hispanica was further subdivided into two subspecies: the form had been included (Sá-Sousa and Harris, 2002). nominal form, in the Iberian Peninsula, and P. h. vauc- The second outcome was the recognition of Podarcis heri, in North Africa. Oliverio et al. (2000) also proposed hispanica as a species complex, since the forms included the elevation of the African populations to the species in this designation do not form a clade relative to P. boc- rank as P. vaucheri on the basis of high genetic distances agei and P. carbonelli (Harris et al., 2002b; Harris and between single individuals from each side of the Strait of Sá-Sousa, 2002; Pinho et al., 2003, 2004). Gibraltar, obtained using the 12S rRNA gene. However, although the existence of multiple forms Genetic studies addressing the phylogeny of the within P. hispanica from the Iberian Peninsula and whole genus Podarcis (Harris and Arnold, 1999; Harris North Africa was well documented both by mitochon- et al., 2005; Oliverio et al., 2000) have shown that all Ibe- drial DNA (mtDNA) and allozyme studies, neither data set convincingly supported relationships between forms, moreover suggesting slightly diVerent evolutionary sce- * Corresponding author. Fax:+351 2 52 66 17 80. narios. This fact was interpreted as either being caused E-mail address: [email protected] (C. Pinho). by the independent evolution of each kind of marker,

1055-7903/$ - see front matter  2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2005.06.012 C. Pinho et al. / Molecular Phylogenetics and Evolution 38 (2006) 266–273 267 leading to diVerent phylogenetic relationships, or by the 2.2. DNA extraction, ampliWcation, and sequencing forms having split during a short period of time, leading to an unresolved phylogeny and short internal branches Total genomic DNA was extracted from small por- (Pinho et al., 2003). To address this question and to tions of alcohol-preserved tail muscle following standard obtain a more reliable estimate of the mitochondrial methods (Sambrook et al., 1989). We ampliWed a portion phylogeny, we extended the sampling of the mitochon- of Wve mitochondrial regions: cytochrome b, 12S rRNA, drial genes studied. Besides 12S rRNA and cytochrome 16S rRNA, ND4-tRNALEU, and the control region. The b, previously studied in other works, we obtained chosen fragments were ampliWed using published prim- sequences for three other mitochondrial DNA regions: ers (Table 2), except the ND4-tRNALEU fragment, for 16S rRNA, NADH dehydrogenase subunit 4 (ND4) and which an additional speciWc primer was designed in the adjacent tRNAs, and the control region in a set of 32 tRNAHIS region (HisR primer, sequence 5Ј-CTAGAGT- individuals representing all known lineages. By sequenc- CACAGTCTAGTGTTTT-3Ј). AmpliWcations of 12S, ing diVerent portions of mitochondrial DNA, we also 16S, cytochrome b, and the control region were carried reduce the probability of putative nuclear copies inXu- out in 25 L volumes, containing 2.5 L of 10£ reaction W V encing the nal estimates of the phylogeny, because the bu er (Ecogen), 3.0 mM MgCl2, 0.4 mM each dNTP, studied genes are scattered along the mtDNA molecule. 0.4 M each primer, 1 U of Ecotaq DNA polymerase (Ecogen) and approximately 100 ng genomic DNA. Polymerase chain reaction (PCR) conditions consisted 2. Materials and methods of a pre-denaturing step of 3 min at 94 °C for and 30–35 cycles of denaturing (94 °C for 30 s), annealing (52 °C for 2.1. Sampling the control region, 50 °C for the rest of the fragments, 30 s) and extension (72 °C for 30 s). A Wnal extension was Thirty-two specimens, including all the known mor- conducted at 72 °C for 3 min. AmpliWcations of the photypes of P. hispanica, P. bocagei, P. carbonelli and the ND4-tRNALEU and ND4-tRNAHIS fragments were con- outgroup P. muralis, were analysed (Fig. 1 and Table 1). ducted in 25 L volumes, containing 2.5 L of 10£ reac- W V Morphological identi cation or prior sequencing of at tion bu er (Ecogen), 3.2 mM MgCl2, 0.4 mM each least one mitochondrial DNA gene allowed the assign- dNTP, 0.2 M each primer, 1 U of Ecotaq DNA poly- ment of individuals to morphotypes/lineages as described merase (Ecogen), and approximately 50 ng genomic in Harris and Sá-Sousa (2002). One of the individuals DNA. AmpliWcation conditions consisted of a pre-dena- included in the study (Gal3) was found not to belong to turing step of 3 min at 94 °C followed by 35 cycles of a any genetic lineage described to date and is herein denaturing step of 30 s at 94 °C, annealing at 54 °C for referred to as “unidentiWed type.” Whenever possible, 30 s, and extension at 72 °C for 40 s. The Wnal extension more than one individual for each form was analysed. was accomplished at 72 °C for 4 min.

