Genetic variation across Tunisian populations ofthe anuran species Discoglossus pictus and

Pelophylax saharicusNabil Amor1*, Guillermo Velo-Antón2,3, Sarra Farjallah1 & Khaled Said1

1

Research Unit: Genetics, Biodiversity and Bio-Resources Development, Biotechnology Institute

of Monastir, 5000 Monastir, Tunisia

2

Department of Ecology and Evolutionary Biology, Cornell University, Corson Hall, Ithaca, NY 14853, U.S.A.

3

Grupo de Ecoloxía Evolutiva e da Conservación, Departamento de Ecoloxía e Bioloxía Animal, Universidade de Vigo,

E.U.E.T. Forestal, Campus Universitario, 36005 Pontevedra, Spain

Received 24 December 2009. Accepted 16 March 2010

The intraspecific variation of mitochondrial cytochrome b sequence was analysed for twowidely distributed Tunisian anuran species, Discoglossus pictus and Pelophylax saharicus. Weobserved low genetic variation within both species (D. p. auritus 0.6% uncorrected geneticdistances, P. saharicus 0.2%) and the absence of genetic structure within both species in thestudied region. There is no correspondence between previously described morphotypes ofP. saharicus in Tunisia and our genetic data. Our results show a lack of phylogeographicstructure in both species despite their fragmented distribution across Tunisia.

Key words: cytochrome b, Discoglossus, genetic variation, Pelophylax, Tunisia.

INTRODUCTIONThe Mediterranean Basin is characterized by acomplex geological and climatic history that hasshaped the distribution and diversity of its biota,resulting in the formation of several areas ofendemism and hotspots (Oosterbroek & Arntzen1992; De Jong 1998; Sanmartin 2003). Identifyingand defining taxa in these areas using molecularmethods may prove pivotal for both discoveringand interpreting broad-scale biogeographicalpatterns and then setting conservation prioritiesat the regional level within these biodiversityhotspots (Blackburn & Measey 2009). Of thesehotspots, North Africa is particularly importantbecause it maintains an outstanding number ofendemic species. Moreover, several studies sup-port the importance of the North African region asa glacial refuge centre, as shown for insects(Gantenbein & Largiadèr 2003), amphibians andreptiles (see Busack & Lawson 2008 for a review)and mammals (Cosson et al. 2005). However, muchremains to be learnt about the distribution, patternsof genetic diversity and taxonomy of most species.

This region is characterized by several geographicand climatic zones that may have played a role ingenetic differentiation. While some patches offorest occur in central Tunisia, widely-forestedareas are limited to the north. Almost one-third ofTunisia’s surface area is covered by the Sahara

desert, and a major portion of the rest of the countryis dominated by an arid climate characterized byannual and seasonal variation in rainfall. Themajor limiting ecological factor for amphibianssurviving in the country, therefore, is water (Waltheret al. 2002).

Both Discoglossus pictus, the painted frog, andPelophylax saharicus, the North African green frog,are widespread in Tunisia, but their populationsare highly fragmented due to the scarcity of suit-able habitats (Amor et al. 2009a). Discoglossus pictusinhabits a wide range of biotopes from mountainousforests to coastal sand dunes. In northwesternTunisia it may be found somewhat distant fromany water, and in southernmost localities it isconfined to oasis areas such as Gafsa and the Ouedel Ferd in Gabès (Busack 2006; Amor et al. 2009a).Pelophylax saharicus is the most abundant species inTunisia (Amor et al. 2009a). It inhabits oases, ponds(natural and artificial) and irrigation ditchesthroughout the country (including northernportions of the Sahara Desert). Despite being themost frequently encountered anurans in Tunisia,little is known about intraspecific genetic variationin P. saharicus and D. p. auritus, and comprehensivegenetic and morphometric studies across Tunisiaare lacking (but see Amor et al. 2009b).

