North-Western Journal of Zoology Vol. 4, No. 1, 2008, pp.119-124 [Online: Vol.4, 2008: 04]

Unexpectedly low mitochondrial DNA variation within the ladder Rhinechis scalaris

Valeria NULCHIS1, Marta BIAGGINI2, Miguel A. CARRETERO3, and D. James HARRIS3,4*

1. Societas Herpetologica Italica - Sezione Sarda, c/o Alea, Via Canepa, 09170 Oristano, . 2. Dipartimento di Biologia Animale e Genetica, Università di Firenze, Via Romana 17, 50125 Firenze, Italy. 3. CIBIO, Centro de Investigação em Biodiversidade e Recursos Genéticos, Campus Agrário de Vairão, P- 4485-661 Vila do Conde, 4. Departamento de Zoologia e Antropologia, Faculdade de Ciências da Universidade do Porto, 4099-002 Porto, Portugal. * Corresponding author: D. James Harris, e-mail: [email protected]

Abstract. The , as an important glacial refugia for much fauna and flora, is a well known biodiversity hotspot. Of the studied herpetofauna almost all that are not recent colonizers of the area show extensive intraspecific genetic variation, often at a level that has implied cryptic speciation. In this study variation within the ladder snake, Rhinechis scalaris, essentially an Iberian endemic, was examined using cytochrome b mitochondrial DNA sequences. Surprisingly no variation across the species range was found. This is an extremely unusual situation, that deserves further investigation into the causes of the low variation.

Key words: Rhinechis scalaris, ytochrome b, mitochondrial DNA, phylogeography, Iberian Peninsula

The ladder snake, Rhinechis scalaris, there has been considerable time for is a large colubrid inhabiting the intraspecific variation to have evolved. Iberian Peninsula except parts of the This is particularly relevant, since north, and Southeast (Arnold molecular phylogeographic assess- 2002). Although historically included ments have illuminated a number of with the other European rat in cryptic species in other Iberian the , it is genetically very including Podarcis (Pinho et al. 2006), differentiated from these, which has Blanus (Vasconcelos et al. 2006) and led to the revalidation of the monotypic Iberolacerta (Carranza et al. 2004a). Di- genus Rhinechis Michaeles, 1833 (Lenk versity has also been considerable in et al. 2001, Utiger et al. 2002). Using most widespread groups molecular dating techniques, Nagy et studied in this region, including Lacerta al. (2004) indicate R. scalaris may have schreiberi (Godinho et al. 2006), Timon split from its closest relatives, a clade lepidus (Paulo 2001), and Psammodromus comprising Elaphe quatuorlineata and algirus (Carranza et al. 2006a). It is well Coronella, approximately 20 million known that Southern European areas years ago. This would suggest that such as the Balkans, Italy and the

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Iberian Peninsula acted as refugia for cols. Polymerase Chain Reaction many species during the climatic primers used in both amplification and fluctuations of the Pleistocene (Hewitt sequencing were GluDG and Cytb2 2000). Further, the findings of highly from Palumbi et al. (1991) and Kocher divergent groups within species from et al. (1989) respectively. Amplification the Iberian Peninsula have led to the conditions were the same as described description of “refugia within refugia” by Harris et al. (1998). Amplified (Gomez & Lunt 2007), with distinct fragments were sequenced on a 310 parts of the Iberian Peninsula acting as Applied Biosystem DNA Sequencing separate refuges and allowing Apparatus. DNA sequences were extensive genetic differentiation to aligned by eye. Three specimens from develop. In this context, the aim of this GenBank (Lenk et al. 2001; Nagy et al. study was to assess levels of diversity 2004) were also analysed. within R. scalaris using Cytochrome b In total 333 base pairs of mitochondrial DNA sequence data to mitochondrial DNA were analysed for assess if this species showed similarly 12 new specimens and three specimens high levels of genetic diversity. from GenBank, although two of these The number and geographic (Lenk et al. 2001) were 16 base pairs locations of the specimens used in this shorter. All the sequences for this study are given in Table 1 and Figure 1. region were identical, except for one C- Most samples were comprised of T transition in one sample from Nagy roadkills. Live individuals were et al. (2004), from . The single released after tail tips were collected. haplotype recovered from the Tissue samples were stored in 100% specimens sequenced in this study has ethanol. Total genomic DNA was been submitted to GenBank (EU497 extracted from tissue samples follow- 634). ing the Sambrook et al. (1989) proto-

Table 1. Localities of samples used, and their position in Figure 1.

