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African Journal of Herpetology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ther20 Genetic variability and relationships within the skinks Eumeces algeriensis and Eumeces schneideri using mitochondrial markers Ana Perera a , Filipa Sampaio a , Sara Costa a , Daniele Salvi a & D. James Harris a b a CIBIO-UP, Centro de Investigação em Biodiversidade e Recursos Genéticos, Campus Agrário de Vairão, 4485-661, Vairão, Portugal b Departamento de Biologia, Faculdade de Ciências da Universidade do Porto, 4099-002, Porto, Portugal Available online: 03 Jun 2011
To cite this article: Ana Perera, Filipa Sampaio, Sara Costa, Daniele Salvi & D. James Harris (2012): Genetic variability and relationships within the skinks Eumeces algeriensis and Eumeces schneideri using mitochondrial markers, African Journal of Herpetology, 61:1, 69-80 To link to this article: http://dx.doi.org/10.1080/21564574.2011.583284
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Short communication Genetic variability and relationships within the skinks Eumeces algeriensis and Eumeces schneideri using mitochondrial markers
1 1 1 ANA PERERA *, FILIPA SAMPAIO ,SARA COSTA , 1 1,2 DANIELE SALVI &D.JAMES HARRIS
1CIBIO-UP, Centro de Investigac¸a˜o em Biodiversidade e Recursos Gene´ticos, Campus Agra´rio de Vaira˜o, 4485-661, Vaira˜o, Portugal; 2Departamento de Biologia, Faculdade de Cieˆncias da Universidade do Porto, 4099-002, Porto, Portugal
Abstract.*The skinks Eumeces algeriensis and Eumeces schneideri are two of the most widespread species of the E. schneideri group. Despite this, data on their intra-specific variation are limited. In this study we analyse the genetic variability of these two species across their distribution range using two mitochondrial fragments, 12S rRNA and 16S rRNA. The results confirm the paraphyly of the group, with E. algeriensis more related to Scincopus than to E. schneideri. Eumeces algeriensis shows relatively low levels of genetic diversity, which is distributed in two main lineages, one across Morocco, and the other restricted to the southern region (Anti-Atlas). These results are discordant with the high diversity found in other species from the same area and suggest a relatively recent expansion of E. algeriensis across most of its current range. Regarding E. schneideri, we find high levels of genetic variability and a complex pattern of genetic diversity, concordant with the phylogeographic patterns found recently in other species from the Middle East region, but not with the current intra-specific taxonomy of this group.
Key words.*Eumeces algeriensis, Eumeces schneideri, North Africa, Middle East, mtDNA, genetic diversity
The genus Eumeces, considered one of the basal groups within the Scincidae (Estes et al. 1988; Wiens et al. 2006), includes more than 50 species spread across the Holarctic region. Long considered monophyletic, Griffith et al. (2000) indicated it may be paraphyletic, and since then, various authors have presented alternatives to split Eumeces into different genera (Schmitz et al. 2004; Smith 2005). The E. schneideri group (sensu Taylor 1935) is distributed across the Saharo-Sindian region from North Africa, Cyprus and the Middle East to southwest Asia. Renamed
Downloaded by [b-on: Biblioteca do conhecimento online UP] at 02:44 11 June 2012 as Novoeumeces by Griffith et al. (2000), most authors rejected this proposal and suggested to maintain the use of Eumeces for the schneideri group, as they contain the type species of the genus (Brandley et al. 2005). Within this group, different authors recognised the existence of a variable number of species (between two and six; Taylor 1935; Lieb 1985; Sindaco & Jeremcenko 2008), with two of them being widely accepted: E. schneideri (Daudin 1802), distributed from eastern Maghreb to West Asia and E. algeriensis Peters 1864, distributed across the western Maghreb. Following Sindaco & Jeremcenko (2008), six subspecies within E. schneideri are
*Corresponding author. Email: [email protected]