Gpb6 Mon8 BTA1, MTA1 Gua1 Ph1 Bur2

Med1 Rua1 MP3 Av2

Albc1 Oro1 Trj1 Mad2

Tie1 Iberian Peninsula Mot1 Phv4 And10

Elv1 And8

PR1 Gal3 Cin1 Pod12 OK1

Bt6

PhM2 LK6

North Africa Ouk7

Js1, Js6

0 300 Km

Fig. 1. Map showing the geographical origin of samples analysed in this study. 268 C. Pinho et al. / Molecular Phylogenetics and Evolution 38 (2006) 266–273 DQ081076DQ081075 DQ081152 DQ081151DQ081079 DQ081108 DQ081086 DQ081155 DQ081107 DQ081085 DQ081162DQ081087 DQ081161DQ081089 DQ081163 DQ081111 DQ081088 DQ081165 DQ081118 DQ081083 DQ081164 DQ081117 DQ081082 DQ081159 DQ081119 DQ081158 DQ081121 DQ081081 DQ081120 DQ081157 DQ081115 DQ081091 DQ081114 DQ081167DQ081094 DQ081113 DQ081092 DQ081170DQ081097 DQ081168 DQ081123 DQ081098 DQ081173DQ081096 DQ081174 DQ081126 DQ081099 DQ081172 DQ081124 DQ081100 DQ081175 DQ081129 DQ081104 DQ081176 DQ081130 DQ081103 DQ081180 DQ081128 DQ081179 DQ081131 DQ081132 DQ081136 DQ081135 a a a a a a a a a a a a a a a a a a a a a 68 DQ081090 DQ081166 DQ081122 12S rRNA12S rRNA 16S and tRNAs ND4 region Control AF469423 AF469425 AF469446 AF469444 AF469443 AF469452 AF469450 AF469454 AF469459 AY134715 AF469435 AF469427 AY134712 AF469416 AY134716 AY134713 AY134714 AY134709 AY134707 AY134717 b a a a a a a a a a a a a a a a a a a a a Cytochrome Cytochrome ). y León, DQ081144 DQ0810 Harris et 2002a,b; al., Harris andSá-Sousa, 2002 BTA1MP3 Tanes, Asturias, Spain Madalena, Portugal DQ081139 AF469424 DQ081064 DQ081077 DQ081153 DQ081109 Av2Albc1PR1 Aveiro, Portugal La Alberca, Castilla y León, Spain Playa del Rompeculos,Andalucía, Spain DQ081141 DQ081142 AY214449 DQ081066 DQ081080Pod12 DQ081156 DQ081140 Granada, Andalucía, Spain DQ081065 DQ081112 DQ081078 DQ081154LK6 DQ081110 AF469428 Le Kef, DQ081147 DQ081071 DQ081101 DQ081177 DQ081133 Gpb6 Malpica, Galicia, SpainRua1Ph1Oro1Trj1 Vila da Rua, PortugalMad2 Vila Real,Portugal Oropesa, Castilla-La Mancha, SpainAnd10Tie1 Trujillo, Extremadura, Spain AF469426 Spain Madrid, Med1 Benatae, Andalucia, Spain Tielmes, Madrid, Spain AF469453 Mot1 Medinaceli, Castilla y León, SpainBt6 AF469445 Ouk7 AF469451 Motilla del Palancar, Castilla-LaMancha, SpainCin1 AF372084 Elv1 AY134677 DQ081143 Bab Taza, Oukaïmeden, Morocco AF469436 Js6Spain Andalucía, Guadalcacin, DQ081067 AY134680 Huelva, Andalucía, Spain AF469460 DQ081084 Jebel Sirwah, DQ081160 Morocco DQ081116 AY134679 AY134678 AY134681 AY134674 AY134682 Gal3MTA1Gua1 Galera, Andalucia, Spain Tanes, Asturias, Spain Guadarrama, Madrid, Spain DQ081146 DQ081149 DQ081150 DQ081070 DQ081073 DQ081074 DQ081095 DQ081105 DQ081171 DQ081106 DQ081181 DQ081182 DQ081127 DQ081137 DQ081138 PhM2 Morocco Mts, Atlas Mid AF372083 vaucheri ed type.ed ) W b morphotype 1 morphotype 2 morphotype Mon8Portugal Montesinho, 3 (NE morphotype Iberia) Bur2 Phv4 sensu stricto Castilla Burgos, Portugal Beja, And8 JebelSirwah AF469447 Puebla de D. Fadrique,Andalucía, Spain Tunisia DQ081145 Js1 AF469455 DQ081069 OK1 DQ081093 Jebel Sirwah, Morocco DQ081169 Oued Kébir, Tunisia DQ081125 AY134672 DQ081148 DQ081072 DQ081102 DQ081178 DQ081134 Indicates gene region previously published ( published previously region gene Indicates Unidenti hispanica . ( a b Specimen typeSpecimen code Sample Locality Nos. Accession GenBank P. bocagei Table 1 Specimentype,locality data and GenBankaccession numberssamplesfor includedthis in study P. carbonelli P. hispanica P. hispanica P. hispanica P. hispanica P P. hispanica P. hispanica P. hispanica P. muralis C. Pinho et al. / Molecular Phylogenetics and Evolution 38 (2006) 266–273 269

