Mitochondrial DNA evolution and population history of

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1 Molecular Ecology (2000) 9, Mitochondrial DNA evolution and population history of Blackwell Science, Ltd the Tenerife skink Chalcides viridanus R. P. BROWN,* R. CAMPOS-DELGADO and J. PESTANO *School of Biological & Earth Sciences, Liverpool John Moores University, Byrom St., Liverpool L3 3AF, UK, Departamento de Genética, Facultad de Ciencias Medicas y de La Salud, Universidad de Las Palmas de Gran Canaria, Las Palmas, Spain Abstract Recent studies of island lizards have suggested that historical vicariance as a result of volcanism may have played an important role in shaping patterns of within-island genetic diversity. The skink, Chalcides viridanus, shows variation in morphology within the volcanic island of Tenerife. Two mitochondrial DNA (mtdna) fragments (from the 12S and 16S rrna regions) were sequenced in individuals from 17 sites to evaluate the relationship between current phylogeography and the geological history of the island. Three main clades were detected. The two most basal clades were restricted to areas representing the ancient precursor islands of Teno and Anaga in the northwest and northeast of Tenerife, respectively. The third clade showed a widespread geographical distribution and provided evidence of a recent rapid expansion after a bottleneck. Within-island cladogenesis appears to have taken place during a recent period of volcanic activity and long after the ancient islands had been united by the eruptions that led to the formation of the Cañadas edifice. Evidence of similar biogeographical histories are found in other species in the Canary archipelago, supporting the volcanism scenario as a potentially widespread cause of within-island differentiation in reptiles. Keywords: evolution, geographical variation, island, lizard, phylogeography, volcanism Received 13 November 1999; revision received 21 February 2000; accepted 21 February 2000 Introduction A large number of studies have demonstrated patterns of vertebrate population differentiation over continental regions that reflect historical fragmentation of gene flow (Avise 1992; Hewitt 1996; Taberlet 1998; Douglas et al. 1999). In terrestrial species, range contractions mediated by global climatic changes appear to be one of the primary causes of this fragmentation. More recently, differentiation over much smaller geographical areas has been highlighted by within-island studies of lizards (e.g. Thorpe & Báez 1987; Brown et al. 1991; Malhotra & Thorpe 1991; Schneider 1996; Thorpe & Malhotra 1996; Báez & Brown 1997). Global climatic scenarios provide unlikely phylogeographical explanations in these cases. Instead, evidence is beginning to favour the hypothesis that historical volcanism is an important and widespread cause of within-island cladogenesis and played a major Correspondence: R. P. Brown. Fax: +(44) ; r.p.brown@livjm.ac.uk role in shaping these patterns of genetic diversity. This is supported by mitochondrial DNA (mtdna) surveys (Pestano & Brown 1999; Thorpe et al. 1996) and the fact that within-island variation has been described on volcanic islands, particularly on Tenerife and Gran Canaria in the Canary archipelago (centre 28 N, 16 W) off northwest Africa. The influence of this mode of differentiation on insect diversity has already been established (Carson et al. 1990). The ability of these phylogeographical patterns to explain within-island polymorphism in phenotypic characteristics is clearly of interest. Cladogenesis in the Tenerife lizard Gallotia galloti is thought to have occurred 0.7 million years ago (Ma), but the resultant mtdna signature is slightly discordant with the morphological variation (Thorpe et al. 1996). Evidence of earlier cladogenesis ( 3 Ma) is found in the Gran Canarian skink, Chalcides sexlineatus, where morphological and phylogeographical patterns are concordant (Pestano & Brown 1999). Parallel geographical variation in morphology is present in the Tenerife skink, C. viridanus (Brown et al. 1991) although it 2000 Blackwell Science Ltd

