Molecular Ecology (2005) 14, 245–254 doi: 10.1111/j.1365-294X.2004.02390.x GeneticBlackwell Publishing, Ltd. exchange across a hybrid zone within the Iberian endemic golden-striped salamander, Chioglossa lusitanica F. SEQUEIRA,*† J. ALEXANDRINO,*§ S. ROCHA,* J. W. ARNTZEN*‡ and N. FERRAND*† *CIBIO/UP-Centro de Investigação em Biodiversidade e Recursos Genéticos, Campus Agrário de Vairão, Rua Padre Armando Quintas, 4485–661 Vairão, Portugal, †Departamento de Zoologia e Antropologia, Faculdade de Ciências, Universidade do Porto, Praça Gomes Teixeira, 4099-002 Porto, Portugal, ‡National Museum of Natural History, PO Box 9517, 2300 RA Leiden, the Netherlands, §Museum of Vertebrate Zoology, University of California, Berkeley, 3101 Valley Life Science Building # 3160, Berkeley, CA 94720– 3160, USA Abstract The study of hybrid zones resulting from Pleistocene vicariance is central in examining the potential of genetically diverged evolutionary units either to introgress and merge or to proceed with further isolation. The hybrid zone between two mitochondrial lineages of Chioglossa lusitanica is located near the Mondego River in Central Portugal. We used mito- chondrial and nuclear diagnostic markers to conduct a formal statistical analysis of the Chioglossa hybrid zone in the context of tension zone theory. Key results are: (i) cline centres are not coincident for all markers, with average widths of ca. 2–15 km; (ii) heterozygote deficit was not observed across loci near the transect centre; (iii) associations of parental allele combinations (‘linkage disequilibrium’ R) were not detected either across loci or across the transect. These observations suggest that the Chioglossa hybrid zone is not a tension zone with strong selection against hybrids but instead one shaped mostly by neutral mixing. The patterns uncovered suggest a complex history of populations over a small scale that may be common in southern Pleistocene refugia. Keywords: allozymes, asymmetric introgression, Chioglossa lusitanica, cline analysis, gene flow, hybrid zone, maximum likelihood, mtDNA Received 03 June 2004; revision received 30 September 2004; accepted 01 October 2004 refugial areas. This pattern of ‘refugia within refugia’ is Introduction becoming especially apparent in the Iberian Peninsula The climatic oscillations of the Quaternary had a profound after phylogeographical studies burgeoned in the region impact on many organisms, subdividing their ranges over (Gomez & Lunt 2004 and references therein). As distinct distinct Pleistocene glacial refugia with the consequent evolutionary units are uncovered in many organisms, it is genetic diversification of evolutionary units that often came important from both evolutionary and conservation per- to meet in hybrid zones (sensu Arnold 1997) after climatic spectives to examine their degree of genetic cohesiveness amelioration (Hewitt 1996, 1999, 2004; Taberlet et al. 1998). in zones of parapatry (i.e. gene flow across hybrid zones). Complete speciation appears not be a common outcome of Here we take advantage of one paradigm in the Iberian Pleistocene vicariance but stages of incipient speciation are refugial historical biogeography, the golden-striped sala- observed at hybrid zones with several degrees of genetic mander Chioglossa lusitanica (described below), to emphasize isolation (Avise et al. 1998; Hewitt 2000). Recent work in how the study of genetic cohesiveness between parapatric western Eurasia suggests that diversification of evolution- evolutionary units is central to make predictions on alter- ary units not only occurred between the three southern native evolutionary outcomes in Quaternary diversifying peninsulas (Iberian, Apennine, Balkans) and the Caucasus taxa (e.g. lineage amalgamation vs. further isolation). but also between smaller range fragments within these The genetic cohesiveness between evolutionary units can be examined in hybrid zones where genetically distinct Correspondence: João Alexandrino, Fax: 1510 6438238, E-mail: populations meet and produce hybrids (Barton & Hewitt [email protected] 1985; Harrison 1990; Arnold 1997). Selection may act on © 2004 Blackwell Publishing Ltd 246 F. SEQUEIRA ET AL. new combinations of alleles generated by recombination existence of a hybrid zone between the northern and southern (Harrison 1990, 1993). This kind of interaction often results forms with some mixing, but sampling was not sufficient in the formation of clines at different loci or phenotypic to describe the genetic structure of the zone in detail traits, the position and width of which are determined by (Alexandrino et al. 2000). a balance between dispersal into the zone and any counter The purpose of our work is to undertake a formal selection acting on hybrids. These cline