Table 2 gamma model of among-site rate variation. In both Primers used in this study searches, stationarity of the Markov Chain was deter- Gene fragment Primers used Reference mined as the point when sampled ln-likelihood values Cytochrome b Cytochrome b1 Kocher et al. (1989) plotted against generation time reached a stable mean Cytochrome b2 Kocher et al. (1989) equilibrium value; “burn-in” data sampled from genera- 12s rRNA 12sa Kocher et al. (1989) tions preceding this point were discarded. All data col- 12sb Kocher et al. (1989) lected at stationarity were used to estimate posterior 16s rRNA 16sL1 Hedges and Bezy (1993) 16sH1 Hedges and Bezy (1993) nodal probabilities and a summary phylogeny. Two ND4-tRNALEU ND4 Arévalo et al. (1994) independent replicates were conducted and inspected for Leu Arévalo et al. (1994) consistency to check for local optima (Huelsenbeck and Control region HisR This study Bollback, 2001). DL3F Crochet et al. (2004) We used the cytochrome b data set to infer divergence DL4R Crochet et al. (2004) times. Molecular clock assumptions were tested using a likelihood-ratio test. The PCR products were enzymatically puriWed and sequenced using the ABI Prism BigDye Terminator Cycle sequencing protocol in an ABI Prism 310 auto- 3. Results and discussion mated sequencer (Applied Biosystems) with the same primers used for ampliWcation. Including the two outgroups, 32 individuals from 12 Sequences were aligned manually using Bioedit v. diVerentiated species/entities were analysed and 5.0.9 (Hall, 1999). The ND4-tRNALEU fragment was sequenced for the 5 mitochondrial regions. Several mea- subdivided into the partial ND4 sequence, tRNAHIS and sures were taken to evaluate the possibility of the pres- tRNASER. The tRNALEU portion was excluded from the ence of nuclear copies among the data. Although the analysis because sequences were incomplete. The cyto- base composition (less than 5% of guanines in the third chrome b, ND4, and tRNASER sequences contained no position, see Harris, 2002) and the absence of stop indels. Alignment of the 12S, 16S, control region, and codons in protein-coding genes conformed to the expec- tRNAHIS required insertions or deletions in Wve, eight, tations of a mitochondrial origin, visual inspection of the two, and two places, respectively. sequences revealed that the ND4 sequence from sample Gal3 seemed to have an excess of amino acid substitu- 2.3. Phylogenetic analysis tions when compared to the others. This was conWrmed by plotting synonymous versus non-synonymous dis- Sequences were imported into PAUP* 4.0b10 (Swo- tances for the complete data set, which showed that for Vord, 2002). For the phylogenetic analysis of the com- all the comparisons involving this particular sample bined data, we used maximum likelihood (ML), there was an excess of non-synonymous substitutions maximum parsimony (MP), and Bayesian inference. (data available from the authors upon request). Also, in When estimating phylogenetic relationships among the phylogenetic analyses of the ND4 gene alone, the sequences, one assumes a model of evolution. We used length of the branch leading to this sample was much the approach outlined by Huelsenbeck and Crandall higher than all the others. Taken together, these observa- (1997) to test 56 alternative models of evolution, tions suggested that this sequence might correspond to a employing PAUP* 4.0b10 and Modeltest 3.06 Posada nuclear pseudogene. As an attempt to conWrm this hypothe- and Crandall (1998), described in detail in Posada and sis, we designed an