species on the rocks: systematics and biogeography of the...

Molecular Phylogenetics and Evolution 85 (2015) 208–220

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Molecular Phylogenetics and Evolution

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Species on the rocks: Systematics and biogeography of the rock-dwellingPtyodactylus geckos (Squamata: Phyllodactylidae) in North Africaand Arabia

http://dx.doi.org/10.1016/j.ympev.2015.02.0101055-7903/� 2015 Elsevier Inc. All rights reserved.

⇑ Corresponding author at: Department of Biology, Villanova University, 800Lancaster Avenue, Villanova, PA 19085, USA.

E-mail address: [email protected] (M. Metallinou).

Margarita Metallinou a,⇑, Jan Cervenka b, Pierre-André Crochet c, Lukáš Kratochvíl b, Thomas Wilms d,Philippe Geniez c, Mohammed Y. Shobrak e, José C. Brito f, Salvador Carranza a

a Institute of Evolutionary Biology (CSIC – University Pompeu Fabra), Passeig Marítim de la Barceloneta 37-49, 08003 Barcelona, Spainb Faculty of Science, Charles University in Prague, Department of Ecology, Vinicná 7, CZ-122 44 Prague, Czech Republicc CEFE UMR 5175, CNRS, Université de Montpellier, Université Paul-Valéry Montpellier, EPHE, 1919 route de Mende, 34293 Montpellier cedex 5, Franced Zoologischer Garten Frankfurt, Bernhard-Grzimek-Allee 1D, 60316 Frankfurt am Main, Germanye Biology Department, Faculty of Science, Taif University, 888, Taif, Saudi Arabiaf CIBIO, Centro de Investigação em Biodiversidade e Recursos Genéticos da Universidade do Porto, Instituto de Ciências Agrárias de Vairão, R. Padre Armando Quintas,4485-661 Vairão, Portugal

a r t i c l e i n f o a b s t r a c t

Article history:Received 28 August 2014Revised 7 February 2015Accepted 16 February 2015Available online 24 February 2015

Keywords:Multilocus phylogenyReptilesArid environmentsAllopatryUndescribed diversityTaxonomy

The understanding of the diversity of species in the Palearctic and the processes that have generated it isstill weak for large parts of the arid areas of North Africa and Arabia. Reptiles are among their mostremarkable representatives, with numerous groups well adapted to the diverse environments. ThePtyodactylus geckos are a strictly rock-dwelling genus with homogeneous morphology distributed acrossmountain formations and rocky plateaus from the western African ranges in Mauritania and the Maghrebto the eastern tip of the Arabian Peninsula, with an isolated species in southern Pakistan. Here, we use abroad sampling of 378 specimens, two mitochondrial (12S and cytb) and four nuclear (c-mos, MC1R,ACM4, RAG2) markers in order to obtain the first time-calibrated molecular phylogeny of the genusand place its diversification in a temporal framework. The results reveal high levels of intraspecific vari-ability, indicative of undescribed diversity, and they do not support the monophyly of one species (P.ragazzii). Ptyodactylus species are allopatric across most of their range, which may relate to their highpreference for the same type of structural habitat. The onset of their diversification is estimated to haveoccurred in the Late Oligocene, while that of several deep clades in the phylogeny took place during theLate Miocene, a period when an increase in aridification in North Africa and Arabia initiated.

� 2015 Elsevier Inc. All rights reserved.

1. Introduction

The biogeography of North Africa and the Sahara-Sahel has beenthe subject of extensive research, often using reptiles as modelorganisms (Carranza et al., 2008; Pook et al., 2009; Metallinouet al., 2012; Rato et al., 2012; Brito et al., 2014). The neighboringArabian Peninsula has received less attention, albeit being veryinteresting from a biogeographical point of view, with diverselandscapes and numerous endemics (Arnold, 1977, 1980a andother articles in the same volumes; Edgell, 2006; Carranza andArnold, 2012; Cox et al., 2012). These studies have addressed inter-esting questions on the origin and relationships between North

African and Arabian reptile faunas, the relative times of speciationevents in Sahara and Arabia and have revealed patterns ofcolonization and diversification in these arid environments.These areas combine a variety of different habitats fromMediterranean and transitional Afro-tropical to very arid desert,all of which have been colonized by different groups of reptiles(e.g. Arnold, 1980b, 1986; Sindaco and Jeremcenko, 2008; Geniezet al., 2011; Metallinou et al., 2012; Sindaco et al., 2013; Šmídet al., 2013a). Notably, they include regions that have been predict-ed to harbor higher species richness than is currently known(Ficetola et al., 2013) and evidence of previously undescribeddiversity is continuously coming to light (e.g. Busais and Joger,2011a; Geniez et al., 2011; Carranza and Arnold, 2012; Šmídet al., 2013b; Metallinou and Carranza, 2013; Badiane et al.,2014; Vasconcelos and Carranza, 2014).

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M. Metallinou et al. / Molecular Phylogenetics and Evolution 85 (2015) 208–220 209

The geckos of the genus Ptyodactylus Goldfuss (1820) extendtheir distribution across a very large area of North Africa and theMiddle East and also include a single known representative fromsouthern Pakistan (Sindaco and Jeremcenko, 2008; Fig. 1). Like othergroups of reptiles from these arid areas [e.g. Acanthodactylus,Chalcides (Sindaco and Jeremcenko, 2008)], they can occupy differ-ent climatic regions, from the mesic Mediterranean strips to ariddeserts, and they also extend to the savannah region of the Sahelsouth of the Sahara desert. Ptyodactylus geckos are easily identifiableby their toe morphology: their slender digits end in a triangular padthat bears multiple divided lamellae in a fan-like pattern with aretractile claw in the distal expansion. They are relatively large(most of the species between 75 and 100 mm of snout-vent length;Heimes, 1987; Werner and Sivan, 1993; Schleich et al., 1996; Baha ElDin, 2006; Gardner, 2013) with slender body, large head andrelatively long limbs. All Ptyodactylus species are rock-climbing spe-cialists and exploit very similar structural habitats: rocky substratessuch as cliffs, boulders and caves (Baha El Din, 2006). They lack anyfemoral or preanal pores and can communicate using sound, whichin most species consists of a series of loud multiple clicks (Werner,1965; Frankenberg, 1974; Werner and Sivan, 1994). Although theirvertical pupil denotes a mainly nocturnal activity, most of thespecies can also have diurnal activity and one species, P. puiseuxiBoutan, 1893, is mainly diurnal, with morning and afternoon activitypeaks (Werner and Sivan, 1994; Disi et al., 2001). Ptyodactylusspecies are mainly allopatric or parapatric, but in the few areaswhere two species coexist, there is a clear temporal or spatial parti-tioning of foraging activity patterns (Baha El Din, 2006; Kratochvíl &Cervenka, pers. obs.).