2 1062 R. P. BROWN, R. CAMPOS-DELGADO and J. PESTANO Fig. 1 The 17 samples site, the three proposed ancient islands (shaded areas) (Ancochea et al. 1990) and altitudes on the island of Tenerife (surface area 2034 km 2 ). appears to be incompatible with the volcanism scenario put forward for G. galloti on the same island. The present work evaluates phylogeographical and morphological concordance in C. viridanus and putative associations between the former and the volcanic history of Tenerife. This is achieved though a detailed analysis of among-population diversity in C. viridanus, based on mtdna sequences from within the 12S and 16S rrna regions. Most of the major volcanic eruption events within Tenerife occurred over 0.6 Ma, which may have left clearly detectable signatures in the studied fragments, given their rates of evolution within the Canary Island skinks (Brown & Pestano 1998). Materials and methods Study species and sample sites Chalcides sexlineatus is a small, ground-dwelling species, which appears to show low vagility. It is most abundant in low-altitude habitats in the north, with densities decreasing in arid southern areas and at altitudes above 1100 m (see Brown et al. 1993). Tail tips were obtained from C. viridanus from 16 selected sites within Tenerife (Fig. 1) and immediately stored in 99% alcohol. Skink morphology has previously been quantified at all these sample sites (Brown et al. 1993). Four individuals were also available from a new site (site 3). The total number of individuals from which sequence data was collected was 85 (n per site = 4 8, except for sites 1 [n = 3] and 14 [n = 1]). Low levels of introgression appear to lead to reduced within-site mtdna diversity and this favours the use of increased numbers of sample sites (increasing the probability that all major lineages have been detected), as opposed to larger sample sizes, when describing within-island phylogeographical patterns (Thorpe et al. 1996; Brown & Pestano 1998). mtdna sequencing Tissue samples were homogenized in a digest buffer and whole genomic DNA was obtained using an organic extraction protocol (Gross-Bellard et al. 1973). The L1091 and H1478 primers (Kocher et al. 1989) were used to amplify a fragment of 384 bp from the 12S rrna region of the mtdna. The polymerase chain reaction (PCR) was performed as follows in the presence of 2 mm MgCl 2 : 35 cycles of denaturation at 94 C for 30 s, annealing at 48 C for 30 s and extension at 72 C for 30 s. 16SaR-L and 16Sd- H primers (Reeder 1995) were used to amplify a fragment of 535 bp from the 16S rrna region of the mtdna, according to the following PCR profile and in the presence of 1.5 mm MgCl 2 : 30 cycles of denaturation at 94 C for 30 s, annealing at 50 C for 45 s and extension at 72 C for 30 s. The light strands were sequenced for all 85 individuals using an ABI 373 sequencer (University of Liverpool sequencing service). Complementary sequences were obtained for 56 and 19 individuals for 16S and 12S rrna, respectively.

3 POPULATION HISTORY OF THE TENERIFE SKINK 1063 Sequence analysis and haplotype relationships The 16S and 12S rrna sequences were aligned separately using clustal w (Thompson et al. 1994). Secondary structures were inferred after alignment with models for Sceloporus undulatus (De Rijk et al. 1999; Van de Peer et al. 1999). Haplotypes were clustered using neighbour joining (NJ) on Kimura 2-parameter distances with a transition : transversion (TS : TV) weighting of 1 : 10 (see Brown & Pestano 1998) using phylip (Felsenstein 1993). Empirical TV : TS ratios were generally lower than this owing to low levels of sequence divergence, so the weighting was based on data from a cross-species study of Chalcides (Brown & Pestano 1998). C. viridanus from La Gomera and El Hierro were used as outgroups (see Brown & Pestano 1998). Maximum parsimony analyses (MP) were carried out using paup (version 4b2a) (Swofford 1998). Bootstrap majority-rule consensus trees (500 replications), based on heuristic searches, were computed for these data sets ( branches collapsed to give polytomies where support < 50%). Among-site nucleotide diversity N ST statistics and a between-site mtdna dissimilarity matrix based on the number of nucleotide substitutions between