ISSN 2156-4574 print/ISSN 2153-3660 online # 2012 Herpetological Association of Africa http://dx.doi.org/10.1080/21564574.2011.583284 http://www.tandfonline.com 70 PERERA ET AL.*Genetic variation in Eumeces
recognised: E. s. aldrovandii from North Africa; E. s. blythianus from Pakistan, Afganistan and Punjab; E. s. pavimentatus from SE Anatolia to Levant; E. s. princeps distributed across East Turkey, Iraq, North and West Iran, Afghanistan, and nearby countries; E. s. zarudnyi from SE Iran and SW Pakistan; and E. s. barani recently described in western Turkey (Kumlutas et al. 2007). Regarding E. algeriensis,two subspecies E. a. algeriensis, widely distributed across Morocco; and E. a. meridionalis, restricted to East Morocco and NW Algeria are recognised (Sindaco & Jeremcenko 2008). However, the taxonomic position of E. a. meridionalis is still under debate (Schleich et al. 1996; Bons & Geniez 1996; Sindaco & Jeremcenko 2008). A recent study by Carranza et al. (2008) including North African specimens from both species highlighted the paraphyly of this group, with Scincopus as sister taxa of E. algeriensis, and E. schneideri closer to Scincus, as already suggested in previous karyological (Caputo et al. 1993, 1994) and morphological studies (Arnold & Leviton 1977; Caputo et al. 1993). Moreover, the study confirmed E. schneideri and E. algeriensis as two different entities with a disjunct distribution in North Africa (Carranza et al. 2008). However, the variability within each unit is still poorly known since, until now, only five individuals for each species have been analysed (Carranza et al. 2008). North Africa and the Middle East are among the most diverse regions in the Western Palaearctic, with a wide variety of geological, geographical and climatic characteristics shaping a diversity of landscapes (McColl 2005). Moreover, both regions were refugia for biota during the Pleistocene climatic fluctuations (De Lattin 1949; Bons 1973; Abed & Yaghan 2000). The phylogeography of various reptiles from the western Maghreb have been studied in recent years, and in general unexpectedly high levels of genetic variation have been reported (Brown et al. 2002; Rato & Harris 2008; Perera & Harris 2010), often supporting the existence of cryptic taxa (Perera et al. 2007; Pinho et al. 2008, Rato et al. 2010). Regarding the Middle East, the few studies existing also indicate that this area is a source of considerable genetic variability for many taxa (Kapli et al. 2008; Gvozdı´k et al. 2010 and references within). Given this, the assessment of genetic diversity within E. algeriensis and E. schneideri is essential to understand better the complexity of patterns and processes underlying the diversity in these two regions. In this study, we analysed 22 individuals from E. algeriensis and E. schneideri from Morocco and the Middle East together with sequences from 14 specimens available in GenBank, and individuals of the genus Scincus and Scincopus. The main goal of this study was to analyse the genetic variability within E. algeriensis and E. schneideri. Additionally, we analysed in a phylogenetic framework the relation- ships between E. algeriensis and E. schneideri and those between them and
Downloaded by [b-on: Biblioteca do conhecimento online UP] at 02:44 11 June 2012 representatives of the genera Scincus and Scincopus. Fresh samples were collected from lizards captured by hand, identified based on their morphological character- istics, and a small piece of the tail taken and stored in 96% ethanol for genetic analysis. All individuals were released immediately afterwards at the same place of capture. Additional tissue samples were obtained from voucher specimens from the National History Museum of Crete (NHMC; Table 1). In total, 15 new samples of E. algeriensis and 7 of E. schneideri (Fig. 1; Table 1) were analysed genetically using 2 different mitochondrial DNA fragments 12S rRNA and 16S rRNA. Additionally, 14 sequences (7 E. algeriensis and 7 E. schneideri) from GenBank were included (Table 1). Finally, in order to investigate Table 1. Samples analysed in this study. For each specimen, locality, GenBank accession numbers (provided after acceptance of the manuscript) and origin is provided.