Taxonomic work on Ptyodactylus has been hampered by thesuperficial similarity of its species (Arnold, 1980a; Baha El Din,2006). Although many Ptyodactylus taxa were described duringthe 19th century (often as ‘‘variations’’ of P. hasselquistii), for manyyears authors followed a conservative approach, treating mostforms as subspecies of P. hasselquistii (Donndorff, 1798)(Anderson, 1898; Loveridge, 1947; Werner, 1965; Kluge, 1967).After the works of Arnold (1986), Heimes (1987) and Schleichet al. (1996), a total of six species were recognized: two fromNorth Africa (P. oudrii Lataste, 1880, P. ragazzii Anderson, 1898),three the Middle East (P. hasselquistii, P. guttatus Heyden, 1827, P.puiseuxi), and one species restricted to south Pakistan (P. homolepisBlanford, 1876). Werner and Sivan (1993, 1994) studied the mor-phology and ecology of the three species occurring within theLevant (P. guttatus, P. puiseuxi and P. hasselquistii) and delineatedwith precision their distribution and sympatric areas. More recent-ly, Baha El Din (2006) ‘‘tentatively’’ elevated P. siphonorhinaAnderson (1896) from Egypt to species rank, hence recognizingseven species in total. Nevertheless, limited access to material fromthe extensive range of the genus hindered further systematic work,paucity even more pronounced in Arabia. Perera and Harris(2010a) and Froufe et al. (2013) demonstrated that P. oudrii andP. ragazzii, respectively, include several geographically concordantgenetic lineages that could correspond to distinct species.However, given the lack of material for genetic and morphologicalanalysis from the complete species’ range, they did not proposeany formal taxonomic changes. A taxonomic study lacking contex-tualization was recently published by Nazarov et al. (2013), com-plicating the picture even more by describing two new speciesbased on very few specimens collected from single localities inJordan (P. ananjevae Nazarov, Melnikov, Melnikova, 2013) andsouth Oman (P. dhofarensis Nazarov, Melnikov, Melnikova, 2013),and another one from four geographically very close localities inthe Hajar Mountains of North Oman (P. orlovi Nazarov, Melnikov,Melnikova, 2013). The lack of information on the distribution ofthese three species (reported only from the type localities or their

vicinity) has forced Gardner (2013) in the most recent field guideto treat them as part of the Ptyodactylus hasselquistii species com-plex until their taxonomy and distribution are clarified.

In this work, we assemble for the first time a geographicallycomprehensive dataset (Fig. 1) including representatives of allbut one (P. homolepis) Ptyodactylus members, integrating a largeamount of new data with information from all previous works onthe genus in one multilocus phylogenetic study. We investigatethe relationships among the currently known forms and the diver-gent lineages reported, assessing the overall diversity of the genus.We reconstruct these relationships within a calibrated timeframe,which allows us to explore the biogeographical history of thiswidely distributed group. Finally, we provide the essential frame-work and taxonomic information for a further systematic assess-ment of the genus Ptyodactylus.

2. Materials and methods

2.1. Taxon sampling and nomenclature

A total of 378 representatives of all but one currently acceptedPtyodactylus species from 220 different localities throughout NorthAfrica and the Middle East were included in the study (Fig. 1). Datafor 26 specimens of P. oudrii (Perera and Harris, 2010a) and 22 of P.ragazzii (Froufe et al., 2013) were retrieved from GenBank (seebelow). Based on recent phylogenetic assessments and availabilityof material, a specimen of Asaccus gallagheri was used as outgroup(Gamble et al., 2008, 2011, 2012; Pyron et al., 2013). Informationon all specimens included in the analyses is provided in Table S1,Supplementary material.

We follow nomenclature from Sindaco and Jeremcenko (2008)and Baha el Din (2006). Like Gardner (2013), as a result of the lackof information on the distribution, unavailability in publicrepositories of the DNA sequences used for the descriptions, andfailure to compare the new species with the relevant material (P.hasselquistii from across its distribution range), we treat the threespecies described recently by Nazarov et al. (2013) as part of theP. hasselquistii species complex until their taxonomic status isrevised (work in progress).

2.2. DNA extraction and sequencing

Genomic DNA was extracted from ethanol-preserved tail, ton-gue or liver tissue using either the DNeasy Blood & Tissue Kit(Qiagen, Valencia, CA, USA) or the SpeedTools Tissue DNAExtraction kit (Biotools, Madrid, Spain). A fragment of the 12SrRNA (12S) mitochondrial gene was PCR-amplified and sequencedin both directions for 329 samples, while 12S sequences weredownloaded from GenBank for the remaining 48 samples. Giventhe very high level of genetic variability detected in the 12S marker(see Results) and the lack of prior systematic information regardingeither morphological or genetic features of the organisms, theGMYC method (see next section) was used to select a subset of145 samples for further gene sampling: of these, 139 samples werenewly sequenced for an additional mitochondrial gene fragment,cytochrome b (cytb), and four nuclear ones: oocyte maturation fac-tor MOS (c-mos), melanocortin 1 receptor (MC1R), acetylcholiner-gic receptor M4 (ACM4) and recombination activating protein 2(RAG2). Available sequences of c-mos and ACM4 for the remaining6 samples were downloaded from GenBank. Primers and PCR con-ditions are listed in Table S2, Supplementary material. Contigassembly and translation to aminoacids was done in Geneious5.3 (Drummond et al., 2010), and heterozygote sites were codedaccording to IUPAC ambiguity codes.

A B150

170

169

149

160

168161

162

157

159158

155154

153

167166

152151

164163

156165

194

193

192

203

214

213212

202

206

201

195

205

204

196

197

200199211

207208

198

210209

216

217

80

215

19

1

17

3 2

18

5

29

4

2827

16 26

1525

14

2412

13

119

2310

2220

821

67

172

186

174 175179180

187173

176

181

183182

188

184

177185

171

Saharan AtlasHigh Atlas

Anti-

Atlas

Tassili

Hoggar

AirMountains

AssabaMountains

WadiRumGulf of Suez

150 3000 Km250 5000 Km

178

30˚N

20˚N

10˚N

10˚W 0˚ 10˚E 20˚E 30˚E 40˚E 50˚E

Clade B

P. guttatus

P. puiseuxi

P. siphonorhinaP. ragazzii

P. togoensis*

P. oudrii

Clade A

P. hasselquistii species complex

C

190189

191

323334

35

494839

50 5152 40

4746453843

44

41 4237

36

143141

144

145 146

147

148

142

130134

133132

131

135117

120118

121

119

6072

53

137

138139

140

136

116

104

115

106

107

108

127

105 123126

125

128129 124

11461

113

30

31

5668

6755

57

6364

62

6665

111

100

101

102

109

99112

110

98

122

54

73

103

58

69

7059

71

818

59597

94

96

93

92

89

79

77

87 88

78

80

86

75

84

76

83

7485

82

90

91

ArabianSea

PersianGulf Gulf

of Oman

Red Sea

Gulfof Aden

Rub al Khali

Dhofar

Hadhramaut

Mountains

Asir

Mountains

Hejaz

Mountains

Gulf of Suez

Hajar

Mountains

250 5000 Km

P. guttatus

P. puiseuxi

P. siphonorhina

P. ragazzii

P. togoensis*

P. oudrii

P. hasselquistiispecies complex

A B

C

Fig. 1. Sampling localities of the specimens included in this study. Type localities of the different species are marked with star symbols of the same color as the species. In theupper left map, known distribution ranges of Ptyodactylus species. Colored ranges are drawn qualitatively based on Baha El Din (2006) and Sindaco and Jeremcenko (2008) andupdated with new records reported herein. Sketched area represents the distribution range of ‘‘P. ragazzii’’, which is herein shown to be polyphyletic. Type localities of species ofthe P. hasselquistii complex described in Nazarov et al. (2013), for which no ranges are known, are represented by grey star symbols. (⁄) P. togoensis, formerly a synonym of P.ragazzii, is herein assigned to the western populations of ‘‘P. ragazzii’’, which is shown to be polyphyletic (see Sections 3.2 and 4.3.1 for more details). Colors refer also to Fig. 2, andlocality numbers to Fig. 2, Supplementary Fig. S1 and Supplementary Table S1.

210 M. Metallinou et al. / Molecular Phylogenetics and Evolution 85 (2015) 208–220


2.3. Phylogenetic analyses and topological tests

Multiple sequence alignments were performed with the onlineapplication of MAFFT v.7 (Katoh and Toh, 2008) applying defaultsettings to parameters (Auto strategy, Gap opening penalty: 1.53,Offset value: 0.0). The models of molecular evolution were selectedunder the AIC information criterion using jModelTest v2.1.3(Guindon and Gascuel, 2003; Darriba et al., 2012). The best-fit par-titioning scheme and models of molecular evolution for the datasetincluding six concatenated loci were selected with PartitionFinderv.1.1.0 (Lanfear et al., 2012) under the same criterion and with thefollowing parameters set: branchlengths: unlinked; models:mrbayes; search: greedy; for the protein-coding genes, the firstand second position were tested as one partition, and the thirdas another (see all models selected in Table 1). For the taxa accept-ed herein, uncorrected p-distances with pairwise deletion for thetwo mitochondrial and four nuclear gene fragments were estimat-ed in MEGA v.5 (Tamura et al., 2011) using the reduced dataset of145 selected samples.