randomly selected pairs of individuals from different sites were computed using the program haplo2 (Lynch & Crease 1990). Only one sample site showed zero nucleotide diversity, so significance of geographical structuring was tested using the program nucleodi (Holsinger & Mason-Gamer 1996). This calculates a hierarchical site structure based on between-site nucleotide diversity, with the (null) hypothesis of no differentiation between daughter nodes tested at each node on the tree. Negative or zero branches were collapsed on the tree. Two different methods were used to test for neutrality of mutations, representing alternative rationales. The test of Tajima (1989) is based on differences between estimates of the product of the mutation rate and the effective population size obtained from: (i) the number of segregating sites; and (ii) the mean number of nucleotide differences between sequences. The test of Fu & Li (1993) is based on comparison of mutations in new external (leading to tips) and old internal (connecting internal nodes) branches of a genealogy. Both of these tests were applied to the data (where haplotype diversity was sufficient). Changes in population size were estimated using a method based on a Metropolis-Hastings genealogy sampler (Kuhner et al. 1998; program: fluctuate). This allowed estimation of Θ = 2N f µ, where N f is the female population size and µ represents the mutation rate per site, and also the population growth rate (denoted as g). Again, this method was only applied when there were more than two haplotypes in the clade to be tested. The significance of the association between among-site mtdna diversities in C. viridanus and Galliotia galloti was computed using matrix correlation with significance determined by randomization (Mantel 1967). A haplotype dissimilarity matrix was calculated for the G. galloti sequences presented by Thorpe et al. (1996). Within-site nucleotide diversity was assumed to be zero for this latter species. Mantel s test was also used to compare between-site nucleotide diversity with between-site morphological differences in C. viridanus colour pattern (four characters; see Brown et al. 1991), scalation (six characters) and body dimensions (nine characters; see Brown et al. 1993). The morphology matrices were either Mahalanobis D 2 distances between site centroids (scalation or body dimensions), or Euclidean distances calculated from site means (colour pattern). Results Sequence divergence The GenBank accession numbers for sequences presented in this paper are AF AF The 12S fragment provided 359 bp of unambiguous sequence containing 18 variable sites, eight of which were parsimony informative within Tenerife alone. All but one of the variable sites corresponded to a loop region of the rrna (although note that the exception coincided with an area of ambiguous alignment). Maximum sequence divergence was 8 bp. The 16S fragment provided 466 bp of homologous unambiguous sequence, containing 20 variable sites (13 of which were parsimony informative). Fourteen variable sites did not appear to be involved in secondary-structure bp interactions. Maximum pairwise divergence was 12 bp. The combined fragments yielded 33 haplotypes. The MP and NJ trees provided similar topologies that supported three ancient cladogenetic events. Two early events first gave rise to a lineage currently confined to sites 8 and 17 in the northwest, and then to a lineage confined to site 10 in the northeast (Fig. 2). These accounted for only three and two of the 33 haplotypes, respectively. A subsequent event led to a relatively recent clade comprising all remaining haplotypes ( central haplotypes). These were geographically widespread, being found everywhere except at sites 10 and 17. The majority of the total nucleotide variation was between populations: N ST = There was considerable differentiation between sites (Fig. 3). The phylogeographical patterns were discordant with the three morphological systems tested in the same species (Table 1). The pattern was also discordant with the phylogeography of Galliotia galloti from the same island (between-matrix correlation, r = 0.031, P = [2000 randomizations]).

