Genetic Variability Within the Oudri's Fan-Footed Gecko Ptyodactylus

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YMPEV 3409 No. of Pages 6, Model 5G ARTICLE IN PRESS 30 October 2009

Molecular Phylogenetics and Evolution xxx (2009) xxx–xxx 1 Contents lists available at ScienceDirect

Molecular Phylogenetics and Evolution

journal homepage: www.elsevier.com/locate/ympev

2 Short Communication

3 Genetic variability within the Oudri’s fan-footed gecko Ptyodactylus oudrii

4 in North Africa assessed using mitochondrial and nuclear DNA sequences

a, a,b 5 Ana Perera *, D. James Harris

6 a CIBIO-UP, Centro de Investigação em Biodiversidade e Recursos Genéticos, Campus Agrário de Vairão, 4485-661 Vairão, Portugal 7 b Departamento de Zoologia e Antropologia, Faculdade de Ciências da Universidade do Porto, 4099-002 Porto, Portugal

8 article info abstract 2410 11 Article history: We analyse for the first time the genetic diversity within Ptyodactylus oudrii across the Maghreb. Two 25 12 Received 12 March 2009 mitochondrial (12s rRNA and 16s rRNA) and two nuclear (C-mos and ACM4) markers are used. The 26 13 Revised 24 August 2009 results confirm the specific status of P. oudrii and show high levels of intraspecific variability, indicative 27 14 Accepted 9 October 2009 of species complex. Lineages found are geographically concordant, and show similar patterns to that 28 15 Available online xxxx found in other species from the region. The study highlights once more the importance of the region 29 as a source of genetic diversity. 30 16 Keywords: Ó 2009 Published by Elsevier Inc. 31 17 Ptyodactylus 18 Maghreb 19 Gecko 20 Genetic variability 21 Nuclear markers 22 Mitochondrial markers 23 32 33 34 1. Introduction Ptyodactylus is one of the most characteristic genera of geckos, 56 easily recognisable by their widely dilated toes formed by two diver- 57 35 The traditional method of delimiting species, based on morpho- gent series of lamellae, similar to a fan (Schleich et al., 1996). Up to 58 36 logical characters changed drastically in the end of the seventies now, six species have been described on the basis of their morphol- 59 37 with the development of molecular techniques (Avise et al., ogy and distribution (Sindaco and Jeremcenko, 2008). Three of them 60 38 1979). Since then many new taxa have been redefined and cryptic are found in the Western Asia region (P. puiseuxi, P. guttatus, P. has- 61 39 species identified based on genetic variation (see for example, Ba- selquistii), one (P. homolepis) is restricted to Pakistan, and the 62 40 ker and Bradley, 2006). Such taxonomic reassessments are more remaining ones (P. ragazzii and P. oudrii) are found in North and Cen- 63 41 evident in groups that have suffered phenomena of morphological tral Africa (Sindaco and Jeremcenko, 2008). This genus has several 64 42 convergence or parallelism or that are morphologically more con- exceptional characteristics, including a high intraspecific morpho- 65 43 servative. A good example among reptiles are geckos. Most of the logical variability, and a wide range of ecological habits (Heimes, 66 44 species are very similar in general morphology and habits, and tax- 1987; Werner and Sivan, 1993, 1994). Such variability has compli- 67 45 onomy is based on some characters, such as digit morphology or cated attempts to settle the taxonomy of this group (Werner and Si- 68 46 pupil characteristics, that have been proved to be homoplastic van, 1994; Sindaco and Jeremcenko, 2008). Morphological studies 69 47 (Russell and Bauer, 2002; Gamble et al., 2008, and references on the three species living in Israel showed relatively high variation 70 48 therein). Studies on gecko phylogeny have revealed up to now at both intra and inter specific levels (Werner and Sivan, 1993). 71 49 the presence of cryptic species that were not detected by tradi- However, with the exception of a general analysis on geckos includ- 72 50 tional morphology based methods (Gamble et al., 2008) or even ing a couple of samples of Ptyodactylus (Gamble et al., 2008), no de- 73 51 species wrongly assigned to a genus (Carranza et al., 2002). Addi- tailed phylogenetic study has been performed to decipher the 74 52 tionally, most of the studiesUNCORRECTED show that geckos have exceptional genetic variation PROOF within this group. 75 53 levels of intraspecific variation in mtDNA (Harris et al., 2004a; Je- The Oudri’s fan-footed gecko P. oudrii is the most recent taxo- 76 54 Q1 sus et al., 2008) that might be a consequence of comparatively fas- nomically redefined species within this genus (Heimes, 1987). 77 55 ter rates of mtDNA evolution (Harris et al., 2004a). Considered a subspecies of P. hasselquistii, it was recently raised 78 to specific level based on electrophoretic and morphological evi- 79 dences as well as its completely different distribution pattern (Hei- 80 * Corresponding author. Fax: + 351 252 661 780. mes, 1987). P. oudrii is a saharien species distributed across the 81 E-mail addresses: [email protected] (A. Perera), [email protected] (D. Maghreb, including South Morocco, Central-Northern Algeria and 82 James Harris).