Both the 12S dataset and the concatenated dataset of the select-ed specimens (see above) were analyzed with maximum likelihood(ML) and Bayesian inference (BI) methods. Models and partitionswere set as shown in Table 1. ML analyses were done withRAxML 7.4.2 (Stamatakis, 2006) as implemented in raxmlGUI(Silvestro and Michalak, 2012), performing 100 ML inferences.Node support was assessed with bootstrap analysis (Felsenstein,1985) of 1000 replicates. The software BEAST v.1.7.5 (Drummondand Rambaut, 2007) was used for BI analyses, where the meanoverall rate was fixed to 1.0. Two individual runs of 4 � 107 gen-erations were performed with a sampling frequency of 4000. Thefollowing models and prior settings were applied, otherwise theywere left by default: Constant Size coalescent tree prior; randomstarting tree; base substitution prior Uniform (0, 100); alpha priorUniform (0, 10). In the analysis of the concatenated dataset, parti-tions and clock models were unlinked and the xml file wasmanually modified to set Ambiguities = ’’true’’ for the nuclear genefragments partition in order to account for variability in theheterozygous positions, instead of treating them as missing data.

Table 1Basic information on genetic markers and datasets.

Dataset N� ind.a Gene fragment N� seq.b

12S 377 12S 377

Six-marker concatenated 145 12S 144cytb 127cmos 134mc1r 122acm4 113rag2 109

7 tips BEAST dating 7 12S 7cytb 6cmos 7mc1r 7acm4 6rag2 6

72 tips BEAST dating 72 12S 72cytb 59cmos 62mc1r 57acm4 53rag2 49

a Number of individuals.b Number of sequences.c Alignment length.d Number of variable positions.e Number of parsimony-informative positions.f Partition scheme and model selected in PartitionFinder or jModelTest (see Section 2

Posterior trace plots and effective sample sizes of the runs weremonitored in Tracer v1.5 (Rambaut and Drummond, 2007) toensure convergence. The results of the individual runs were com-bined in LogCombiner discarding 10% of the samples and the ultra-metric tree was produced with TreeAnnotator (both provided withthe BEAST package).

The general mixed Yule-coalescent model (GMYC) (Pons et al.,2006; Fujisawa and Barraclough, 2013) analysis was performedusing the SPLITS package (Ezard et al., 2009) in R (RDevelopment Core Team, 2014) in order to objectively identifythe divergent lineages in the Ptyodactylus phylogeny. We usedthe latest version of GMYC (Fujisawa and Barraclough, 2013) andtested both the single-threshold and the multiple-thresholdapproach on the 12S ultrametric tree obtained (see above). Basedon the results of this analysis, one or a few specimens of eachGMYC entity were selected for further gene sampling.

The Approximately-Unbiased (AU) (Shimodaira, 2002) and theShimodaira-Hasegawa (SH) (Shimodaira and Hasegawa, 1999)tests were employed to test the monophyly of ‘‘P. ragazzii’’. Per-sitelog likelihoods for the unconstrained (best) ML tree and a treewhere all samples identified as ‘‘P. ragazzii’’ were forced in a mono-phyletic group were estimated in RAxML as above. P-values wereestimated in CONSEL (Shimodaira and Hasegawa, 2001).

2.4. Estimation of divergence times

Absolute divergence times were estimated in a Bayesian frame-work with the application of previously calculated mean rates ofmolecular evolution for the two mitochondrial markers 12S (mean:0.00755, stdev: 0.00247, substitutions per site per million years)and cytb (mean: 0.0228, stdev: 0.00806) (Carranza and Arnold,2012). The mean rate of the mitochondrial 12S marker calculatedby Carranza and Arnold (2012) coincides with the mean rate calcu-lated for the geckos of the genus Stenodactylus based on partiallydifferent calibration points (mean: 0.00701) (Metallinou et al.,2012). We performed two sets of analyses applying only the 12Srate, and applying both 12S and cytb rates, to compare the results.As a result of the high level of hidden diversity uncovered in this

Len.c Var.d PIe Scheme and modelf

403 216 209 GTR + I + G

406 211 202 12S+(1st + 2nd)cytb: GTR + I + G395 206 198 3rd cytb: GTR + I + G415 55 46 nuclears: GTR + I + G668 94 84431 44 31410 46 35

390 131 77 GTR + G395 149 66 TrN + G415 22 7 HKY + G668 47 14 GTR + G431 16 6 HKY + G410 19 6 HKY

405 209 186 GTR + I + G394 202 189 GTR + I + G415 51 39 HKY + G668 85 70 GTR + I + G431 37 23 TrN + I410 39 23 GTR + G

).


study (see below), which complicates an accurate knowledge of thespecies limits, we chose to conduct concatenated gene tree ana-lyses testing two different combinations of the number of termi-nals to explore if this affected the results of the calibrationanalyses. A first combination including one representative fromeach one of the seven species considered in the present study(see Fig. 1), and a second including one representative from eachone of the lineages identified by the GMYC analysis (72 specimensin total). These concatenated datasets were analyzed partitioningper gene fragment with BEAST v.1.7.5, performing three runs of10 � 107 generations with a sampling frequency of 10,000. Meanrates for the mitochondrial markers were used for calibration(Uncorrelated Lognormal clock) with a normal prior as explainedabove, models as shown in Table 1, a Yule prior was selected forthe tree and the rest of the parameter priors and the xml filemodifications were as in previous BEAST analyses. An additionalrun was performed for all the analyses sampling entirely fromthe prior in order to ensure that the data were sufficientlyinformative.

2.5. Biogeographical analyses

A Bayesian discrete phylogeographic approach was used for thereconstruction of ancestral areas in BEAST 1.7.5, using the BayesianStochastic Search Variable Selection (BSSVS) (Lemey et al., 2009).The same dataset as in the 72-clade dating analysis was analyzed(see above). Geographical traits were attributed to the specimensincluded in the dataset based on their locality information and afive-region delimitation (Fig. 3) based on the biogeographical par-titioning of Africa with multivariate methods (Linder et al., 2012)and relevant biodiversity and herpetological literature (Šmídet al., 2013a; Brito et al., 2014). An exponential prior was usedfor the discrete location state rate (locations.clock.rate) withmean = 1.0 and offset = 0, and two runs of 20 � 107 generationswith a sampling frequency of 20,000 were performed. All other set-tings were as above.

3. Results

3.1. Taxon sampling and data matrices

The 12S dataset included 377 terminals (excluding the out-group) and 403 base pairs (bp), while the six-marker concatenateddataset included 145 terminals and a total of 2725 bp. Table 1 listsinformation on all markers and datasets used in the phylogeneticand dating analyses with their corresponding numbers of variableand parsimony informative positions, as well as models of molecu-lar evolution selected for each one. The uncorrected p-distances forthe mitochondrial and nuclear gene fragments between and withinthe seven species considered in the present work are summarizedin Table S3 of the Supplementary material.

3.2. Phylogenetic analyses and topological tests

The ML and BI analyses of the 12S dataset yielded almost iden-tical results, except for few, not well-supported, nodes (Fig. S1,Supplementary material). The level of genetic variability withinPtyodactylus is very high and this is reflected in the results of theGMYC analysis that recognized 72 different lineages with thesingle threshold approach and 116 with the multiple thresholdone (see Table S1). The result of the likelihood ratio test wassignificant (p < 0.05) in both cases indicating that the null model(i.e. single population) could be rejected. Herein, we consider theresults of the former estimation (72 clades), as it is probable that

non-uniform geographic sampling in our dataset is causingoversplitting (Talavera et al., 2013).