Locality: Country: Coordinates: Vila Cova, Barcelos, district Portugal 41º 32' N, 8º 42' W Castelo Branco, Portugal 39º 52' N, 7º 07' W Resende, district Portugal 41º 06' N, 7º 58' W Sintra, Lisboa district Portugal 38º 47' N, 9º 23' W Motilla del Palancar, Cuenca province Portugal 39º 34' N, 1º 55' W Touro, , Portugal 40º 53' N, 7º 45' W Laroya, Almeria province Spain 37º 17' N, 2º 19' W Fermoselle, Zamora province Spain 41º 18' N, 6º 24' W Ribeira de Pena, Portugal 41º 31º N, 7º 48' W Vale de Algoso, Vimioso, Bragança district Portugal 41º 29' N, 6º 32' W Fornillos, Zamora province Spain 41º 22' N, 6º 19' W Monte La Algaida, Sevilla province Spain 36º 50' N, 6º 18' W

North-West J Zool, 4, 2008 mtDNA variation within Rhinechis scalaris 121

Figure 1. Map showing the sampling locations of R. scalaris sequenced for this study. Codes are given in Table 1.

Such low diversity within a wide- is also high in the relatively closely spread snake is unexpected, especially related Coronella austriaca partially given the very high levels of using the same gene region (Santos et intraspecific variation typically seen in al. 2008). Diversity is low in the Iberian herpetofauna from the Iberian Peninsula in the snakes Malpolon Peninsula. Although sampling was monspessulanus, Hemorrhois hippocrepis limited, they do cover a wide area and (Carranza et al. 2006b) and Macro- so this is unlikely to be the reason that protodon brevis (Carranza et al. 2004b; diversity is so low. In the viperine Vasconcelos & Harris 2006), but these snake, Natrix maura, there is high species all show extensive variation in differentiation between samples in North Africa, and seem to be relatively Southern Iberia and the rest of the recent colonizers of the Iberian Peninsula (Guiking et al. 2006; Barata et Peninsula. The low diversity within R. al. in press). This southern region scalaris would then require that it too shows no differentiation in R. scalaris. was found in North Africa but has Diversity within the Iberian Peninsula either gone unnoticed or went extinct if

North-West J Zool, 4, 2008 122 Nulchis, V. et al. a similar hypothesis for this species tion for the low diversity could be the was made. A different hypothesis phenomenon of a “selective sweep”, would be that R. scalaris suffered an where direct or indirect selection leads extreme range decline during the last to a near fixation of a mitochondrial glacial periods, and lost most genetic DNA lineage within a species diversity. Current populations would (reviewed in Ballard & Whitlock, 2004). then reflect a recent expansion from a It is now more clearly understood how single refugium. A similar situation the history of the mitochondria may occurs in the Wall lizard Podarcis not always reflect the history of the bocagei, but this species is found only in species, and assessment of nuclear the Northwest Iberian Peninsula – markers is clearly needed to fully other Podarcis species in the south of understand the situation in R. scalaris. the Iberian Peninsula still contain The species does however seem to extensive genetic variation (Pinho et al. stand as the exception to the rule of 2007). In comparison with the other very high intraspecific mitochondrial common Iberian large snakes Malpolon variation in Iberian herpetofauna. monspessulanus and Hemorrhois hippocrepis, R. scalaris displays early breeding due to summer spermato- Acknowledgements. This project was supported by grants from Fundação para a genesis (Pleguezuelos & Feriche in Ciência e Tecnologia POCTI/BIA-BDE/74349 press); a stenophagous diet restricted to /2006, SFRH/BPD/27025/2006 and SFRH/BPD/ endotherms and in particular 26738/2006. Thanks to JC Brito and all the mammals, all actively hunted students from CIBIO who participated during fieldwork. (Pleguezuelos et al. 2007); and high optimal temperatures despite partial nocturnal activity (Blázquez 1995). References

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