4 1064 R. P. BROWN, R. CAMPOS-DELGADO and J. PESTANO Fig. 2 Maximum parsimony trees displaying relationships, between haplotypes and phylogeography. Values on internal nodes are bootstrap values (branches collapsed to give polytomies where bootstrap support < 50%). A neighbour joining tree also provided support for the central (C), northwestern (NW) and northeastern (NE) clades shown here. Table 1 Mantel tests of association between mitochondrial DNA (mtdna) and morphology matrices (males [M] and females [F], separated where appropriate) Matrix Association Scalation (M) r = 0.100, P = Scalation (F) r = 0.353, P = Body dimensions (M) r = 0.075, P = Body dimensions (F) r = 0.105, P = Colour pattern r = 0.017, P = When applied to all Chalcides viridanus Tenerife haplotypes, neither Tajima s (1989) (D = 1.418, P > 0.10) nor Fu & Li s (1993) (D* = 1.148, P > 0.10, F* = 1.464, P > 0.10) tests of neutrality were significant. These results changed slightly when the central haplotypes alone were analysed (Tajima s D = 1.875, P < 0.05; Fu & Li s D* = 1.950, P > 0.10; Fu & Li s F* = 2.262, P < 0.10), suggesting possible deviations from expectations under neutrality. Estimations based on central haplotypes, using Metropolis-Hastings simulations, suggested a large value of 2N f µ, i.e. Θ = and rapid population growth: g = (log-likelihood ), respectively. Discussion Is there an association between the geological history of Tenerife and the genesis of the northeast, northwest and central lineages? Potassium-Argon (K-Ar) dating of rocks in Tenerife indicates three ancient areas of the island: Teno in the northwest, Anaga in the northeast and Roque del Conde in the southwest. The ages of these areas are estimated at 7.4 million years (Myr), 6.5 Myr and 11.6 Myr, respectively (Ancochea et al. 1990). Volcanic eruptions in the central Cañadas edifice united these ancient islands (although Teno and Roque del Conde may represent parts of the same island). These central eruptive

5 POPULATION HISTORY OF THE TENERIFE SKINK 1065 Fig. 3 Hierarchical relationships among sites (Holsinger & Mason-Gamer 1996). Polytomies arise where branch lengths 0 have been collapsed. episodes occurred principally in three cycles between 3.5 and 2.7 Ma, Ma and Ma, each giving rise to a new Cañadas edifice (Ancochea et al. 1999). The ancient islands were probably united at the beginning of the first cycle. Broad estimates of the timing of cladogenesis are useful in assessing whether this fits in with the pattern of divergence in Chalcides viridanus. Independent of their reliability, there is considerable heuristic value in applying molecular clocks when distinguishing between putative causal events that were well separated in time (Zamudio & Greene 1997). A ball-park figure of 1.5% pairwise sequence divergence can be estimated based on the estimated date of colonization of El Hierro from La Gomera (see Brown & Pestano 1998). This estimate is in agreement with published sequence divergence rates (e.g. Mindell & Thacker 1996). If this is correct then the central and northeastern lineages diverged 0.9 Ma while the divergence of the northwestern lineage occurred 1.1 Ma, i.e. long before the ancient islands were joined. Whether they involved vicariance or colonization events is unknown. Colonization probably took place 4 Ma (Brown & Pestano 1998) (close to the time when the precursor islands were joined) so a considerable time period subsequently appears to have elapsed before the described within-island cladogenesis. Molecular clocks are problematical (Hillis et al. 1996) and so spatial correlations provide stronger evidence of a relationship between cladogenesis and geology. The northeastern lineage is located within the known extension of the ancient island of Anaga, while the northwestern lineage is located within Teno. Application of the hypergeometric distribution provides an insight into the probability of this pattern arising by chance. For our sampling regime, if one lineage is confined to two of the 17 sample sites (as for the northwestern lineage), and another to one of 17 sites (as for the northeastern lineage), then the probability that the former will comprise two of the four Teno sites and the latter one of the five Anaga sites is P = The considerable postcolonization range expansion by the central clade contrasts sharply with the spatially limited northeastern and northwestern clades. Star-like relationships within the former, and