The levels of intraspecific variability in the 12S mitochondrialgene ranged between 1.49% and 11.31%, for the eastern clade of‘‘P. ragazzii’’ and the P. hasselquistii species complex, respectively.The levels of interspecific variability for the same marker rangedbetween 9.4% for the comparison between P. siphonorhina and P.guttatus, and 20.4% for the comparison between the two clades of‘‘P. ragazzii’’ (see below) (Table S3, Supplementary material).

In order to ensure a good representation of the geneticvariability and good geographical coverage in further multilocusanalyses, a total of 145 specimens were selected based on theresults of the GMYC analysis and taking into account the geo-graphical distribution of the localities. Of the 72 GMYC entitiesrecovered, only seven (six single specimens clades and one includ-ing two specimens) were not represented in the multilocus datasetas a result of the failure to amplify the nuclear gene fragments forthese samples (see Table S1, Supplementary material). Thesummary of the results of ML and BI analyses of this concatenateddataset are presented in Fig. 2. The topologies inferred were, onceagain, almost identical, with the main difference being the groupthat branches first in each case: in the ML analysis this was theclade formed by the West African populations of ‘‘P. ragazzii’’, likein the analyses of the 12S dataset with both methodologies, but inthe BI analysis it was P. oudrii, although the clade grouping allother species was not supported (posterior probability, pp: 0.60).

The species ‘‘Ptyodactylus ragazzii’’ as traditionally recognized(i.e. including both East and West African populations, see below)was not recovered as monophyletic in any of the analyses carriedout. The samples from East Africa (Ethiopia and Djibouti), closestto the type locality in present-day Eritrea and Ethiopia (Fig. 1), andthe samples from the western part of the species’ distribution(Mauritania, Mali, Burkina Faso, Benin, Niger, Algeria) form twowell-supported clades that branch in different places of the phy-logeny, making ‘‘P. ragazzii’’ polyphyletic (Figs. 1 and 2). The resultsof the topological tests indicate that, based on our multilocus data-set, the monophyly of this species can be clearly rejected (AU:0.006, SH: 0.011). In order to resolve the polyphyly of ‘‘P. ragazzii’’,we herein separate the western populations as a distinct species,to which we tentatively apply the name P. togoensis, which wasintroduced as a ‘‘variety’’ of P. hasselquistii from Northern Togo byTornier (1901) and synonymized with P. ragazzii by Werner(1919). The type locality of this form is found approximately115 km west of our southernmost sample from this area (see Fig. 1).

The clade of P. siphonorhina and P. guttatus, and their sisterrelationship with P. puiseuxi (all together forming clade C) arewell-supported in all analyses. Lastly, all the 12S and concatenateddataset analyses recovered the P. hasselquistii species complex asmonophyletic, even though support for this relationship was lowin ML analyses. Within this highly variable group, there are 37GMYC lineages (see Table S1, Supplementary materials), whichare divided into two well-supported clades. Clade A is distributedacross most of the species’ range, including Sudan and Egypt,across the Nile Valley and the northwestern coast of the Red Sea;northern, central and the western coast of the Arabian Peninsula;some inland parts of the Asir Mountains in southern Arabia andacross the Hajar Mountain range in Oman and the U.A.E. (Fig. 1).Clade B is distributed across the Asir Mountains (from Taif inSaudi Arabia down to southwestern Yemen), the coastal parts ofsouthern Yemen and the Hadhramaut, east to Dhofar in southOman (Fig. 1).

3.3. Divergence time estimation analyses

As shown in Table 2, the differences between the estimatedages using one representative of each species considered in the

ZFMK-E38_Egypt_1

NHMC.80.3.49.1_Syria_180

BEV.T3757_Jordan_13

JEM650_Yemen_136

Poud-P2_Morocco_157

SPM002962-105_Jordan_7

ZFMK 60844_Syria_178

NHMC.80.3.47.15_Egypt_2

IBE-S10231_Saudi Arabia_58

J 06/04_Jordan_174

SUD12/2010-50_Sudan_84

Prag-3038_Mauritania_200


DNA44_Ethiopia_190

Poud-P5_Morocco_161


DJI-02_Djibouti_189

S10632_Algeria_192

Jor44_Jordan_8

Poud-P24_Morocco_155

TW1008_Jordan_175

JOR003_Jordan_15





TW1009_Jordan_172

UAE37_UAE_40

TW1007_Jordan_175

Poud-908_Algeria_150


NHMC.80.3.48.6_Syria_183

AO138_Oman_141


BEV.T519_Algeria_193

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JEM122_Yemen_133

BEV.10369_Egypt_79

SPM002388-87_Mali_

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990J_Syria_179


PTY12_Morocco_162

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JEM508_Yemen_117

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BEV.7211_Egypt_93


BEV.8802_Israel_4

JOR042_Jordan_8

07-12M_Morocco_154



NHMC.80.3.47.8_Jordan_6

JOR100_Jordan_173

07-09M_Morocco_153

ZFMK 70963_Oman_38

BEV.7210_Egypt_215


DJI-01_Djibouti_189


DJI-03_Djibouti_189


SPM002378-2_Egypt_76

TW1000_Morocco_

JOR071_Jordan_12

BEV.9002_Egypt_77


SPM002970-1_Egypt_76




BEV.8105_Morocco_152

BEV.T3039_Benin_195Prag-429_Burkina Faso_196

BEV.T3958_Jordan_95

JEM132_Yemen_132

BEV.2279_Niger_203

TW1005_Jordan_11


SPM001852_Egypt_

OM04/2010-54_Oman_142

BEV.8486_Israel_94

NHMC.80.3.47.9_Jordan_14

BEV.8870_Mali_197

Jor35_Jordan_5

TW1006_Jordan_9

TW1001_Morocco_

Prag-349_Niger_202

JEM424_Yemen_130IBE-S10297_Saudi Arabia_125

BEV.T394_Egypt_3

06-14b_Israel_171



Prag-430_Burkina Faso_196

BEV.10359_Egypt_78

BEV.10156_Algeria_194

JEM110_Yemen_140JEM115_Yemen_138



JOR128_Jordan_16

JEM37_Yemen_60

Jor10_Jordan_15

AO60_Oman_37

JOR040_Jordan_5

OM7_Oman_39

JEM515_Yemen_118

Prag-409_Niger_201

JOR036_Jordan_5


IBE-S993_Morocco_159

OM04/2010-56_Oman_142

BEV.9000_Egypt_75


SPM002952-48_Egypt_216

JEM639_Yemen_137



UAE22_Oman_32

BEV.8998_Egypt_74

BEV.12105_Lebanon_177

JEM62_Yemen_143

BEV.7244_Egypt_92



SUD12/2010-98_Sudan_82

JOR050_Jordan_10

TMHC179_Ethiopia_191

UAE41_Oman_36

SUD12/2010-31_Sudan_83

ZFMK87104_Saudi Arabia_30

11022012A_Morocco_160


NHMC.80.3.49.15_Jordan_176

TW1011_UAE_34



JEM559_Yemen_119IBE-S10085_Saudi Arabia_113


TW1030_UAE_33

ZFMK 87179_Saudi Arabia_61

96

94

99

97

78

100

100

100

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9983

Clade A

Clade B

Clade C

P. oudrii

P. hasselquistiicomplex

P. siphonorhina

P. guttatus

P. puiseuxi

P. ragazzii

0.1

P. togoensis*

100

100

100100

99

88

100

100

100

96

99

Fig. 2. ML tree of the genus Ptyodactylus inferred using the six-locus concatenated dataset. Numbers next to the node refer to bootstrap support (values above 70), blackcircles indicate posterior probability in BI analysis above 0.95 and empty circles mark nodes that are not recovered in the latter analysis. The tree was rooted using Asaccusgallagheri. (⁄) P. togoensis, formerly a synonym of P. ragazzii, is herein assigned to the western populations of ‘‘P. ragazzii’’, which is shown to be polyphyletic (see Sections 3.2and 4.3.1 for more details). Codes of the tips represent Specimen Number, followed by Country of origin, followed by locality code, as illustrated in Fig. 2. Detailed informationon all samples is listed in Supplementary Table S1. Inset picture shows a specimen of P. cf. hasselquistii from Oman.