Previous studies on populations of these anuransin Tunisia include faunistic surveys (Boulenger1891; Mayet 1903; Gauthier 1928; Schneider 1978;

African Zoology 45(1): 121–128 (April 2010)

*Author for correspondence. E-mail: nabil.amor@gmail.com

Nouira 2001; Romdhane & Missaoui 2001; Siciliaet al. 2007) and broad-scale taxonomic studies(Discoglossus: Fromhage et al. 2004; Zangari et al.2006; Pelophylax: Arano et al. 1998; Buckley et al. 1994,1996; Harris et al. 2003). More recently, cytogeneticstudies of both species (Amor et al. 2007), and amorphometric study of P. saharicus in Tunisia(Amor et al. 2009b) have also been published. Theformer study did not find any significant variationin chromosome banding patterns in Tunisianspecimens of the two species, but the latter studysupported the existence of three morphotypes inP. saharicus related to the three main biogeograph-ical regions (northern, central and southernmorphotypes).

The aims of this study were: 1) to estimategenetic diversity in Tunisian populations of bothanuran species; 2) to characterize patterns ofgenetic structure in both taxa and to identifypotential historical barriers to gene flow, and 3) totest for correspondence between genetic data andthe three previously described morphotypes ofP. saharicus.

MATERIALS & METHODS

Samples and laboratory proceduresWe collected tissue samples from 22 Discoglossus

and 35 Pelophylax, sampled at 10 and 13 localitiesacross Tunisia respectively (Fig. 1). Sampling local-ities are listed in Table 1. Total genomic DNA wasextracted from ethanol-preserved tissues using a

standard proteinase K/phenol chloroform extractionprotocol (Sambrook et al. 1989). Partial sequencesof the mitochondrial cytochrome b gene (cytb)were obtained through amplification by polymerasechain reaction (PCR) using primers MVZ15 andMVZ16 (Moritz et al. 1992). We performed amplifi-cations in a 12.5 µl volume, containing MgCl2

(2.5 mM), buffer reaction (1x, Biotools Edmont,Alberta, Canada), dNTPs (0.2 mM), forward andreverse primers (0.2 mM each), Taq polymerase(Biotools, 5 U/ml) and 1 µl DNA template(10–20 ng). Amplification was carried out using thefollowing conditions: initial denaturation for2 min at 94°C, followed by 40 cycles (denaturationfor 60 s at 94°C, annealing for 60 s at 50°C andextension for 60 s at 72°C) and a final extension for10 min at 72°C.

Double-strand templates were cleaned usingsodium acetate and ethanol to precipitate the PCRproducts and then re-suspended in 20–25 µl ofddH2O. Sequencing was carried out using an ABIPRISM 3700 DNA sequencer, following the ABIPRISM BigDye Terminator Cycle Sequencingprotocol.

Data analysisWe used the software ClustalX (Thompson et al.

1997) to align and edit sequences and MEGAversion 3.1 (1000 bootstrap replicates, Kumar et al.2004) to estimate nucleotide and amino acidcomposition and estimates of mean uncorrectedgenetic distances within the two species. Haplotype

122 African Zoology Vol. 45, No. 1, April 2010

Fig. 1. Maps showing sampling localities for Discoglossus pictus and Pelophylax saharicus.

Amor et al.: Genetic variation across Tunisian populations of two anuran species 123

Table 1. Sampling localities, including latitude and latitude coordinates, voucher codes, and haplotypes observed ineach population studied.

ID Species Locality Region Haplotype Voucher Latitude Longitude

1 D. pictus 14 km E Tabarka North D9 MVZ235683 36°57’30.60”N 08°52’33.60”E2 D. pictus 14 km E Tabarka North D1 MVZ235684 36°57’30.60”N 08°52’33.60”E3 D. pictus 14 km E Tabarka North D8 MVZ235685 36°57’30.60”N 08°52’33.60”E4 D. pictus 14 km E Tabarka North D2 MVZ235689 36°57’30.60”N 08°52’33.60”E5 D. pictus 3 km S Fernana North D6 MVZ235686 36°36’58.80”N 08°41’9.00”E6 D. pictus 2 km S Fernana North D1 MVZ235687 36°40’22.80”N 08°42’7.20”E7 D. pictus Kélibia North D10 GVA340 36°50’58.75”N 11°6’49.49”E8 D. pictus Kélibia North D11 GVA342 36°50’58.75”N 11°6’49.49 E9 D. pictus Kélibia North D5 GVA344 36°50’58.75”N 11°6’49.49”E