internal primer in the tRNAHIS region Crandall (2001). Once a model of evolution was chosen, (HisR, see Section 2), which was used to amplify a frag- it was used to estimate a tree using ML (Felsenstein, ment of about 750 bp when used together with primer 1981) with random sequence addition (10 replicate heu- ND4 (Arévalo et al., 1994). The sequence of Gal3 ristic searches). The MP analysis was also performed obtained using these primers, showed several heterozy- with random sequence addition (10 replicate heuristic gous-like positions which were interpreted as resulting search), and support for nodes was estimated using the from an overlap of the mitochondrial and the nuclear non-parametric bootstrap technique (Felsenstein, 1985) sequence. From this, we were able to reconstruct the with 100 replicates. The Bayesian analysis was imple- putative mitochondrial sequence. When plotting synony- mented using MrBayes (Huelsenbeck and Ronquist, mous versus non-synonymous distances in this gene 2001), which calculates Bayesian posterior probabilities using this corrected sequence we did not Wnd the bias using a Metropolis-coupled, Markov chain Monte Carlo that had previously been found. The putative nuclear (MC-MCMC) sampling approach. Bayesian analyses copy sequence has been deposited in GenBank, Acces- were conducted with random starting trees, run 0.5 £ 106 sion No. DQ081063. generations, and sampled every 100 generations using a Aligned sequences of the combined mitochondrial general-time-reversible model of evolution with a DNA genes were 2425 bp long. From these, 306 bp 270 C. Pinho et al. / Molecular Phylogenetics and Evolution 38 (2006) 266–273 correspond to cytochrome b, 381 bp to 12S rRNA, same tree as that derived from ML, only the ML tree is 509 bp to 16S rRNA, 419 bp to the control region, 675 bp depicted in Fig. 2, but MP bootstraps and Bayesian pos- to ND4, 69 bp to tRNAHIS and 66 bp to tRNASER. The terior probabilities have been overlaid onto this estimate length of the sequences used in this study was approxi- of phylogeny. mately the same for all the samples, with the exception of The clade formed by the Northeastern type of the above mentioned Gal3, for which sequences lack P. hispanica and the newly found P. hispanica form from around 30 bp of the ND4 gene and the complete tRNA Galera, in the Baza Depression of SE Spain, is the sister genes due to the procedures described above. Of these taxon to all other Iberian and North African Podarcis. 2425 bp, 597 characters were variable and 530 parsimony The remaining taxa are split into two very well sup- informative. The most appropriate model of evolution ported clades: one comprising all the Western Iberian for the data was the GTR model with an estimate of forms and one including all Southern Iberian and North invariable sites (0.67) and a discrete approximation of African morphotypes. Within the Wrst clade, there is the gamma distribution (4 rate categories,  D 1.67). strong support for two groups: one that encompasses Using this model we recovered a single ML tree of P. carbonelli and morphotype 2 of P. hispanica (sensu ¡ln10,181 (Fig. 2). Maximum parsimony analysis recov- Sá-Sousa et al., 2002) and another that comprises P. boc- ered four equally parsimonious trees, the strict consen- agei and morphotype 1 of P. hispanica. This morphotype sus of which diVered from the ML tree only in being less is further subdivided into two geographically separated well resolved. Since Bayesian analysis recovered the and well-supported clades, one corresponding to the