1055-7903/$ - see front matter Ó 2009 Published by Elsevier Inc. doi:10.1016/j.ympev.2009.10.020

Please cite this article in press as: Perera, A., James Harris, D. Genetic variability within the Oudri’s fan-footed gecko Ptyodactylus oudrii in North Africa assessed using mitochondrial and nuclear DNA sequences. Mol. Phylogenet. Evol. (2009), doi:10.1016/j.ympev.2009.10.020 YMPEV 3409 No. of Pages 6, Model 5G ARTICLE IN PRESS 30 October 2009

2 A. Perera, D. James Harris / Molecular Phylogenetics and Evolution xxx (2009) xxx–xxx

83 Western Tunisia (Bons and Geniez, 1996). This region has several species, we included two individuals from P. hasselquistii and three 111 84 geological, topographic, and climatic characteristics well differen- from P. ragazzii, plus the only four Ptyodactylus sequences available 112 85 tiated from the rest of the continent (Bons and Geniez, 1996), giv- from GenBank (two C-mos and two ACM4 sequences from P. has- 113 86 ing rise to complex patterns of diversity (Harris et al., 2004a; selquistii and P. guttatus; GenBank Accession Numbers EU293658, 114 87 Perera et al., 2007; Rato and Harris, 2008). In effect, most of the EU293659, EU293681 and EU293682; Gamble et al., 2008). Mito- 115 88 reptiles studied phylogeographically in this area have revealed chondrial sequences of Phyllodactylus xanti (Accession Numbers 116 89 unexpected levels of mitochondrial DNA variation and the exis- AY763273 and AY763284, Weiss and Hedges, 2007) and Tarentola 117 90 tence, in many cases, of cryptic taxa (Pinho et al., 2006; Mendonça mauritanica (Accession Numbers AY828481 and AY828456, Harris 118 91 and Harris, 2007; Perera et al., 2007; Barata et al., 2008). In partic- et al., 2004b) were used as outgroups. 119 92 ular, the two geckos from this region analysed up to now, Tarentola Total genomic DNA was extracted from small pieces of tail using 120 93 (Harris et al., 2004a) and Saurodactylus (Rato and Harris, 2008), standard saline methods (Sambrook et al., 1989). Fragments of two 121 94 show high levels of genetic variability. mitochondrial regions (12s rRNA and 16s rRNA) and two nuclear 122 95 In this study, we analyse for the first time the genetic variation regions (C-mos and ACM4) were analysed. Primers used in both 123 96 within the Oudri’s fan-footed gecko P. oudrii across the Maghreb. amplification and sequencing were 12sa and 12sb for the 12s rRNA 124 97 We use both mitochondrial (12s rRNA and 16s rRNA) and nuclear (Kocher et al., 1989), 16sa and 16sb for the 16s rRNA (Hedges et al., 125 98 markers (C-mos and ACM4) with different rates of evolution to 1991), TgF and TgR for the ACM4 (Gamble et al., 2008), and G73 126 99 (i) assess the genetic diversity within Ptyodactylus and to confirm and G74 for the C-mos (Gamble et al., 2008). PCR mix was carried 127 100 the recent taxonomic differentiation between P. oudrii and P. has- out in a 25 ll total volume, following conditions described in Har- 128 101 selquistii, ii) analyse the genetic variation within P. oudrii and the ris et al. (1998) for the mitochondrial markers and Gamble et al. 129 102 geographic distribution of the lineages, and (iii) corroborate the (2008) for the nuclear ones. Amplified fragments were sequenced 130 103 possible existence of faster rates of mtDNA evolution observed in on a 310 Applied Biosystem DNA sequencing apparatus. 131 104 gecko species (Harris et al., 2004a; Jesus et al., 2006; Rato and Har- 105 ris, 2008). 2.2. Phylogenetic analyses 132