Table 2Comparison of divergence time estimations.

Node 72 clades 7 species

12Sonly

12S + cytba 12Sonly

12S + cytb

Root 25.66(16.10–38.03)

27.07(17.66–37.96)

24.43(12.61–39.18)

26.53(13.45–42.23)

togoensis 14.68(8.44–22.11)

15.47(9.52–22.42)

– –

oudrii 12.02(7.09–17.77)

12.69(8.06–18.10)

– –

hasselquistii + ragazzii + CladeC

18.09(10.96–26.23)

19.10(12.51–26.75)

18.93(9.70–30.20)

20.59(10.46–32.71)

ragazzii + Clade C 16.37(10.08–24.15)

17.29(11.44–24.71)

15.64(7.85–24.78)

17.01(8.91–27.37)

Clade C 9.57(5.83–14.26)

10.10(6.38–14.38)

8.68(4.45–13.76)

9.47(4.90–15.13)

siphonorhina + guttatus 6.02(3.58–9.00)

6.36(3.87–9.09)

5.35(2.64–8.60)

5.83(2.89–9.44)

puiseuxi 5.52(3.13–8.25)

5.83(3.54–8.53)

– –

siphonorhina 2.96(1.61–4.50)

3.13(1.85–4.60)

– –

guttatus 2.72(1.43–4.26)

2.88(1.62–4.39)

– –

hasselquistii complex 16.20(9.62–23.40)

17.11(11.18–24.12)

– –

Clade A 11.34(6.74–16.53)

11.99(7.66–16.83)

– –

Clade B 8.90(5.40–13.24)

9.40(5.81–13.32)

– –

a This set of dates is referred to in the text.


present study (Fig. 2) and one representative of each of the 72GMYC lineages (Table S1, Supplementary materials) are not sub-stantial. The posterior mean rates for the mitochondrial markerswere 0.0258 for cytb and 0.0104 for 12S. Given the relatively highvalue of the standard deviation applied to the calibration rates ofboth 12S and cytb genes, the values of the 95% highest probabilitydensities (HPDs) were quite wide for all the nodes in the phylogeny(see Table 2). Also, the ages estimated applying solely the 12S rateor both 12S and cytb rates were quite similar, with the latter beingslightly older. Therefore, herein, we indicate the ages estimatedusing both rates and the 72-clades dataset, where more nodesare represented. This estimation suggests that diversification inthe genus Ptyodactylus started in the Late Oligocene (�27 Ma ago).

3.4. Ancestral area reconstruction

The results of the ancestral area reconstruction analysis usingthe BSSVS approach are presented in Fig. 3, on an annotated treewithin a temporal framework. Node ages were very similar tothe dating-only analysis of the same dataset. The area inferred tohave the highest posterior probability is attributed to each ances-tral node of the tree and in most cases posterior area probabilitywas above 0.80. The ancestral area for the P. hasselquistii radiationwas Arabia, and that of the clade grouping P. ragazzii and Clade Cwas most probably somewhere in northeastern Africa or the

Levant region. The ancestor of these two sister clades was mostprobably Arabian, but could also have been in the latter region.Finally, for the two most basal nodes in the phylogeny, westernNorth Africa sums the highest probability as the ancestral area,but inferences are far from conclusive.

4. Discussion

This study represents the first comprehensive systematic workon the genus Ptyodactylus, providing an insight on the phylogenyand systematics of this widely distributed group of North Africaand Arabia, based on multilocus molecular data. Taxonomic sam-pling has been shown to be very important in phylogenetic, speciesdelimitation, diversification and other studies (Dimitrov et al.,2012; Talavera et al., 2013; Ruane et al., 2014). Here, the samplingcovers the largest part of the known distribution range of the spe-cies (Heimes, 1987; Werner and Sivan, 1994; Sindaco andJeremcenko, 2008; see Fig. 1). For three of the members of thisgroup, P. puiseuxi, P. siphonorhina and P. ragazzii (see Results),DNA sequences are provided for the first time. Moreover, weinclude here an exhaustive taxonomic sampling of P. hasselquistii,for which DNA sequences from only three specimens were previ-ously available, and we investigate the patterns of high geneticdiversity in this and other species.

4.1. Habitat, morphology and allopatry in the genus Ptyodactylus

Despite the broad distribution of the genus Ptyodactylus (Fig. 1),all of its members present conserved morphologies, in terms of sizeand shape, and none has clearly adapted to different structuralhabitats that are very diverse across their range (Arnold, 1977,1980a; Werner and Sivan, 1994). In contrast to Ptyodactylus, sever-al other geckos from the same geographical areas, like Tarentola,Stenodactylus, Pristurus or members of the Arid clade ofHemidactylus (sensu Carranza and Arnold, 2006) comprise speciesthat are adapted to living on different substrates and make differ-ent habitat use (Carranza et al., 2000; Carranza and Arnold, 2006;Arnold, 2009; Metallinou et al., 2012). Even the highly conservedNorth African genus Tarentola presents a ground-dwelling memberwith very distinct morphology (Carranza et al., 2002), and thestrictly climbing geckos of the genus Asaccus include some small-sized members with rather distinct morphologies (Arnold andGardner, 1994; Gardner, 1994). This suggests that a strong selec-tive pressure might be acting to maintain this morphology forthe rock-climbing Ptyodactylus while, at the same time, this con-figuration of characters may hinder their versatility to exploit dif-ferent substrates. Indeed, it has been shown that habitat use iscorrelated with morphological evolution in rock-dwelling lizardsof the family Scincidae (Goodman et al., 2008) and that theextreme morphological divergence between two closely relatedspecies of the gecko genus Rhoptropus, a sand-dwelling and a rock-dwelling one, is related to locomotor performance on different sub-strates (Higham and Russell, 2010).

This morphological conservatism observed in Ptyodactylus hasprobably played a critical role in shaping the diversification anddistribution patterns of the members of the genus. All the speciesare largely allopatric or parapatric and very few areas of sympatryexist where a maximum of two species are present (Baha El Din,2006; Disi et al., 2001; Sindaco and Jeremcenko, 2008; Wernerand Sivan, 1993, 1994; see also Fig. 1). This contrasts with the alsonocturnal geckos of the genus Stenodactylus and the members ofthe Arid clade of Hemidactylus, which present very large areas ofsympatry among species, including up to three sympatric speciesand, in the case of Hemidactylus, up to four sympatric species insome areas of Socotra Island (Carranza and Arnold, 2012; Gómez-

region

Maghreb

E Sahel

Arabia

NE Africa & Levant

W Sahel

0510152025

JEM37_Yemen_60

991J_Syria_182

BEV8105_Morocco_152

Poud_P22_Morocco_158

IBE_S10109_Saudi_Arabia_73

BEV8870_Mali_197

SPM001852_Egypt_

NHMC80_3_48_6_Syria_183



ZFMK_60844_Syria_178


UAE41_Oman_36


IBE_S4198_Egypt_81



JEM62_Yemen_143

ZFMK_70963_Oman_38


SUD12/2010_98_Sudan_82

BEV7210_Egypt_215

UAE37_UAE_40


AO125_Oman_145

ZFMK_70961_Oman_47

07_09M_Morocco_153


BEV7244_Egypt_92

BEV_T519_Algeria_193

JOR128_Jordan_16


JEM466_Yemen_131

DNA44_Ethiopia_190

JEM424_Yemen_130


BEV_T702_Algeria_149

JEM559_Yemen_119


JEM650_Yemen_136

11022012A_Morocco_160

JEM113_Yemen_139

MHNG_2678_90_Mali_206

TW1009_Jordan_172

IBE_S993_Morocco_159


TMHC179_Ethiopia_191



SPM002962_105_Jordan_7TW1006_Jordan_9

JEM110_Yemen_140

DJI_02_Djibouti_189

Prag_429_Burkina_Faso_196

TW1011_UAE_34

BEV_T3039_Benin_195

BEV_T3958_Jordan_95


Prag_349_Niger_202

JOR036_Jordan_5

Prag_3128_Mauritania_199

BEV10156_Algeria_194Prag_409_Niger_201



JEM508_Yemen_117


ZFMK_60930_Syria_181


Poud_908_Algeria_150

06_14b_Israel_171

SPM002952_48_Egypt_216

P. togoensis*

P. oudrii

P. ragazzii

P. puiseuxi

P. siphonorhina

P. guttatus

P. h

asse

lqui

stii complex

A

B

C

D

E

Fig. 3. BEAST chronogram with ancestral area reconstruction using the BSSVS approach. Brach colors refer to areas as pictured in the lower left inset map. Branch width isproportional to the ancestral area posterior probability. Nodes that receive less than 0.80 probability are accompanied by a pie chart detailing the probability of each inferredarea (highest to lowest clockwise). Codes of the tips represent Specimen Number, followed by Country of origin, followed by locality code, as illustrated in Fig. 1. Detailedinformation on all samples is listed in Supplementary Table S1.