10 D. pictus Oued Ezzarga North D3 GVA377 36°38’34.22”N 09°12’59.33”E11 D. pictus Kairouan Central D3 GVA304 35°40’0.00”N 10°5’57.72”E12 D. pictus Kairouan Central D1 GVA394 35°40’0.00”N 10°5’57.72”E13 D. pictus Kairouan Central D7 GVA395 35°40’0.00”N 10°5’57.72”E14 D. pictus Kasserine Central D11 GVA397 35°10’25.93”N 08°49’36.37”E15 D. pictus Monastir Central D11 GVA302 35°45’33.04”N 10°48’49.35”E16 D. pictus Monastir Central D12 GVA350 35°45’33.04”N 10°48’49.35”E17 D. pictus Gafsa South D6 GVA390 34°25’17.94”N 08°47’11.96”E18 D. pictus Gafsa South D1 GVA392 34°25’17.94”N 08°47’11.96”E19 D. pictus Gafsa South D4 GVA393 34°25’17.94”N 08°47’11.96”E20 D. pictus Gafsa Oasis N South D6 GVA300 34°23’21.00”N 08°46’15.00”E21 D. pictus Gafsa Oasis S South D6 GVA301 34°23’18.00”N 08°47’50.00”E22 P. saharicus Nabeul North P1 NA111 36°27’32.37”N 10°44’7.44”E23 P. saharicus Nabeul North P5 NA112 36°27’32.37”N 10°44’7.44”E24 P. saharicus Nabeul North P5 NA113 36°27’32.37”N 10°44’7.44”E25 P. saharicus Nefta Oasis South P3 MVZ 235691 33°52’3.00”N 07°52’46.20”E26 P. saharicus Nefta Oasis South P3 GVA339 33°52’3.00”N 07°52’46.20”E27 P. saharicus Oued Ezzarga North P5 GVA287 36°38’34.22”N 09°12’59.33”E28 P. saharicus Oued Ezzarga North P7 GVA381 36°38’34.22”N 09°12’59.33”E29 P. saharicus Oued Ezzarga North P4 GVA382 36°38’34.22”N 09°12’59.33”E30 P. saharicus Oued Ezzarga North P7 GVA385 36°38’34.22”N 09°12’59.33”E31 P. saharicus Oued Ezzarga North P6 NA131 36°38’34.22”N 09°12’59.33”E32 P. saharicus Oued Ezzarga North P4 NA132 36°38’34.22”N 09°12’59.33”E33 P. saharicus Ghar Dimaou North P1 NA123 36°26’59.37”N 08°25’43.60”E34 P. saharicus Ghar Dimaou North P1 NA118 36°26’59.37”N 08°25’43.60”E35 P. saharicus Ghar Dimaou North P1 GVA294 36°26’59.37”N 08°25’43.60”E36 P. saharicus Kelibia North P1 NA114 36°50’58.75”N 11°6’49.49”E37 P. saharicus Kelibia North P3 NA115 36°50’58.75”N 11°6’49.49”E38 P. saharicus Tunis North P1 NA116 36°52’21.00”N 10°10’54.00”E39 P. saharicus Tunis North P1 NA117 36°52’21.00”N 10°10’54.00”E40 P. saharicus Kairouan Central P2 GVA293 35°40’0.00”N 10°5’57.72”E41 P. saharicus Kasserine Central P2 GVA296 35°10’25.93”N 08°49’36.37”E42 P. saharicus Monastir Central P1 NA125 35°45’33.04”N 10°48’49.35”E43 P. saharicus Monastir Central P1 NA126 35°45’33.04”N 10°48’49.35”E44 P. saharicus Monastir Central P5 NA127 35°45’33.04”N 10°48’49.35”E45 P. saharicus Monastir Central P1 GVA297 35°45’33.04”N 10°48’49.35”E46 P. saharicus Cheneni South P3 NA128 33°52’59.00”N 10°4’39.68”E47 P. saharicus Cheneni South P1 NA130 3°52’59.00”N 10°4’39.68”E48 P. saharicus Cheneni South P1 GVA295 33°52’59.00”N 10°4’39.68”E49 P. saharicus Gafsa Oasis N South P1 NA121 34°23’21.00”N 08°46’15.00”E50 P. saharicus Gafsa Oasis N South P1 NA122 34°23’21.00”N 08°46’15.00”E51 P. saharicus Gafsa Oasis S South P1 NA119 34°23’18.00”N 08°47’50.00”E52 P. saharicus Gafsa Oasis S South P1 NA120 34°23’18.00”N 08°47’50.00”E53 P. saharicus Gafsa Oasis S South P1 GVA292 34°23’18.00”N 08°47’50.00”E54 P. saharicus Gafsa Oasis S South P1 NA118 34°23’18.00”N 08°47’50.00”E55 P. saharicus Tozeur South P1 NA119 33°55’20.59”N 08°7’58.93”E56 P. saharicus Tozeur South P1 NA124 33°55’20.59”N 08°7’58.93”E