a mean pairwise divergence below 0.5%, suggest that this expansion may have been recent following a bottleneck around years ago, i.e. towards the end of the last major eruptive cycle. A recent selective sweep or bottleneck would be expected to largely remove old mutations, leaving only new, lowfrequency mutations. Tajima s neutrality test (Tajima 1989) supports this hypothesis. Three lineages of the gecko Tarentola delalandii on the same island also show evidence of population growth following a relatively recent bottleneck, suggesting a common effect caused by a geological event during the last eruptive cycle (T. Gübitz, personal communication). The Tenerife lacertid lizard Gallotia galloti shows a discordant pattern of phylogeographical variation, but it is still interpreted in terms of the ancient islands of Anaga and Teno that form part of Tenerife (Thorpe et al. 1996). Unlike C. viridanus, the ancient G. galloti lineages are widespread, indicating considerable dispersal out of Anaga and Teno. Levels of sequence divergence are comparable between G. galloti and C. sexlineatus, raising the possibility of a common causal event. Phylogeographical patterns in Pimelia beetles also show similarities with C. viridanus (Juan et al. 1996). Why are phylogeographical patterns discordant with geographical variation in generalized morphology? More detailed analysis reveals that generalized patterns may mask more subtle concordance. For example, site 10 individuals had northeastern haplotypes and (unlike other individuals) possessed green tail pigment. However, they showed little differentiation in other characteristics. Differential introgression between nuclear and mtdna provides an alternative explanation of this disparity, as does local selection pressures for different morphologies, overriding the historical effects that have shaped the mtdna variation (e.g. Juan et al. 1996; Thorpe et al. 1996). The differential selection pressures component of this argument

6 1066 R. P. BROWN, R. CAMPOS-DELGADO and J. PESTANO was also previously put forward as an explanation of variation in characters such as blue-tail coloration (a trait of considerable functional significance in lizards ([Arnold 1984; Cooper & Vitt 1986]), which is found at high frequency in Chalcides in the arid southern regions of Tenerife and also predominates in Chalcides from the arid south of Gran Canaria (Brown et al. 1991). Acknowledgements This work was funded by a Royal Society research grant, a Canary Islands Government fellowship to J. P. and by Liverpool John Moores University. We are grateful to Angie Rosin (University of Liverpool) for her assistance in sequencing, Marcos Báez for help obtaining fieldwork permits and Thomas Gübitz for suggestions, discussion and tissue samples. References Ancochea E, Fuster JM, Ibarrola E et al. (1990) Volcanic evolution of the island of Tenerife (Canary Islands) in the light of new K-Ar data. Journal of Volcanology and Geothermal Research, 44, Ancochea E, Huertas MJ, Cantagrel JM et al. (1999) Evolution of the Canadas edifice and its implications for the origin of the Canadas caldera (Tenerife, Canary Islands). Journal of Volcanology and Geothermal Research, 88, Arnold EN (1984) Evolutionary aspects of tail shedding in lizards and their relatives. Journal of Natural History, 18, Avise JC (1992) Molecular population structure and the biogeographic history of a regional fauna: a case history with lessons for conservation biology. Oikos, 63, Báez M, Brown RP (1997) Testing multivariate patterns of within-island differentiation in Podarcis dugesii from Madeira. Journal of Evolutionary Biology, 10, Brown RP, Pestano J (1998) Phylogeography of Canary Island skinks inferred from mtdna sequences. Molecular Ecology, 7, Brown RP, Thorpe RS, Báez M (1991) Parallel within-island evolution in lizards on neighbouring islands. Nature (London), 352, Brown RP, Thorpe RS, Báez M (1993) Patterns and causes of morphological population differentiation in the Tenerife skink, Chalcides viridanus. Biological Journal of the Linnean Society, 50, Carson HL, Lockwood JP, Craddock EM (1990) Extinction and recolonization of local-populations on a grow shield-volcano. Proceedings of the National Academy of Sciences of the USA, 87, Cooper WE Jr, Vitt LJ (1986) Blue tails and autotomy: enhancement of predator avoidance in juvenile skinks. Zeitscrift für Tierpsychology, 70, De Rijk P, Robbrecht E, de Hoog S et al. (1999) Database