Fig. 2. Estimate of relationships derived from a ML search using the GTR model with an estimate of invariable sites (0.67) and a discrete approxima- tion of the gamma distribution (4 rate categories,  D 1.67). Bayesian analysis produced the same estimate of relationships. The tree was rooted using P. muralis. Bayesian posterior probabilities are given above nodes, ML and MP bootstraps, respectively, below nodes. When all three were identical one value is given in a box. (-) Indicates bootstrap values lower than 50%. (¤) Indicates branches where diVerent methods yielded diVerent topologies. C. Pinho et al. / Molecular Phylogenetics and Evolution 38 (2006) 266–273 271

Northwestern part of its distribution, whereas the other Strait, occurred ca. 5.3 million years ago (Mya), after the seems to be conWned to the Spanish central system and Messinian salinity crisis, which began ca. 5.6 Mya and surrounding areas. The second clade is composed of allowed land connection between Iberia and North both Iberian and North African forms. As sister taxon to Africa (Hsü et al., 1977). However, according to the phy- all the other forms in this clade is P. hispanica sensu logeny published by these authors, based on cytochrome stricto, from SE Iberia. The other forms can be subdi- b and 12S rRNA gene sequences, P. hispanica sensu vided into two groups: one including P. (hispanica) vauc- stricto was not the sister taxon to all North African heri, which is further subdivided into Iberian and North forms, but it clustered within this group. These relation- African forms, and another including sequences of P. ships were weakly supported (less than 50% bootstrap hispanica from Tunisia and the Jebel Sirwah form from support). Moreover, the authors used a calibration for Southern Morocco. the molecular clock based on the two genes combined Our estimates of phylogeny support most of the derived from geckos (Carranza et al., 2000). In this work, topology of previous works with less included characters we chose to base our analysis on cytochrome b distances (Harris et al., 2002b). However, bootstrap values have only, since more calibrations have been accomplished signiWcantly increased, approaching 100% in branches for this gene than for any of the others analysed. A com- where previous analyses had retrieved less than 50%. pilation of these (Paulo, 2001) suggested a standard rate Therefore, besides conWrming the monophyly of previ- of substitution of around 2.0% per million years for ously described groups, almost all the relationships cytochrome b gene evolution in lacertids. The likelihood- between those forms are now well supported. There is, ratio test performed on the cytochrome b data set therefore, strong support for the monophyly of P. hispa- showed no signiWcant diVerence between the log-likeli- nica morphotype 1, which is formed by two very diver- hood of phylogenetic trees with (¡ln 1776) and without gent lineages (around 9% cytochrome b divergence). The (¡ln 1760) the molecular clock enforced. Although the close relationship between this morphotype and P. boca- ML estimate of phylogeny based on the full data set gei, as well as that between P. hispanica type 2 and P. diVers in topology from that obtained using only the carbonelli, is also conWrmed through the analysis of this cytochrome b data, we forced the latter to Wt the topol- data set. This is relevant in understanding the evolution- ogy of the former. Since this tree was not signiWcantly ary patterns within the Iberian Podarcis because the cur- less likely using a Shimodaira and Hasegawa (1999) test rent knowledge on the distribution of the lineages points than the best estimate of phylogeny derived from cyto- to the fact that the only forms with distributions that chrome b (p D 0.112), we calculated divergence times overlap extensively are precisely these sister taxa from cytochrome b distances only. Our estimates of phy- (Geniez, 2001; Sá-Sousa, 2000). logeny place P. hispanica sensu stricto as the sister taxon In this study, we describe a previously undetected to all North African forms. The mean uncorrected pair- lineage of Podarcis from the Iberian Peninsula. This line- wise distance between this form and all other lineages in age, represented in our work by sample Gal3, has been this group, computed using Mega v2.1 (Kumar et al., found only in the Xat, arid depression between Sierra de 2001) is 0.1101 § 0.0129 (minimum 0.088, maximum Cazorla and Sierra de Baza, in the Betic region, South- 0.13). Using the above-mentioned rate of substitution, eastern Spain. Previous works including more samples this yields a divergence time around 5.50 § 0.65 Mya, from Southeastern Spain (Harris et al., 2002b) only roughly coincident with the opening of the Strait of detected P. hispanica sensu stricto. Therefore, taken Gibraltar. Our estimates of relationships would then together, our data seem to indicate that the distribution indicate that Podarcis (h.) vaucheri crossed back across of this lineage may be very