Díaz et al., 2012; Metallinou et al., 2012; Šmíd et al., 2013a).Similarly, the diurnal geckos of the genus Pristurus also exhibitlarge areas with up to three sympatric species (Arnold, 2009;Sindaco and Jeremcenko, 2008).

4.2. General biogeographic patterns of Ptyodactylus

The characteristic and exclusive toe morphology of Ptyodactylusamong phyllodactylid geckos is accepted as a strong support forthe monophyly of the genus (Loveridge, 1947), which has also beenrecovered recently by molecular data, although not all the repre-sentatives of the genus were included (Gamble et al., 2008, 2011,2012; Pyron et al., 2013). Heimes (1987) in his revision ofPtyodactylus hypothesized that the Middle East acted as the centerof diversification of the genus, based on the relative species rich-ness, and from there they spread westwards to North Africa andthe Sahel and eastwards to Pakistan through the Strait ofHormuz. He did not provide, however, any insight into the sistergroup of Ptyodactylus. Recent molecular studies on the phylogeny

of Phyllodactylidae (Gamble et al., 2008, 2011, 2012; Pyron et al.,2013) have also failed to give an unequivocal answer to this ques-tion. In our phylogenetic and biogeographical reconstruction, thetwo west North African lineages are the first to branch out, withP. togoensis as a sister clade to the remaining Ptyodactylus (Figs. 2and 3). The ancestral area reconstruction favors a western NorthAfrican (either Maghreb or Sahel) origin and basal branching forPtyodactylus, but only slightly more than an eastern one, so resultswith the present data should be considered inconclusive. The pres-ence of several long branches in the chronogram (Fig. 3) may beindicative of unsampled and/or extinct clades, which would furthercomplicate ancestral inference.

The divergence time estimations (Table 2) indicate thatPtyodactylus have a rather old origin in the Late Oligocene.According to our results, Ptyodactylus started diversifyingapproximately 27 Ma ago (17.7–38, 95% HPD). This suggests thattheir similarity in terms of morphology and habitat use is not theresult of a recent origin and rapid dispersal of the genus acrossits current range but it is rather the result of a high degree of


conservatism regarding these two aspects. The estimated time isvery similar to that inferred for the onset of diversification inStenodactylus (29.5 Ma [20.7–39.2, 95% HPD]) (Metallinou et al.,2012), and for the members of the Arid clade of Hemidactylus(29.1 Ma [19.2–40.3, 95% HPD]) (Šmíd et al., 2013a). This was atime of high geological instability in North Africa and Arabia as aresult of the onset of major seismic and volcanic events in theAfar region, affecting eastern Ethiopia, northeast Sudan and south-west Yemen (Menzies et al., 1992). These volcanic and tectonicevents triggered the formation of some of the most relevant andcomplex physiographic features in the contact zone betweenAfrica and Arabia, like the Gulf of Aden, the Red Sea and the eleva-tion of the Afro-Arabian rift-flanks to heights above 3600 m(Menzies et al., 1992; Bosworth et al., 2005; Autin et al., 2010),which had a major impact on the fauna and flora of the area (e.g.Pook et al., 2009; Thiv et al., 2010; Vargas-Ramírez et al., 2010;Portik and Papenfuss, 2012).

Diversification within the main clades of Ptyodactylus started inthe middle Miocene, and may be related to the progressive aridifi-cation of North Africa, which took place at the time (Flower andKennett, 1994). This phenomenon has been shown to have had aprofound effect on the diversification of biota (Douady et al.,2003; Guillaumet et al., 2008; Thiv et al., 2010; Gonçalves et al.,2012; Metallinou et al., 2012). In the cases of rock-dwelling ani-mals like Ptyodactylus or Agama (Gonçalves et al., 2012), the expan-sion of continuous sands probably acted as a vicariant agent,restricting their populations into areas of hard substrate (Britoet al., 2014; Fig. 1), although less in Agama, which are much moregeographically widespread (Leaché et al., 2014) than Ptyodactylus,probably due to their higher tolerance to temperature and sub-strate types. It is interesting to notice that the same expansion ofsand surfaces that promoted vicariance in the rock-dwellingPtyodactylus, facilitated dispersal in sand-adapted species, likethe geckos of the genus Stenodactylus and the scincids of the genusScincus and some sand-diving species of the genus Chalcides(Carranza et al., 2008; Metallinou et al., 2012). The situation ofPtyodactylus in Arabia is very similar to that of western NorthAfrica, with populations tightly linked to rocky environments andwith large desert areas like the Rub al Khali or flat arid plainsacting as barriers to dispersal (Fig. 1). Diversification in this areastarted approximately 12 Ma ago in clade A and 9.4 Ma ago in cladeB; dates that coincide with the allopatric speciation of the fivemorphologically similar Arabian species of the genus Stenodactylus(clade B in Metallinou et al., 2012). However, since little informa-tion is available on the geological and climatic history of theinterior of Arabia, the biogeographic patterns in both Ptyodactylusand Stenodactylus are very difficult to interpret.

4.3. Phylogeny and systematics of Ptyodactylus

The samples analyzed in this study fall within six major cladesin the Ptyodactylus phylogeny. Three of them, P. oudrii, P. togoensisand P. ragazzii, are distributed in North Africa and the Sahel; twoare found in northeastern Africa, the Levant and Arabia (clades Aand C) and one is exclusive to southern and southwestern Arabia(clade B) (Figs. 1 and 2). The monophyly of each one of these cladesreceives very high support in all the analyses, but not all relation-ships among them are equally supported (Fig. 2). The sister rela-tionship between P. ragazzii and clade C is supported in ML butnot BI analyses. Also, there is a topological difference betweenthe results of ML and BI analyses of the concatenated datasetregarding the first clade to branch in the phylogeny. In the follow-ing sections, we describe the major findings and phylogeneticstructure in each one of the aforementioned clades and explorethe inter- and intraspecific relationships of the Ptyodactylusmembers.

4.3.1. Polyphyletism of ‘‘Ptyodactylus ragazzii’’One of the most important results of our study is that the taxon

known as ‘‘P. ragazzii’’ is composed of two deep lineages thatbranch independently in the phylogeny (Figs. 1 and 2). As men-tioned above, this is confirmed by a topological test in which thealternative topology of monophyly is clearly rejected based onour dataset. The systematic issue caused by this non-monophylyis resolved herein by resurrecting the name Ptyodactylus togoensisTornier (1901) for the West African populations. Ptyodactylusragazzii was described on the basis of specimens from at least threelocalities in present-day Eritrea and Ethiopia (Anderson, 1898).Despite the distance of a few hundred kilometers between these(approximate) and our sampled localities (Fig. 1), given the extend-ed distribution of ‘‘P. ragazzii’’ across the Sahel, it can be at presentjustified to attribute P. ragazzii to our samples from Eastern Africa,pending further investigations in the area. It should be noted thatalthough the spelling for this name is ‘‘ragazzi’’ in the speciesdescription (Anderson, 1898, p. 62), a corrigendum issued simulta-neously with the original work confirms ‘‘ragazzii’’ as the correctspelling and this is accepted as valid according to theInternational Code of Zoological Nomenclature (Article 32.5).