106 2. Materials and methods Corrected sequencesPROOF were opened in MEGA4 software (Tamura 133 et al., 2007) Sequences were ﬁrst aligned using ClustalW with de- 134 107 2.1. DNA extraction, ampliﬁcation and sequencing fault parameters and then adjusted by hand, taking into account 135 known secondary structures (Kjer, 1995). 136 108 A total of 29 samples of P. oudrii from 12 localities were ana- Aligned mtDNA sequences were concatenated. Combined 137 109 Q3 lysed (Table 1 and Fig. 1). In order to evaluate the relative position mtDNA sequences were 885 bp (371 for 12s rRNA and 514 for 138 110 of P. oudrii and its taxonomic value relative to other Ptyodactylus 16s rRNA). Prior to phylogenetic analysis, mtDNA sequences were 139

Table 1 Codes, species, sampling localities and GenBank Accession Numbers for the samples included in this study. ‘‘–” indicates samples that were not analysed genetically.

Code Species Locality Country GenBank Accession Numbers 12s 16s c-mos ACM4 P1 P. oudrii Batna Algeria * * * * P2 P. oudrii Erfoud Morocco * * * * P3 P. oudrii Tayahiat Morocco * * * * P4 P. oudrii Tiguert Morocco * * * * P5 P. oudrii Talsinnt Morocco * * * * P6 P. oudrii Tayahiat Morocco * * * * P7 P. oudrii Batna Algeria * * * * P8 P. oudrii Tayahiat Morocco * * * * P9 P. oudrii Talsinnt Morocco * * – – P10 P. oudrii Hilala Morocco * * * * P11 P. oudrii Ida Ougnidif Morocco * * – – P12 P. oudrii Ida Ougnidif Morocco * * * * P13 P. oudrii Ammelne Morocco * * * * P14 P. oudrii Ida Ougnidif Morocco * * * * P15 P. oudrii Ammelne Morocco * * * * P16 P. oudrii Ida Ougnidif Morocco * * * * P17 P. oudrii Fask Morocco * * * * P18 P. oudrii Amerzgane Morocco * * * * P19 P. oudrii Amerzgane Morocco * * * * P20 P. oudrii Ñit Morrhad Morocco * * – – P21 P. oudrii Ñit Morrhad Morocco * * * * P22 P. oudrii UNCORRECTEDÑit Morrhad Morocco * * * * P23 P. oudrii Ñit Morrhad Morocco * * * * P24 P. oudrii Bou Gafer Morocco * * * * P25 P. oudrii Bou Gafer Morocco * * – – P26 P. oudrii Bou Gafer Morocco * * – – P27 P. oudrii Agouim Morocco * * * * P28 P. oudrii Saharian Atlas Algeria * * * * P29 P. oudrii Aurès Algeria * * – – P30 P. hasselquistii Mughsayl Oman * * * * P31 P. hasselquistii Oman Oman * * * * P32 P. ragazzii Egypt Egypt * * – – P33 P. ragazzii Mali Mali * * – – P34 P. ragazzii Hoggar Algeria * * – –

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Fig. 1. General map of the area of study and geographic location of the samples of P. oudrii analysed. Small map indicates the distribution of the other Ptyodactylus species included in the study (modiﬁed from Sindaco and Jeremcenko, 2008). Black circles: P. ragazzii, dark grey squares: P. guttatus, light grey squares: P. hasselquistii and white circles: P. oudrii.