GenBank accession numbers
Species Code Locality 12S rRNA 16S rRNA Origin Eumeces algeriensis algeriensis EA1 Taroudant, Morocco JF931196 JF931174 This study FIA ORA FHERPETOLOGY OF JOURNAL AFRICAN Eumeces algeriensis algeriensis EA2 Agadir - Tiznit, Morocco JF931197 JF931175 This study Eumeces algeriensis algeriensis EA4 Oulad Brahim, Morocco JF931198 JF931176 This study Eumeces algeriensis algeriensis EA5 Volubilis, Morocco JF931199 JF931177 This study Eumeces algeriensis algeriensis EA6 Moulouya Valley, Morocco JF931200 JF931178 This study Eumeces algeriensis algeriensis EA7 Moulouya Valley, Morocco JF931201 JF931179 This study Eumeces algeriensis algeriensis EA8 Ouazzane, Morocco JF931202 JF931180 This study Eumeces algeriensis algeriensis EA9 Mechra Ben Abhou, Morocco JF931203 JF931181 This study Eumeces algeriensis algeriensis EA10 Mechra Ben Abhou, Morocco JF931204 JF931182 This study Eumeces algeriensis algeriensis EA11 Agadir Tiznit, Morocco JF931205 JF931183 This study Eumeces algeriensis algeriensis EA12 Tirhmi, Morocco JF931206 JF931184 This study Eumeces algeriensis algeriensis EA13 Bin-El-Ouidanne, Morocco JF931207 JF931185 This study Eumeces algeriensis algeriensis EA14 Gorges near Guelmin, Morocco JF931208 JF931186 This study
Eumeces algeriensis algeriensis EA15 Between Casablanca and Rabat, Morocco JF931209 JF931187 This study 71 2012 61(1) Eumeces algeriensis algeriensis EA18 Near Oued Ouahar, Morocco JF931210 JF931188 This study Eumeces algeriensis algeriensis GB1 Massa, Morocco EU278021 EU278086 Carranza et al. (2008) Eumeces algeriensis algeriensis GB2 Essaouira, Morocco EU278020 EU278085 Carranza et al. (2008) Eumeces algeriensis algeriensis GB3 Ras el Ma, Morocco EU278019 EU278084 Carranza et al. (2008) Eumeces algeriensis GB4 Morocco EU278018 EU278083 Carranza et al. (2008) Eumeces algeriensis GB13 Morocco EU278017 EU278082 Carranza et al. (2008) Eumeces algeriensis GB14 AY308345 AY308196 Schmitz (2003) Eumeces algeriensis GB15 AY308344 AY308195 Schmitz (2003) Eumeces schneideri pavimentatus1 ES10 Al Jaboul lake, Syria JF931211 JF931189 This study2 (NHMC 80.3.84.3) Eumeces schneideri pavimentatus1 ES11 Azraq, Jordan JF931212 JF931190 This study2 (NHMC 80.3.84.5) 1 2
Downloaded by [b-on: Biblioteca do conhecimento online UP] at 02:44 11 June 2012 2012 11 June at 02:44 UP] online do conhecimento Biblioteca by [b-on: Downloaded Eumeces schneideri pavimentatus ES13 Um Quays, Jordan JF931213 JF931191 This study (NHMC 80.3.84.12) 2P 72
Table 1 (Continued )
GenBank accession numbers
Species Code Locality 12S rRNA 16S rRNA Origin
Eumeces schneideri pavimentatus1 ES14 Dana Nature Reserve, Jordan JF931214 JF931192 This study2 (NHMC 80.3.84.13) ERERA Eumeces schneideri pavimentatus1 ES17 Lattakia Beach, Syria JF931215 JF931193 This study2 (NHMC 80.3.84.4)
Eumeces schneideri pavimentatus1 ES18 Petra, Jordan JF931216 JF931194 This study2 (NHMC 80.3.84.10) AL ET Eumeces schneideri pavimentatus GB7 Coastal dunes after Karatas, Turkey EU278003 EU278070 Carranza et al. (2008) . Eumeces schneideri pavimentatus GB8 Karaotlak in the Euphrates Valley, Turkey EU278002 EU278069 Carranza et al. (2008) * eei aito in variation Genetic Eumeces schneideri princeps ES19 Echegnatzor, Armenia JF931217 JF931195 This study Eumeces schneideri princeps GB5 Igdir, Turkey EU278006 EU278073 Carranza et al. (2008) Eumeces schneideri schneideri GB6 Egypt EU278004 EU278071 Carranza et al. (2008) Eumeces schneideri schneideri GB16 Egypt EU278005 EU278072 Carranza et al. (2008) Eumeces schneideri GB11 AY308361 AY308212 Schmitz (2003) Eumeces schneideri GB12 West Asia AB028800 AB028812 Honda et al. (2000)