on the structure of large subunit ribosomal RNA. Nucleic Acids Research, 27, Douglas ME, Minckley WL, DeMarais BD (1999) Did vicariance mold phenotypes of western North American fishes? Evidence from Gila River Cyprinids. Evolution, 53, Felsenstein J (1993) PHYLIP (phylogeny inference package), Version 3.5c. Distributed by the author. University of Washington, Seattle. Fu YX, Li WH (1993) Statistical tests of neutrality of mutations. Genetics, 133, Gross-Bellard M, Oudet P, Chambon P (1973) Isolation of high-molecular-weight DNA from mammalian cells. European Journal of Biochemistry, 36, Hewitt GM (1996) Some genetic consequences of ice ages, and their role in divergence and speciation. Biological Journal of the Linnean Society, 58, Hillis DM, Mable BK, Moritz C (1996) Applications of molecular systematics: the state of the field and a look to the future. In: Molecular Systematics (eds Hillis DM, Moritz C, Mable BK), pp Sinauer Associates, Sunderland, MA. Holsinger KE, Mason-Gamer RJ (1996) Hierarchical analysis of nucleotide diversity in geographically structured populations. Genetics, 142, Juan C, Ibrahim KM, Oromi P, Hewitt GM (1996) Mitochondrial DNA sequence variation and phylogeography of Pimelia darkling beetles on the island of Tenerife. Heredity, 77, Kocher TD, Thomas WK, Meyer A et al. (1989) Dynamics of mitochondrial DNA evolution in animals: amplification and sequencing of conserved regions. Proceedings of the National Academy of Sciences of the USA, 86, Kuhner MK, Yamato J, Felsenstein J (1998) Maximum likelihood estimation of population growth rates based on the coalescent. Genetics, 149, Lynch M, Crease TJ (1990) The analysis of population survey data on DNA sequence variation. Molecular Biology and Evolution, 7, Malhotra A, Thorpe RS (1991) Microgeographic variation in Anolis oculatus, on the island of Dominica, West Indies. Journal of Evolutionary Biology, 4, Mantel N (1967) The detection of disease clustering and a generalized regression approach. Cancer Research, 27, Mindell DP, Thacker CE (1996) Rates of molecular evolution: Phylogenetic issues and applications. Annual Review of Ecological Systematics, 27, Pestano J, Brown RP (1999) Geographical structuring of mtdna in Chalcides sexlineatus within the island of Gran Canaria. Proceedings of the Royal Society of London, Series B, 266, Reeder TW (1995) Phylogenetic relationships among phrynosomatid lizards as inferred from mitochondrial ribosomal DNA sequences: substitutional bias and information content of transitions relative to transversions. Molecular Phylogenetics and Evolution, 4, Schneider CJ (1996) Distinguishing between primary and secondary integradation among morphologically differentiated populations of Anolis marmoratus. Molecular Ecology, 5, Swofford DL (1998) PAUP*: Phylogenetic Analysis Using Parsimony (*and Other Methods), Version 4. Sinauer Associates, Sunderland, MA. Taberlet P (1998) Biodiversity at the intraspecific level: the comparative phylogeographic approach. Journal of Biotechnology, 64, Tajima F (1989) Statistical method for testing the neutral mutation hypothesis by DNA polymorphism. Genetics, 123, Thompson JD, Higgins DG, Gibson TJ (1994) clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research, 22, Thorpe RS, Báez M (1987) Geographic variation within an island:

7 POPULATION HISTORY OF THE TENERIFE SKINK 1067 univariate and multivariate contouring of scalation, size, and shape of the lizard Gallotia galloti. Evolution, 41, Thorpe RS, Malhotra A (1996) Molecular and mophological evolution within small islands. Philosphical Transactions of the Royal Society of London, Series B, 351, Thorpe RS, Black H, Malhotra A (1996) Matrix correspondence tests on the DNA phylogeny of the Tenerife Lacertid elucidate both historical causes and morphological adaptation. Systematic Biology, 45, Van de Peer Y, Robbrecht E, de Hoog S et al. (1999) Database on the structure of small subunit ribosomal RNA. Nucleic Acids Research, 27, Zamudio KR, Greene HW (1997) Phylogeography of the bushmaster (Lachesis muta: Viperidae): implications for neotropical biogeography, systematics, and conservation. Biological Journal of the Linnean Society, 62, R. P. Brown lectures in statistics, evolution and ecology and carries out research into the evolution and ecology of island reptiles. J. Pestano and R. Campos-Delgado are both forensic geneticists with research interests in population genetics of human and animal populations.