(h) and nucleotide (p) diversity (Nei 1987) wereestimated for each species using the softwareDnaSP ver. 4.0 (Rozas et al. 2003).

Because we encountered few phylogeneticallyinformative sites in the cyt b fragment in bothspecies, we used a phylogenetic network approachto visualize alternative genealogical pathwaysamong haplotypes from both species. We con-structed a statistical parsimony network (Templetonet al. 1992) using the software TCS ver. 1.21 (Clem-ent et al. 2000). This software implements a statisti-cal parsimony algorithm to reconstruct a haplotypenetwork in which the number of mutational stepsconnecting haplotypes is minimized. In contrast todichotomous phylogenetic trees, networks allowfor persistent ancestral nodes and reticulationevents. Such a network is able to illustrate simulta-neous alternative evolutionary pathways, withoccurrence of reticulations visualizing ambiguousor uncertain domains (Posada & Crandall 2001).Moreover, the position of a haplotype in a networkimplies information regarding its age and olderhaplotypes are thought to have a greater likeli-hood of being located internally in the network(Posada & Crandall 2001).

We used a spatial analysis of molecular variance(AMOVA), as implemented in ARLEQUIN ver.3.11 (Excoffier et al. 2005) to analyse genetic struc-ture in both species. We grouped populationsbased on the three main bioclimatic regions inTunisia (northern, central and southern localities;Table I). The Aures Mountains separate northernand central populations, whereas the OrbataMountains and associated arid areas separatecentral from southern populations. Because thethree previously described morphotypes inP. saharicus (Amor et al. 2009b) correspond withbioclimatic regions, this analysis also accounts forthe association of morphotypes with patterns ofgenetic structuring in this species. Levels of signifi-cance of statistics characterizing variation at differ-ent hierarchical levels were assessed through10 000 permutations.

RESULTSFor D. pictus, the 21 aligned sequences comprised730 base pairs (bp). We identified 12 parsimony-informative sites within 21 variable positions,defining 12 haplotypes. Of the 713 sites includedin the analysis of P. saharicus (35 sequences), ninewere variable and eight parsimony-informative,defining seven haplotypes. For both species, noinsertions, deletions, or stop codons were ob-

served, and base content was typical of mtDNA,suggesting the sequences obtained are functionalmtDNA copies rather than nuclear pseudogenes.

For both species, there is a very strongly biasedtransition/transversion ratio (K Discoglossus =16.8; K Pelophylax = 31.7), which is characteristic ofmitochondrial genes, and a very low value of thealpha parameter related to rate heterogeneity (αDiscoglossus = 0.16; α Pelophylax = 0.18), indicatinga very strong heterogeneity of substitution ratesbetween sites (Arctander 1995). All sequenceshave been deposited in GenBank (accession nosGU799090–GU799108).

Genetic diversity in D. pictus (Pi = 0.006; Hd =0.913) was higher than in P. saharicus (Pi = 0.002;Hd = 0.684). Intraspecific pairwise divergencevalues were also higher in D. pictus (mean = 0.6 %,S.D. = 0.2%) than in P. saharicus (mean = 0.2%,S.D. = 0.1%).