Scincus albifasciatus SC14 Around Ayoun el Atrous, Mauritania EU278007 EU278074 Carranza et al. (2008) Eumeces Scincus albifasciatus SC17 Bouˆ Derga, Mauritania EU278011 EU278078 Carranza et al. (2008) Scincus mitranus SC11 El Ain, UAE EU278015 EU278080 Carranza et al. (2008) Scincus mitranus SC12 UAE AY649133 AY649174 Brandley et al. (2005) Scincus scincus SC3 Unknown AY315515 AY712942 Schmitz et al. (2005) Scincus scincus SC4 AY218025 AY217996 Whiting et al. (2003) Scincus conirostris SC6 Jabal Dannah, UAE EU278016 EU278081 Carranza et al. (2008) Scincopus fasciatus SCP2 ca.30km NW Rosso, Mauritania AY649132 AY649173 Brandley et al. (2005) Scincopus fasciatus SCP3 AY308453 AY308302 Schmitz (2003)
1Individuals whose subspecific taxonomy could not be assessed and were predicted based on geographical location following Eiselt (1940). 2Samples obtained from specimens deposited in the Natural History Museum of Crete. Voucher codes are detailed in parenthesis. Downloaded by [b-on: Biblioteca do conhecimento online UP] at 02:44 11 June 2012 2012 11 June at 02:44 UP] online do conhecimento Biblioteca by [b-on: Downloaded AFRICAN JOURNAL OF HERPETOLOGY 61(1) 2012 73
Figure 1. Distribution map of the Eumeces algeriensis and E. schneideri samples included in the study. The small maps show the exact location of the samples for E. algeriensis and E. schneideri. Different symbols represent different lineages (see text for more details). Samples with approximate location are marked with (*), while samples of unknown location are not shown (see Table 1 for more details).
the paraphyly of Eumeces suggested in previous studies (Carranza et al. 2008 and references within), sequences from two Scincopus and seven Scincus (Table 1) were selected from GenBank and incorporated into the analysis. Genomic DNA was extracted from the tail tips following standard saline method (Sambrook et al. 1989). Two mitochondrial DNA fragments, 12S rRNA and 16S rRNA were amplified through polymerase chain reaction (PCR). Primers used were 12sa and 12sb for the 12S rRNA (Kocher et al. 1989) and 16sH and 16sL for the 16S rRNA (Palumbi 1996). In both cases, the PCR mix was carried out in a 25ml total volume, with the following conditions: an initial cycle of 928C for 2 min, followed by 30 cycles of 928C for 30 s, 508C for 40 s and 728C for 45 s, and a final cycle of 728C for 5 min. Amplified products were sequenced on an ABI 3730XL automated sequencer using commercial sequencing facilities (Macrogen Inc., Seoul, Downloaded by [b-on: Biblioteca do conhecimento online UP] at 02:44 11 June 2012 South Korea). All sequences obtained were aligned using the ClustalW software (Thompson et al. 1994) implemented in MEGA4 (Tamura et al. 2007). Resulting fragments of 373 base pairs (bp) for 12S rRNA and 503 bp for 16S rRNA were concatenated obtaining a final fragment of 876 bp. Chalcides striatus (GenBank accession numbers EU277995 and EU278067) and C. minutus (GenBank accession numbers EU277974 and EU278063) were included as outgroups (Carranza et al., 2008). Poorly aligned positions of the concatenated sequences were detected and eliminated using the Gblocks software (online version 0.91b; Castresana 2000) considering a relaxed 74 PERERA ET AL.*Genetic variation in Eumeces