140 analysed with Gblocks software (online version 0.91b, Castresana, informative sites. The most appropriate model of evolution for 180 141 2000) using a relaxed selection of blocks to eliminate poorly the data was the TVMef + G model. The analysis of mtDNA frag- 181 142 aligned positions (Talavera and Castresana, 2007). MtDNA se- ments identified two well supported phylogenetic groups (Fig. 2). 182 143 quences were analysed using maximum-parsimony (MP), maxi- The first one includes all samples of P. oudrii. The second lineage 183 144 mum likelihood (ML) and Bayesian inference methods. MtDNA groups the P. ragazzii and P. hasselquistii samples. These results re- 184 145 sequences were analysed using jModelTest 0.1.1 (Posada, 2008) veal wide genetic divergence between P. oudrii and P. hasselquistii, 185 146 to choose the best model of evolution under the corrected Akaike not even being sister taxa (Fig. 2), and thus support the elevation of 186 147 Information Criterion (AICc, Posada, 2008), and then they were im- P. oudrii to specific rank by Heimes (1987) based on morphological 187 148 ported into PAUP*4.0b10 (Swofford, 2002) for phylogenetic analy- and electrophoretic characteristics and different distribution. 188 149 sis. MP analysis was carried out using heuristic searches involving P. hasselquistii and P. ragazzii despite being included in the same 189 150 tree bisection and reconnection (TBR) branch swapping, with 100 main lineage are grouped into two monophyletic clades (Fig. 2). 190 151 replicates. For the ML analysis, the best model of nucleotide substi- The divergence between them is 19.3% (uncorrected distances for 191 152 tution obtained was used to estimate a tree with a 10 replicate 12s rRNA), similar to the values existing between the two species 192 153 heuristic search with100 random addition sequences. Support for and P. oudrii (18.4% and 17.2%, respectively, uncorrected p-dis- 193 154 nodes was assessed by bootstrap analysis (Felsenstein, 1985) with tances for 12s rRNA). One interesting result is the extremely high 194 155 1000 and 100 pseudo-replicates respectively. Bayesian analysis level of divergence within the two samples of P. hasselquistii from 195 156 was performed with MrBayes v.3.1.2. (Huelsenback and Ronquist, Oman (15.4% uncorrected genetic distance for 12s rRNA), much 196 157 2001). Analysis started with a randomly generated tree and was higher than the ones found within the other species included in 197 158 run for 1 Â 107 generations sampling one tree each 100 genera- this study (4.2% in P. ragazzii and 7.4% in P. oudrii, uncorrected val- 198 159 tions. Two independent replicates were conducted to determine ues for 12s rRNA, Fig. 2). Such surprising high divergence within 199 160 that analyses were not trapped at local optima (Huelsenbeck and this species is corroborated by the nuclear markers (Fig. 3). The 200 161 Bollback, 2001). Convergence of the MCMC chains was explored divergence value within P. hasselquistii for C-mos and ACM4 com- 201 162 graphically using the online program AWTY (Wilgenbusch et al., bined is 1.2%, contrasting with the 0.6% divergence observed with- 202 163 2004) and previous trees to stationarity (30,000) were burned-in. in P. oudrii, although such differences are evident in C-mos (1.6% 203 164 The remaining 70,000 trees were used to assess posterior probabil- against 0.4%, respectively), but less so in ACM4 (0.8% against 204 165 ities for nodal support. 0.7%, respectively). On the other hand, nuclear markers show also 205 166 For the nuclear markers, 29 C-mos sequences of 339 bp and 23 no variation between P. guttatus and P. hasselquistii from Egypt 206 167 ACM4 sequences of 339 bp were generated. In order to assess the (Fig. 3), while Werner and Sivan (1994) referred to P. hasselquistii 207 168 levels of genetic variation in the nuclear markers, we analysed both from Oman as P. guttatus. Taking into account all these evidences, 208 169 fragments using a Median Joining Network with the software Net- it is clear that the systematics of this group needs to be carefully 209 170 work 4.5.1.0. (ÓFluxus Technology; Bandelt et al., 1999). Heterozy- reassessed, especially in the Western Asia region, as other authors 210 171 gotes were represented as independentUNCORRECTED samples in the Median have suggested ( PROOFWerner and Sivan, 1994; Sindaco and Jeremcenko, 211 172 Joining Tree. Distribution of the samples in the lineages was similar 2008). This area has been indicated as the geographic origin of this 212 173 in both cases (not shown) and therefore we concatenated both nu- group (Heimes, 1987) and holds three of the six currently recogni- 213 174 clear fragments in a single sequence of 678 bp to estimate a com- sed species (Sindaco and Jeremcenko, 2008). Unfortunately, due to 214 175 bined Median Joining Network. the small number of samples available from this region, the genetic 215 variability found in this study is only orientative. More detailed 216 176 3. Results and discussion sampling throughout this region is needed to evaluate the com- 217 plete genetic variation within these groups in order to provide fur- 218 177 After eliminating poorly aligned positions a fragment of 838 bp ther evidence to state species limits. 219 178 of mtDNA (362 bp 12s rRNA and 476 bp 16s rRNA) was analysed Regarding P. oudrii, the mtDNA show that populations from 220 179 from which 486 were invariable, 352 were variable and 267 were Algeria and Morocco form well supported monophyletic clades 221