Within D. pictus, we identified two main haplo-groups, separated by five mutational steps. Haplo-types in the first group (D1, D2, D4, D7, D9, D11and D12) occur throughout Tunisia, from south-ern to northern populations, whereas haplotypesin the second group (D3, D5, D6, D8 and D10) areconfined to the central and northern regions.Haplotypes D1, D6 and D10 are slightly morecommon within studied samples. Among D. pictushaplotypes, the highest genetic diversity occurs inthe population of Tabarka, where we found fourhaplotypes (Table 1; Fig. 2).

The analysis of P. saharicus samples allows us todistinguish seven haplotypes (Fig. 2). Networkrepresentation shows haplotype 2 (P2) as the mostcommon, including 18 individuals from southernto northern Tunisian populations. Three mutationsdifferentiate P2 from haplotype P6, including twosamples from Oued Ezzarga, and two mutationsfrom haplotype P4. However, P8, P7, P5, and P3differ only by one mutational step from P2. In oursample, haplotypes P6 and P7 are restricted to thepopulation of Oued Ezzarga (northwest) and P5 toCheneni (south). Also, samples from the southern-most populations all have the P2 haplotype, exceptthose from Nefta, which have haplotype P4.

According to AMOVA results there is no evidenceof genetic structuring related to bioclimatic regionsin any of the two species (Table 2). Most of thevariation was explained by differences betweenindividuals within populations in both species (76%and 77% for Discoglossus and Pelophylax, respec-tively). Also there was a significant component ofvariation related to differences between popula-

124 African Zoology Vol. 45, No. 1, April 2010

tions within bioclimatic regions in Pelophylax(21%) (Table 2).

DISCUSSIONOur results on genetic variation and differentia-tion across Tunisian populations of Discoglossuspictus and Pelophylax saharicus are in accordancewith the low values of genetic diversity previouslyreported by other studies conducted on both

species across their ranges (Harris et al. 2003;Fromhage et al. 2004; Zangari et al. 2006; Amor et al.2007). Despite our increased sample sizes andgeographical coverage, including fragmentedhabitats for both taxa, we observed extremely lowgenetic variation and no genetic structure withinboth species.

In this study, the sample size for Pelophylax wasalmost double the sample size for Discoglossus

Amor et al.: Genetic variation across Tunisian populations of two anuran species 125

Fig. 2. Cytochrome b networks for Discoglossus pictus and Pelophylax saharicus.

Table 2. AMOVA results for Discoglossus pictus and Pelophylax saharicus populations in Tunisia. Weassessed hierarchical patterns of genetic structure at three levels (among bioclimatic regions, amongpopulations within bioclimatic regions and among individuals within populations) in both species.d.f. = degrees of freedom; SS = sum of square deviations; P = P-value (99%).

AMOVA TotalVariation source d.f. SS variance variation (%) P

DiscoglossusAmong climatic regions 2 4.93 –0.34 –14.97 0.75Among populations 5 17.36 0.84 38.66 0.10Within populations 13 21.70 1.66 76.31 0.04

Total 20 44.00 2.18

PelophylaxAmong climatic regions 2 2.00 0.01 1.18 0.33Among populations 10 8.28 0.13 21.31 0.04Within populations 22 10.75 0.49 77.51 0.03

Total 34 21.03 0.63

but D. pictus showed higher values for bothgenetic variation (12 haplotypes) and intraspecificsequence divergence (0.6%) than P. saharicus(seven haplotypes and 0.2% genetic divergence).Moreover, while Discoglossus haplotypes are splitup into two major haplogroups separated by fivemutational steps, but not geographically related,in Pelophylax there is also no genetic structure, butall samples are included in a single haplogroup,which is widespread across Tunisia (Fig. 2). Differ-ences in habitat requirements and populationsizes between the two species might explain thesedifferences. Thus, while P. saharicus inhabits oases,ponds (natural and artificial) and irrigation ditchesthroughout the country (including northernportions of the Sahara desert), and populationdensity is often very high, D. pictus inhabits a widerange of biotopes from mountainous forests tocoastal sand dunes in the northern Tunisia, but insouthernmost localities it is confined to areas suchas the oases of Gafsa and population densities arevery low (Amor et al. 2009a). As a result of thehigher sensitivity of Discoglossus to environmentalchanges due to natural and human causes, it mayface more severe restrictions to gene flow. How-ever, further studies, including additional sampling,genetic analyses and ecological studies on habitatspecialization, are needed in order to test whetherthese differences reflect historical or contemporarydifferences in broad scale patterns of gene flow.