selection of blocks (Talavera & Castresana 2007). A final set of sequences of 863 bp length was used in the phylogenetic analyses. Two different phylogenetic approaches, maximum likelihood (ML) and Bayesian inference (BI) were used for the concatenated fragment, implementing the two genes using a partitioned analysis. The best models of nucleotide substitution for each gene were calculated in jModelTest 0.1.1 (Posada 2008) under the Akaike Information Criterion (AIC; Posada 2008). ML was carried out in TREEFINDER (Jobb et al. 2004). We used a partitioned analysis and the bootstrap analysis option with 1000 replicates to assess the nodal support for the ML tree (BP). Bayesian inference was conducted in MrBayes v.3.1.2. (Huelsenbeck & Ronquist 2001). Analysis started with a randomly generated tree and was run for 1 107 generations using a partitioned dataset and the most appropriate evolutionary model for each partition, sampling one tree each 100 generations. Two independent replicates were conducted in parallel (Huelsenbeck & Bollback 2001) using the default heating values. Convergence of the MCMC chains was assessed using the online program AWTY (Wilgenbusch et al. 2004) and the data sampled from generations preceding the stationarity were discarded. The remaining trees were used to assess posterior probabilities (BPP) for nodal support. In addition to the tree-building methods, we analysed the genealogical relation- ships among E. algeriensis and E. schneideri haplotypes across their ranges constructing a Median-Joining network (Fig. 2) using the program Network v.4.5 (# Fluxus Technology; Bandelt et al. 1999). Poorly aligned positions from subsets of the data (E. algeriensis and E. schneideri) were eliminated using Gblocks following the same procedure as described previously. Uncorrected p-distances were calculated using the software MEGA4 (Tamura et al. 2007). The amount of genetic variation within E. algeriensis and E. schneideri was estimated as haplotype (h) and nucleotide (p) diversity and as average number of nucleotide differences (K) using the software DnaSP v. 5 (Librado & Rozas 2009). The hypothesis of past population expansion was tested by means of statistics based on the distribution of mutation frequencies such as Tajima’s D (Tajima 1989) and R2 (Ramos-Onsins & Rozas 2002), and on haplotype distribution such as FS (Fu 1997). Of the 863 bp analysed, 227 positions were variable and 206 were parsimony- informative. The best evolutionary models were TIM2 G for 12S rRNA and TPM2uf I G for 16S rRNA. Trees obtained in ML and BI phylogenetic approaches had similar topology, with the exception of minor branches in the E. schneideri clade (Fig. 3). Our results confirm the paraphyly of Eumeces, with E. algeriensis and Scincopus fasciatus, as sister taxa, and E. schneideri related to them. The relationship between
Downloaded by [b-on: Biblioteca do conhecimento online UP] at 02:44 11 June 2012 Scincopus and E. algeriensis is well supported (BP/BPP 89/99; Fig. 3). This result was already reported in previous studies (Griffith et al. 2000; Schmitz et al. 2004; Carranza et al. 2008; Giovannotti et al. 2009). Divergence between S. fasciatus and E. algeriensis was estimated to be 10.5% uncorrected p-distance (12S and 16S rRNA concatenated), similar to the values found previously for 12S rRNA (10.8%; Carranza et al. 2008). Within E. algeriensis from Morocco, genetic diversity is low (h 0.589, p 0.008, K 6.654), but two divergent lineages geographically congruent and well supported (BP/BPP 99) are observed. The first one is widespread across Morocco and includes most of the individuals analysed, while the second is restricted to AFRICAN JOURNAL OF HERPETOLOGY 61(1) 2012 75
Figure 2. Median Joining Network based on the mitochondrial fragment (12S rRNA and 16S rRNA concatenated). (a) Median Joining Network for E. algeriensis based on a 863 bp fragment; (b) Median Joining Network for E. schneideri based on 861 bp. Grey circles represent different haplotypes with size proportional to the sample frequency, and black circles represent missing haplotypes (extinct or not sampled).