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Fig. 2. Phylogram derived from the Bayesian analyses of the 875 bp mtDNA fragments (12s rRNA and 16s rRNAPROOF concatenated; see the section Phylogenetic analyses in Section 2 for more details). MP and ML analyses produced the same estimate of relationships. Bootstrap support for MP and ML (above) and Bayesian probabilities (below) are given for each respective node. Nodes 100% supported by all three methods are represented by a square. (–) indicates nodes with less than 50% support. Divergence values (uncorrected p genetic distances) between the main P. oudrii clades are given (%). The tree was rooted using Tarentola mauritanica and Phyllodactylus xanti.

Fig. 3. Haplotype (median joining) network showing the relationships of Ptyodactylus species, inferred from 678 bp nuclear sequences (C-mos and ACM4 fragments concatenated). Each species is represented with a colour: light grey (P. oudrii), dark grey (P. hasselquistii) and stripped grey (P. guttatus). Heterozygotes are represented by lower case letters after the sample number and were included in the median joining network as independent samples. UNCORRECTED 222 (Fig. 2). In Morocco, three main mtDNA subclades are identified Arnold et al., 2007) that may represent a species complex (Perera 232 223 (Fig. 2). The first lineage includes individuals from Southern Mor- et al., 2007). 233 224 occo (Antiatlas and Jbel Ouarkziz). The second one groups samples High levels of intraspecific genetic divergence have already 234 225 from Western High Atlas and surrounding areas (Jbel el Sahro). And been reported for several gecko species (Harris et al., 2004a; Rato 235 226 finally, a third lineage is formed by individuals from the Eastern and Harris, 2008). In P. oudrii this could be either due to an in- 236 227 High Atlas. The levels of mtDNA variation found within this species creased rate of mtDNA evolution as in other geckos (Harris et al., 237 228 (7.4% divergence values for 12s rRNA) is much higher than the var- 2004a, 2004b; Jesus et al., 2006; Rato and Harris, 2008) or to the 238 229 iation observed within other groups such as Lacerta perspicillata, existence of cryptic species. The differentiation of the Algerian line- 239 230 considered part of the genus Scelarcis by some authors (between age and to a lesser extent the Antiatlas one is corroborated by the 240 231 5.2% and 6.6% uncorrected p-distance for 12s; Harris et al., 2003; nuclear markers, although the differentiation patterns of Eastern 241