shapes are rela- population genetic analysis of the hybrid zone between the tively independent of the type of selection acting against two evolutionary units of C. lusitanica. Here, we apply hybrids which is usually unknown (endogenous, exogen- maximum likelihood tension zone models (Szymura & ous or both; Kruuk et al. 1999). The ‘coincidence’ of the cline Barton 1986, 1991; Barton & Gale 1993; Phillips et al. in centres and the ‘concordance’ of cline widths are expected press) to quantify cline width, cline concordance/coincidence when a hybrid zone first forms from secondary contact. and genotypic disequilibria at five diagnostic loci (four After several generations of hybrid formation and back- allozymes and mtDNA) across a transect perpendicular crossing through continued dispersal into the zone, selec- to the Mondego River. We will use the above hybrid zone tion against hybrid genotypes, epistatic effects between properties not only to learn about the genetic differences loci and genome-wide linkage disequilibria will generate a between the populations involved but also to investigate if steep step in allele frequencies that will result in coincidence a barrier to gene exchange (i.e. hybrid counter selection) and concordance among clines for independent traits (i.e. between differentiated gene pools is in place. If selection tension zones; Barton 1983; Barton & Hewitt 1985; Barton is operating against hybrids, we expect to observe cline & Gale 1993; Gavrilets 1997; Barton & Shpak 2000). If selec- widths that are narrow relative to dispersal and significant tion against hybrids is strong, the centre of the zone will act genetic disequilibria in the centre of the zone. as an effective barrier to the introgression of both nega- tively selected and neutral alleles, but positively selected Materials and methods alleles could still cross such a barrier (Piálek & Barton 1997). A very different multilocus cline shape pattern can arise if Collection of samples selection is acting only weakly on loci, which will free clines to act independently of each other rather than as sin- Sampling was carried out in central Portugal within and gle nonrecombining unit. Each cline may then respond to around the putative contact zone between the southern independent forces such as locus-specific selection and/or and the northern population groups of Chioglossa lusitanica drift with the resulting pattern being noncoincidence (Alexandrino et al. 2000). This area (ca. 50 × 60 km) is located and nonconcordance of clines (Barton & Bengtsson 1986; in the southern part of the range and comprehends the Harrison 1990; Barton & Gale 1993; Searle 1993). A narrow mountain ranges of Buçaco (North), Lousã (West), Muradal hybrid zone with coincident and concordant clines and (South) and Estrela (East), and is crossed by two main rivers strong selection against hybrids is more likely to effectively — Mondego and Zêzere (Table 1 and Fig. 1). Salamanders maintain isolation and further promote divergence between were collected at 14 sites between 1998 and 2003 and diverged forms than a hybrid zone with wider clines asso- the tail end of each individual was removed and frozen at ciated with neutral mixing. −70 °C for genetic analysis. Sample size ranged from 15 to The golden-striped salamander, Chioglossa lusitanica 47, including samples reported upon earlier (sites 1, 3, 5, 7, (Salamandridae), is an Iberian endemic species in which at 11, 12 and 13; Alexandrino et al. 2000). least two local refugia have been identified from protein and mitochondrial (mt) DNA data. The salamander in- Markers habits the banks of small streams over mountainous areas characterized by high rainfall and mild summer and Allozyme electrophoresis. We conducted allozyme electro- winter temperatures (Arntzen 1981; Teixeira et al. 2001). phoresis of products encoded by the seven allozyme loci Groups of populations at either side of the Mondego River reported to be polymorphic in C. lusitanica by Alexandrino in central Portugal are genetically distinct (Alexandrino et al. (1997, 2000): alcohol dehydrogenase (Adh), phospho- et al. 2000, 2002) and have slightly different ecological pro- glucomutase (Pgm-1), phosphogluconate dehydrogenase files, with climatic conditions in the south that is harsher (Pgd) and four peptidases (Pep-A, Pep-B, Pep-C and Pep-D). than in the north (Arntzen & Alexandrino 2004). Divergence Only four of these (Adh, Pep-C, Pep-D and Pgm-1) were time estimates from genetic data (Alexandrino et al. 2000) diagnostic between northern and southern C. lusitanica in combination with the extrapolation of bioclimatic and were therefore useful for hybrid zone analysis. models (Teixeira et al. 2001; Teixeira & Arntzen 2002) suggest that the two evolutionary units may