the southern region (Anti-Atlas) with the exception of a southern locality Downloaded by [b-on: Biblioteca do conhecimento online UP] at 02:44 11 June 2012 (Agadir-Tiznit) where individuals from both clades are found (EA2 and EA11). It is noticeable the low intra-group genetic diversity in both clades (widespread lineage: h 0.350, p 0.001, K 0.500; southern lineage: h 0.733, p 0.003, K 2.267). The Median-Joining network also illustrates this, with only eight different haplotypes distributed in two groups (five from the widespread lineage, and three from the southern one), separated by 18 mutational steps from each other (Fig. 2). These results are unexpected, as other studies in the region report, in general, high levels of variability (Rato & Harris 2008; Rato et al. 2010) and occasionally, the existence of differentiated lineages in the Anti-Atlas region (Perera & Harris 2010). The low 76 PERERA ET AL.*Genetic variation in Eumeces
Figure 3. Phylogenetic tree based on the 863 bp mtDNA fragment (12S rRNA and 16S rRNA concatenated). Topology of the tree corresponds to the maximum likelihood (ML) analysis. Numbers separated by slash near the nodes represent the bootstrap support (BP) values for the ML analysis, and the Bayesian posterior probability values (BPP), respectively. * and ** refer to BP and BPP values above 95 and 98 respectively, while values with full support for both BP and BPP are represented by a dark rectangle. (1) indicates individuals whose subspecific taxonomy was not clear and that were designated on the basis of their geographical location following Eiselt (1940).
variability within E. algeriensis reported here might be tentatively explained as the result of a recent expansion of this species across most of its current range, facilitated by the higher dispersal abilities related to the large body size of these skinks. This hypothesis is supported by both significant large negative values of the Tajima’s D
(Tajima’s D 1.831; PB0.05), and FS (FS 1.790; PB0.02) statistics, although the latter was marginally significant. By contrast, the low but not significant value of
the R2 statistic (R2 0.140; P 0.05), did not support a past sudden expansion scenario. The finding of a locality in the south where both lineages co-occur also supports a fast expansion scenario and might indicate that both lineages were Downloaded by [b-on: Biblioteca do conhecimento online UP] at 02:44 11 June 2012 previously confined to the south of the Atlas, and in the relatively recent past only one of them expanded northwards to occupy the current range. With all this information, and taking into account the wide distribution of this Moroccan lineage and the shape of the network (Fig. 3), we can tentatively hypothesize a past geographic-demographic expansion for this lineage, although further data are necessary to fully test this hypothesis. Finally, our sampling did not include any individual from the subspecies E. a. meridionalis. This subspecies has been reported from a few localities across the High Plateaux from East Morocco and NW Algeria (Bons & Geniez 1996; AFRICAN JOURNAL OF HERPETOLOGY 61(1) 2012 77
Sindaco & Jeremcenko 2008), and has been considered by authors as part of E. algeriensis (Caputo et al. 1993), E. schneideri (Eiselt 1940) or a unique species (Schleich et al. 1996). The two subspecies present in Morocco occupy similar habi- tats and have the same colour pattern and size, differing only on scalation characters (Schleich et al. 1996). Future analysis of specimens of E. a. meridionalis will be fundamental to clarify the relationships within these two groups and to confirm the validity of this form. Regarding E. schneideri, considerable within group diversity is observed (h 0.945, p 0.017, K 14.363; Fig. 3) with a remarkable genetic differentiation between haplotypes (Fig. 2). The phylogenetic tree reveals three well-supported lineages corresponding to the three subspecies of E. schneideri included in this study, although the relationships among them are not well supported (Fig. 3). One group is constituted by E. s. princeps from northeast Turkey and South Armenia. A second group is formed by E. s. schneideri from Egypt, plus the sample from West Asia and another of unknown origin (Figs. 1 and 3). Finally, the third group is constituted by E. s. pavimentatus, that includes several lineages with some geographical concor- dance. Eumeces s. pavimentatus from Syria (ES10) and Turkey (GB7 and GB8) show closely related haplotypes. Interestingly, despite the fact that sample GB8 fits within the E. s. princeps