A. Perera, D. James Harris / Molecular Phylogenetics and Evolution xxx (2009) xxx–xxx 5

242 and Western High Atlas populations is less evident (Fig. 3). Inter- of the region. The taxonomy of this group needs to be redefined, 305 243 estingly, Heimes (1987) found distinct colour patterns in individu- pending assessment of contact zones between lineages and detailed 306 244 als from Antiatlas and Algerian Atlas relative to other P. oudrii. analyses of their morphological variation. 307 245 Although skin colour can change depending on ecological charac- 246 Q2 teristics such as temperature (Arnold and Ovenden, 2002), such Acknowledgments 308 247 variation is mostly reduced to a lightening or darkening of the 248 main pattern, thus considerable variation in colour pattern We thank people who helped with sampling collection in Mor- 309 249 supports the hypothesis of cryptic species within P. oudrii. Thus, occo, and to J.C. Brito (CIBIO/UP) and specially S. Larbes (Tizi-Ouzou 310 250 it will be important in the future to combine detailed morpholog- University) for providing tissue samples from Algeria from his pri- 311 251 ical analyses to confirm putative differentiation patterns between vate collection. Thanks to S. Carranza for helpful comments and for 312 252 these lineages and to look for possible contact zones. providing outgroup material from Oman (permit number 08/2005) 313 253 The pattern of differentiation between Moroccan and Algerian and to S. Fahd (Tetouan University) and N. Naïm (Moroccan Forest 314 254 populations have been already observed in other reptile species, Ministry) for arranging permits to work in Morocco. We also 315 255 such as Acanthodactylus (Fonseca et al., in press)orTarentola (Har- thanks to A. Van der Meijden for helping with German translations. 316 256 ris et al., 2004a), suggesting the existence of common vicariance Partial funding was obtained by the FCT project PTDC/BIA-BDE/ 317 257 phenomena affecting the phylogeographic history of the species 74349/2006. A. Perera was supported by a FCT post-doctoral grant 318 258 inhabiting these regions (reviewed in Barata et al., 2008). The Mag- SFRH/BPD/26546/2006. 319 259 hreb is a biogeographically complex area and some barriers such as 260 the Atlas Mountains or the Moulouya River have molded the evo- References 320 261 lutionary history of some species, such as agamas (Brown et al., 262 2002) or tortoises (Álvarez et al., 2000). During the last million Álvarez, Y., Mateo, J.A., Andreu, A.C., Diaz-Paniagua, C., Diez, A., Bautista, J.M., 2000. 321 263 years the Maghreb suffered important ecological changes derived Mitochondrial DNA haplotyping of Testudo graeca on both continental sides of 322 the Straits of Gibraltar. J. 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The other three lin- evidence from 12S rRNA sequence data. Amphibia-Reptilia 24, 386–390. 368 Harris, D.J., Batista, V., Lymberakis, P., Carretero, M.A., 2004a. Complex estimates of 369 296 eages, distributed in Morocco, are geographically congruent. There evolutionary relationships in Tarentola mauritanica (Reptilia: Gekkonidae) 370 297 is a well defined southern group supported by nDNA and mtDNA, derived from mitochondrial DNA sequence. Mol. Phylogenet. Evol. 30, 855–859. 371 298 while the two remaining groups, one in east and the other in west Harris, D.J., Batista, V., Carretero, M.A., Ferrand, N., 2004b. Genetic variation in 372 373 299 High Atlas, are well differentiated mitochondrial lineages, but not Tarentola mauritanica (Reptilia, Gekkonidae) across the Strait of Gibraltar derived from mitochondrial and nuclear DNA sequences. Amphibia-Reptilia 25 374 300 by these slow evolving nuclear markers. 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