Froufe et al. (2013) analyzed samples of P. togoensis (under thename P. ragazzii) and discussed the presence of three major lin-eages in their phylogeographic study: a western lineage from theTagant plateau and the Assaba mountains, a southern one fromthe western Sahel and an eastern one from the Aïr mountains inNiger. In our study, the second lineage is shown to extend furthernorthwest reaching the edge of the sands of the Sahara desert(localities 206 & 197 in Fig. 1). Our samples from the Hoggar andTassili mountains are assigned to the eastern lineage mirroringthe pattern uncovered in the lizard Agama tassiliensis from thesame mountain formations (Gonçalves et al., 2012). Interestingly,with the addition of new samples in this study, the geographicaldistance between the western and the southern lineages nowequals that between the southern and the eastern one.Nevertheless, the genetic distance between the last two is muchsmaller (data not shown), and in the phylogenetic tree these lin-eages present very little divergence, while the western cladeappears to be the most divergent (Fig. 2). The presence of sandydeserts immediately to the east of the Assaba Mountains couldbe responsible for this isolation, but without samples from theapparently more suitable area to the south a possible connectioncannot be discarded.

Apart from this specific area where further sampling is neededin order to confirm this pattern, the greater Sahel area from wherethere are abundant records of ‘‘P. ragazzii’’ (Sindaco andJeremcenko, 2008; Fig. 1) should be explored in order to establisha possible contact zone between P. togoensis and P. ragazzii. Giventhe presence of large unsampled areas within the distribution ofthe genus Ptyodactylus in the Sahara-Sahel, additional overlookedgenetic diversity should be expected.

4.3.2. New data on Ptyodactylus oudriiAnother Ptyodactylus species that exhibits deep genetic

intraspecific divergence is P. oudrii from the Maghreb, which ledPerera and Harris (2010a) to postulate the existence of a speciescomplex. Here, we include previous sequence data (with codes‘‘Poud’’, Fig. 2 and Fig. S1, Supplementary material) and provideexpanded taxonomic and genetic sampling that confirm the exis-tence of four major lineages with distinct geographic distributionwithin this group: an eastern lineage from the Saharan AtlasRange, two lineages from the High Atlas Range (one in the westand one in the east), and one from the Anti-Atlas. Our additionalsamples extend the range of the eastern lineage, previously knownonly from the eastern part of the Saharan Atlas Range in Algeria, toEastern Morocco. This range expansion opens the possibility that a


contact zone exists between the eastern lineage and the lineageinhabiting the eastern parts of the High Atlas (locality 160 inFig. 1). The two other lineages inhabit the Western High Atlas –again with a possible contact zone with the previous lineage –and the Anti-Atlas.

The Saharan Atlas lineage is the most divergent one and speci-mens from the gap of 250 km that separates it from the easternHigh Atlas lineage would help circumscribe their limits.Ptyodactylus geckos are rock-dwellers, so this area with plenty ofrocky formations does not apparently present any barrier to theirdispersal. Instead, we hypothesize that reproductive rather thandispersal barriers exist between members of these two lineages.Importantly for future taxonomic considerations, the type localityof P. oudrii is found only 35 km north of our sampling locality169 (Fig. 1) included in the Saharan Atlas lineage.

The three lineages distributed across the High Atlas and theAnti-Atlas show a longitudinal geographical pattern of highdivergence (Figs. 1 and 2). Several groups of reptiles like Timontangitanus (Perera and Harris, 2010b), the high altitude lizards ofthe genus Atlantolacerta (Barata et al., 2012a), the geckos of thegenus Quedenfeldtia (Barata et al., 2012b), the agamid Agamaimpalearis (Brown et al., 2002; Gonçalves et al., 2012) and theterrapin Mauremys leprosa (Fritz et al., 2005, 2006) also presentrelatively high levels of genetic variability across the High Atlas andthe Anti-Atlas, although there is not a clear pattern across all taxa.

According to our divergence time estimations, the four deep lin-eages identified in P. oudrii originated approximately 12.7 Ma ago,a date much older than that inferred for some of the other reptilespecies mentioned above (A. impalearis, M. leprosa, T. tangitanus)and that roughly coincides with some estimations of the majoruplift of the Atlas Mountains (Gomez et al., 2000; Piqué et al.,2002). Nevertheless, this geological process was extended in timeand very complex (Frizon de Lamotte et al., 2008), and it is difficultto build specific scenarios on how the landscape of the AtlasMountains and processes during its formation have affected thediversity.

To conclude, the P. oudrii complex constitutes a very interestingmodel to explore diversification processes in the biodiversity-richAtlas Mountains, once its species limits become well defined.With the new data on the range of the lineages presented here,we pinpoint the crucial areas for completing the taxonomic sam-pling across potential contact zones, which will enable a fine-scalegenetic multilocus approach focused on the degree of reproductiveisolation between the lineages, ideally combined with a morpho-logical assessment of the different forms.

4.3.3. Ptyodactylus guttatus species group (Clade C)This clade represents a small radiation from the Levant that

originated approximately 10 Ma ago (Table 2) and resulted in threemainly allopatric or parapatric species (Figs. 1 and 2; Werner andSivan, 1994; Baha El Din, 2006). Thanks to its geographic locationat the crossroad of three continents, the Levant has long been con-sidered an area of great biogeographic interest (Werner and Sivan,1993; Šmíd et al., 2013a,b). The realization of this significancetogether with the relatively good accessibility of the area have con-tributed to a better knowledge of its faunal elements (Disi et al.,2001; Baha El Din, 2006; Sindaco and Jeremcenko, 2008), whichis also reflected on the taxonomy of the Ptyodactylus geckos.With the revisionary works of Werner and Sivan (1993, 1994)and Baha El Din (2006), the three species of this group, P. puiseuxi,P. guttatus and P. siphonorhina, became relatively well known froma morphological, ecological and physiological point of view.Genetic data on this group are presented here for the first timeand do not contradict the current taxonomical hypothesis basedon morphology and ecology.

Ptyodactylus siphonorhina was «tentatively elevated to the speci-fic level» recently (Baha El Din, 2006; p. 71) based on morpho-logical and ecological evidence that differentiated it from itssupposed sister species, P. guttatus. Although it had previouslybeen considered a nomen nudum by Werner and Sivan (1994), fol-lowing Wermuth (1965), or a synonym of P. guttatus by Heimes(1987), this latter author already observed different patterns inprotein electrophoresis analyses between P. guttatus from NorthAfrica and from Jordan and interpreted it as an old separationbetween the two populations (Heimes, 1987). Here, the geneticdistinctiveness of P. siphonorhina is confirmed based on a limited,but very well distributed across the species’ range, number of sam-ples which also includes a sample from very close to its type local-ity (Figs. 1 and 2). The two sister species are mainly distributed oneither side of the Gulf of Suez but they are sympatric in parts of thenorthern Eastern Desert (Baha El Din, 2006). Although, like allother Ptyodactylus, both species are mainly found on vertical rockysurfaces, boulders, under ledges and in caves, they have differentecological preferences that support their specific distinctiveness.While P. siphonorhina is found on rocks in highly arid open desert,P. guttatus is mainly found on hills, in areas of modest aridity (BahaEl Din, 2006). Despite its relatively large distribution range, thelevel of intraspecific genetic variability of P. siphonorhina is moder-ate (4.2% in the 12S; Table S3, Supplementary materials), whilethat of P. guttatus, which shows a more restricted distributionrange (Fig. 1), is even lower (2.4%). In this latter species, only thepopulation from Wadi Rum in Jordan seems to be genetically dis-tinct (Figs. 1 and 2).