Our study shows geographically unstructuredpatterns of genetic variation in both anuran species(Table 2), which is unexpected considering thepresence of major present-day barriers to naturaldispersal, like Chott el Jerid (5360 km2); the largestdepression in Tunisia separating the southern-most area from the other regions (Gueddari et al.1983; Millington et al. 1989), where no significantgenetic discontinuities were found. This is unlikelyto be the result of poor resolution in the markerselected for the study. In fact, the mitochondrialcytochrome b gene has been extensively used inamphibians to successfully uncover patterns ofgenetic structure and to delimit geographic bound-aries within species in both Discoglossus (García-París & Jockusch 1999; Martínez-Solano 2004) andPelophylax (Lymberakys et al. 2007; Canestrelli &Nascetti 2008). Therefore, the observed low geneticdiversity and lack of genetic structure in these twoanurans across Tunisia can be subject to multipleinterpretations: 1) lack of any major historicalisolation events in Tunisia, 2) shallow evolutionaryhistories for both species in Tunisia due to recent

and strong bottlenecks, and 3) reduction of geneticdiversity by selective sweeps in a strong positiveselection scenario. The first scenario might beignored because both species show limited geneticdiversity in this study.

For P. saharicus, the low genetic divergences andabsence of geographic genetic structure found inthis study (Table 2) are not in correspondence withmorphometric variation as reported in a previousstudy (Amor et al. 2009b). Results from geneticanalyses suggest that environmental factors maybe driving morphological differences betweenpopulations. In fact, climate change and habitatfragmentation have often been invoked as keypressures affecting the different levels of bio-diversity (Opdam 2004). Several studies haveshown that the principal response of species to thesefactors is a spatial one, which may be comple-mented by adaptive genetic responses by thepopulation (Walther et al. 2002; Tryjanowski et al.2003). Moreover, in amphibians, which are seden-tary and ectothermic, it seems that the action ofsuch pressures is more important than in endo-thermic animals (Bull & Burzacott 2002; Tryjanowskiet al. 2003).

In conclusion, this genetic survey in Tunisianpopulations of Discoglossus and Pelophylax revealedlittle genetic variation and geographic unstruc-tured genetic patterns within both species, despitetheir fragmented habitats. However, a morecomprehensive genetic study covering the distri-bution ranges of both species across North Africaand including unlinked molecular markers (bothmtDNA and nucDNA) would be required to inferthe evolutionary histories of both species, includ-ing historical estimates of gene flow patterns(Velo-Antón et al. 2008). Furthermore, the decreaseof suitable habitat in Tunisia is leading to theextinction of both species on a broad geographicscale (Amor et al. 2009a). Thus, protection-orientedaction, placing a priority on localized populations,must be implemented in the near future to preserveTunisian anurans.

ACKNOWLEDGEMENTS

We thank Iñigo Martínez-Solano and DavidBuckley for support and valuable suggestions onearlier drafts of the manuscript. We thank theMuseum of Vertebrate Zoology (University ofCalifornia, Berkeley) for access to tissue samplesfrom their collection. Guillermo Velo-Antón wassupported by a grant from Galician Government(Consellería de Innovación, Industria e Comercio,

126 African Zoology Vol. 45, No. 1, April 2010

Xunta de Galicia) and is currently supported by apostdoctoral fellowship from the Spanish Ministeriode Ciencia e Innovación (Ref: 2008-0890).

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Responsible Editor: P. leF. N. Mouton

128 African Zoology Vol. 45, No. 1, April 2010