restricted. The same happens the Strait from North Africa to the Iberian Peninsula with the lineage from Jebel Sirwah, in Southern around 2.81 Mya. This scenario thus represents an alter- Morocco, which is known only from one locality. In our native biogeographical hypothesis to that suggested by estimates of phylogeny, the newly described lineage clus- Harris et al. (2002b). ters with the Northeastern type of P. hispanica with 79% The divergence time between the western Iberian bootstrap support in the ML analysis. The clustering of clade (comprising P. bocagei, P. hispanica type 1, P. car- these two forms may imply that in the past the ancestor bonelli and P. hispanica type 2) and the Southern Ibe- of this group had a widespread distribution throughout rian/North African clade, estimated from uncorrected eastern Iberia, and that the Betic form might be a relic of pairwise distances of cytochrome b, is about that ancient distribution. 7.02 § 0.65 Mya. Around 7.8–7.6 Mya, a salinity crisis Harris et al. (2002b) proposed a biogeographic sce- (the Tortonian salinity crisis; Krijgsman et al., 2000) nario for the colonization of North Africa by Podarcis in caused the formation of land bridges between the main- which these lizards crossed the Strait of Gibraltar twice land and small islets in what is now the Betic region. after its formation, both times from the Iberian Penin- These areas may have been colonized by lizards and sula to North Africa. This analysis thus discarded the could have acted as speciation hotspots, as has been sug- hypothesis of vicariance caused by the opening of the gested in other lacertids (Paulo, 2001). Later when the 272 C. Pinho et al. / Molecular Phylogenetics and Evolution 38 (2006) 266–273 two continents became connected (around 5.6 Mya), Felsenstein, J., 1981. Evolutionary trees from DNA sequences: a maxi- these were the lizards that colonized North Africa. This mum likelihood approach. J. Mol. Evol. 17, 368–376. W scenario of a greater similarity between Southern Span- Felsenstein, J., 1985. Con dence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783–791. ish and North African forms than that between South- Geniez, P. 2001. Variation géographique des lézards du genre Podarcis ern Iberian and other regions in the Peninsula has also (Reptilia, Sauria, Lacertidae) dans la péninsule Ibérique, l’Afrique been reported in other herpetofaunal species that exist du Nord et le sud de la . Mémoire présenté pour l’obtention on both sides of the Strait of Gibraltar (e.g., Pleurodeles, du diplôme de l’École Pratique des Hautes Etudes, Montpellier. (Batista et al., 2004; Veith et al., 2004)). Hall, T.A., 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp. Ser. 41, 95–98. Harris, D.J., 2002. Reassessment of comparative genetic distance in rep- 4. Conclusions tiles from the mitochondrial cytochrome b gene. Herpetol. J. 12, 85–86. Harris, D.J., Arnold, E.N., 1999. Relationships and evolution of wall Podarcis Although previous works suggested that Iberian and lizards (Reptilia: Lacertidae) based on mitochondrial DNA sequences. Copeia 1999 (3), 719–754. North-African Podarcis were a species complex, the phy- Harris, D.J., Batista, V., Carretero, M.A., Pinho, C., Sá-Sousa, P., logenetic relationships between these forms were weakly 2002a. Mitochondrial DNA sequence data conWrms the presence of supported. By increasing the extent of sequencing , Pérez-Mellado, 1981 in Southern Spain. Herpe- included in the analysis, we conWrm the existence of tozoa 15 (3/4), 188–190. cryptic forms and have now obtained a robust phylog- Harris, D.J., Carranza, S., Arnold, E.N., Pinho, C., Ferrand, N., 2002b. Complex biogeographical distribution of genetic variation within eny for these lizards. We also suggest that, unlike previ- Podarcis wall lizards across the Strait of Gibraltar. J. Biogeogr. 29, ous inferences had reported, the opening of the Strait of 1257–1262. Gibraltar and other geological events may well have Harris, D.J., Sá-Sousa, P., 2001. Species distinction and relationships of been important factors in the diversiWcation process. the western Iberian Podarcis lizards (Reptilia, Lacertidae) based on morphology and mitochondrial DNA sequences. Herpetol. J. 11, 129–136. Harris, D.J., Sá-Sousa, P., 2002. Molecular phylogenetics of Iberian Acknowledgments wall lizards (Podarcis): is Podarcis hispanica a species complex?. Mol. Phylogenet. Evol. 23, 75–81. This work was partially Wnanced by FCT (Fundação Harris, D.J., Pinho, C., Carretero, M.A., Corti, C., Böhme, W., 2005. Determination of genetic diversity within the insular Podar- para a Ciência e a Tecnologia) research projects POCTI/ cis tiliguerta using mtDNA sequence data, with a reassessment of 34547/BSE/2000 and POCTI/41912/BSE/2001. C. Pinho the phylogeny of Podarcis. Amphibia-Reptilia, 26, in press. is supported by a PhD grant (SFRH/BD/4620/2001) and Hedges, S.B., Bezy, R.L., 1993. Phylogeny of Xantusiid lizards: concern D.J. 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