The third taxon of this small radiation of Levantine Ptyodactylusis P. puiseuxi. Our dense sampling surrounds its type locality inpresent-day Israel (between loc. 171 and 185, Fig. 1). Distributedfurther north than P. guttatus, these two species can be distin-guished on the basis of morphology and present a mainly parapatricdistribution in Israel, with just a single site of sympatry that hasbeen considered a consequence of a recent human introductionof P. guttatus into the range of P. puiseuxi (Werner and Sivan,1993). Interestingly, in the same work, few specimens from thecontact zone presented intermediate states for some charactersand were hypothesized to be hybrids. Hybridization experimentsconfirmed that F1 hybrids could be obtained, albeit with low success,but not F2 (Werner and Sivan, 1996). Among the samples analyzedherein, there is no indication of presence of a hybrid, as bothP. guttatus and P. puiseuxi present unique alleles for all loci (datanot shown). Apart from their morphology, the two species alsodiffer in their behavior, ecology, physiology and biology, withdifferences in the activity patterns (P. puiseuxi diurnal and P. guttatusdiurnal-nocturnal) and vocalization being the most relevant ones(Werner and Sivan, 1994). Of all three species of clade C, P. puiseuxiis the one that presents the highest level of intraspecific diversitybased on our dataset (Table S3, Supplementary material) withthe samples from Syria grouping in a divergent clade (Figs. 1 and2 and S1 Supplementary material). Importantly, three names, cur-rently synonymized with P. puiseuxi, were proposed for specimenscoming from a single locality, at the ruins of Palmyra in Syria,which coincides with locality 182 of our sampling (Figs. 1 and 2):Ptyodactylus bischoffsheimi Boutan, 1893, P. montmahoui Boutan,1893 and P. barroisi Boutan, 1893. Bibliographic research revealsthat two more names have been placed under the synonymy ofP. puiseuxi: P. lobatus sancti-montis Barbour, 1914 (type locality8 km south of locality 177; Figs. 1 and 2) and P. lobatus syriacusPeracca, 1894 (part) (Werner and Sivan, 1994) (type localitybetween localities 27 (P. guttatus) and 187 (P. puiseuxi) of oursampling (Figs. 1 and S1 Supplementary material). Since P. lobatussyriacus had also been placed under the synonym of P. guttatus, theallocation of this name in the current systematics of Ptyodactylusis unclear and remains to be established. To conclude, further


sampling mainly in the area between the two main genetic andgeographical clusters (in northern Lebanon and eastern Syria)would be needed to clarify if the divergent lineage from Syriamay represent a different form.

4.3.4. Ptyodactylus hasselquistii species complexPreliminary information on the morphological variability of P.

hasselquistii in Arabia became available with the work of Arnold(1980a, 1986) on the understudied herpetofauna of thePeninsula. He perceived it as intraspecific geographical variationand outlined a draft pattern of differentiation between animalsfrom northwest Arabia along the Hejaz mountains, from the Asirmountains, from Dhofar and the Hajar Mountains and from centralnorthern Arabia (Arnold, 1986). It therefore became evident that asystematic revision with comprehensive material of the speciesrange was warranted for a proper delimitation of the differentforms of this complex.

In this study, taxonomical sampling in the P. hasselquistii com-plex is one of the main deliverables in order to sufficiently docu-ment the genetic variability and provide the necessary materialfor a study using species delimitation methods (Metallinou et al.,unpubl. results). Our data uncovers for the first time the existenceof very high levels of genetic divergence within the P. hasselquistiispecies complex (11.3% in 12S and 18.2 in cytb; Table S3,Supplementary material), which is also supported by the identifi-cation of 37 GMYC lineages, two more than among all the remain-ing species of the genus included in our study. We consistentlyrecover two deep clades both in the mitochondrial and concatenat-ed phylogenies (Figs. 2 and S1, Supplementary material) that wename clades A and B. The genetic distance between these twoclades (14.1% in the 12S) is much higher than other interspecificdistances in the genus (Table S3, Supplementary material). Theonset of the diversification in the P. hasselquistii species complexdates back to approximately 17 Ma ago, while in clades A and B,12 Ma and 9.4 Ma ago, respectively (Table 2). These last ages arecomparable with the age of clade C (10.1 Ma) that comprises theradiation of the three species from the Levant (see above).

Among our sampling, the type locality of P. hasselquistii (Cairo,Egypt) is represented in our analyses by locality 81 belonging toclade A (Figs. 1 and 2). Two of the taxa described recently byNazarov et al. (2013) can also be tentatively allocated to this cladethanks to the proximity with some of our specimens. The typelocality of P. orlovi is found 28 km southwest of locality 38 and ofP. ananjevae, 52 km east of locality 5 (Figs. 1 and 2). Regarding P.dhofarensis, its type locality (Wadi Ayun) coincides with locality145 (Figs. 1 and S1, Supplementary material) of a sample com-prised in clade B. Nevertheless, as a result of the high level ofgenetic diversity in the P. hasselquistii species complex and theunavailability in public repositories of the cytochrome oxidase Isequences of the type specimens by Nazarov et al. (2013), no con-clusions regarding the limits of these taxa can be drawn at thispoint but will be the subject of a separate in-depth study.

The mountains of the Arabian Peninsula harbor a great diversityof flora and fauna (Arnold, 1972, 1977, 1980a and other articles inthe same volumes) and current systematic studies in reptiles areuncovering an important overlooked diversity in this area (e.g.Arnold and Gardner, 1994; Gardner, 1994; Busais and Joger,2011a; Carranza and Arnold, 2012; Metallinou and Carranza,2013; Šmíd et al., 2013a, b; among others). However, given the dif-ficult accessibility and political instability in the region, there arestill several parts that remain largely unexplored. It is thereforeexpected that unrecognized diversity is comprised within thePtyodactylus species complexes, the delimitation of which willrequire comprehensive analyses of genetic and morphological data(Metallinou et al., unpubl. results) similar to the work carried outon the geckos of the genus Hemidactylus of the Arabian Peninsula

(Busais and Joger, 2011a,b; Carranza and Arnold, 2012; Šmídet al., 2013a,b, 2015).

Acknowledgments

The authors would like to thank the people who donatedsamples or helped in the field: F. Amat, M. Aoutchiki, E.N. Arnold, O.Chaline, M. Cheylan, L. Chirio, A. Cluchier, D. Donaire, D. Escoriza,A. Foucart, M. Geniez, E. Gómez-Díaz, F. Hulbert, Y. Mansier, D.Modry, O. Peyre, J. Renoult, V. and X. Rufray, S. Scholz, M. Siol, F.Speyser, J. Viglione, and especially S. Baha El Din for theP. hasselquistii sample from the type locality, J. Šmíd for P. hasselquistiisamples from Sudan, P. Lymberakis for several P. puiseuxi samples,A. Nistri and T. Mazuch for P. ragazzii samples from Djibouti andEthiopia, respectively. We would like to thank E. Planas for invalu-able advice and help with the illustrations. Special thanks are dueto the members of the Nature Conservation Department of theMinistry of Environment and Climate, Sultanate of Oman fortheir help and support and for issuing collecting permits (Refs.:08/2005; 16/2008; 38/2010; 12/2011). We thank A.K. Nasher forsupport and Environment Protection Agency, Sana’a, Republic ofYemen for permits (Ref. 10/2007). We are thankful to theDeanship of academic research at Taif University for funding thesample collection in Saudi Arabia (Grant no. 1-433-2108). Thiswork was supported by Grant CGL2012-36970 from theMinisterio de Economía y Competitividad, Spain (co-funded byFEDER). M.M. was supported by a FPU predoctoral grant from theMinisterio de Educación, Cultura y Deporte, Spain (AP2008-01844). JCB is supported by Fundação para a Ciência e Tecnologia(IF/00459/2013). We thank two anonymous reviewers for helpfulcomments on an earlier version of the manuscript.

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.ympev.2015.02.010.

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