distribution according to Kumlutas et al. (2007), based on our mitochondrial DNA sequences, it is E. s. pavimentatus. This result expands the distribution range (Kumlutas et al. 2007) of the subspecies in Turkey. Another group is formed by the samples from Jordan and Syria, and finally, the remaining samples from Jordan formed a group with some geographical congruence, although not well supported (Fig. 3). The Median-Joining network confirmed the high variability within E. schneideri, with 10 different haplotypes in 14 individuals, and a distance of 34 mutational steps between the most divergent haplogroups (3.2% uncorrected p-distance; Fig. 2). The two E. s. princeps samples share the same haplotype, which was well separated from the remaining E. schneideri samples. Interestingly, the genetic differentiation between E. s. pavimentatus and E. s. schneideri haplogroups (16 23 mutational steps) was similar to that observed within E. s. pavimentatus haplotypes from different regions of Jordan (15 17 mutational steps) or Syria (22 mutational steps) (Fig. 2). Several E. schneideri taxa (species/subspecies depending on the authors) have been described for the Middle East region, mostly based on morphological features (Eiselt 1940; Go¨c¸men et al. 2002; Kumlutas et al. 2007). Our results, although limited in their interpretation we could not assign to the subspecies level some of the individuals provide interesting information regarding the genetic variability of Eumeces in this region. Genetic differentiation within E. schneideri is quite high and not completely congruent
Downloaded by [b-on: Biblioteca do conhecimento online UP] at 02:44 11 June 2012 with the current intra-specific taxonomy. Indeed, while E. s. princeps forms a well supported and differentiated entity, E. s. pavimentatus has a more complex structure, reflected in its major haplotypic diversity and genetic differentiation. Interestingly, Werner (1988) observed ecological differences between E. schneideri and E. pavimentatus, one being more linked to Mediterranean habitats and the other more associated to desert areas, respectively. Although samples from Jordan and Syria were assigned to E. s. pavimentatus based on geographical location (following Eiselt (1940) and Sindaco & Jeremcenko (2008)), the genetic differentiation observed suggests the occurrence of several undescribed entities from Syria and Jordan. In addition, the distribution of the lineages found within this region has some 78 PERERA ET AL.*Genetic variation in Eumeces
geographical structure (north, central and southern Middle East) concordant with patterns observed in Hyla frogs (Gvozd´ık et al. 2010). Studies in this region that are now emerging, confirm the complexity of this area (Gvozd´ık et al. 2010; Kornilios et al. 2010) and our results also point in this direction. The Middle East is a crossroad of Palearctic, Oriental and Afrotropic ecozones and has been considered a refugia for the herpetofauna during Pleistocenic climatic oscillations (Hewitt 1999; Fritz et al. 2008; Gvozd´ık et al. 2010). It will be interesting in the future to increase the sampling in this region and surrounding areas, to complete the picture of the variation of Eumeces across this region. In conclusion, our results support once more the paraphyly of Eumeces, suggesting the need of a taxonomic reassessment for this genus. Moreover, this preliminary assessment of the genetic diversity within E. algeriensis and E. schneideri has revealed surprising patterns. The former shows a relatively low genetic variability in North Africa and the existence of two widely divergent lineages, which might suggest a history of a recent expansion. Regarding the latter, complex patterns, not completely congruent with the currently described subspecies, have been retrieved. Our results confirm the need of a deeper assessment in the Middle East region in order to understand the exact distribution of the different subspecies and their genetic differentiation.
ACKNOWLEDGEMENTS
We are grateful to all the people from CIBIO that collaborated in the sampling in Morocco. This study was partially funded by the FCT project PTDC/BIA-BDE/ 74349/2006. AP is supported by a FCT grant SFRH/BPD/26546/2006, DS by a FCT grant SFRH/BPD/66592/2009 and SC and FS did the study under the FCT program BII/CIBIO/INTBIO/2009. We are especially grateful to Petros Lymberakis from the National History Museum of Crete for the donation of Eumeces samples.
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Received: 30 October 2010; Final acceptance: 19 April 2011