0 Birkhfiuser Verlag, Base], 1998 J. evol. biol. I I (1998) 481 494 1010-061X/98/040481-14 $ 1.50 +0.20/O 1 Journal of Evolutionary Biology

Evolution and biogeography of the genus (Sauria: Gekkonidae) in the , inferred from mitochondrial DNA sequences

M. Nogales,’ M. Lopez,’ J. Jimenez-Asensio,2 J. M. Larruga,’ M. Hernandez’,* and P. Gonzalez2

1Department of Zoology, University oJ’ La Lugunu, E-38271 Tenerijk, Cunury Islunds, Spain 2Department of’ Genetics, University of La Laguna, E-38271 Tenerijk, &nary Islands, Spain, e-mail address: [email protected]

Key brords: mtDNA; Turentolu; Gekkonidae, phylogeny; biogeography; Canary Islands.

Abstract

Sequences from fragments of the 12s ribosomal RNA and cytochrome b mito- chondrial genes were used to analyze phylogenetic relationships among of genus Turentolu from the Canary Islands. A surprisingly high level of within island differentiation was found in T. delulundii in Tenerife and T. boettgeri in Gran Canaria. Molecular differentiation between populations of T. ungustimentulis on Lanzarote and Fuerteventura, and between Moroccan and Iberian Peninsula T. muuritunicu, also indicate that at least two subspecies should be recognized within each of them. Phylogenetic relationships among these reveals a higher level of differentiation and a more complex colonization pattern than those found for the endemic genus Gullotiu. Lack of evidence for the presence of T. boettgeri bischoffi on the island of Madeira does not seem to support the origin of T. delulundii, T. gomerensis and the canarian subspecies of T, boettgeri from this island, whereas molecular data confirms that T. ungustimentulis is a sister species of the continental T. muuritunicu. Several independent colonization events from the continent and the extinction of some species are probably responsible for the current distribution of Turentolu in the Canary Islands.

* Author for correspondence

481 482 Nogales et al.

Introduction

The Canary Islands are situated about 100 km at the closest point to the north-western coast of Africa. These volcanic islands have geological ages rang- ing from one to 35 million years (Carracedo, 1984) and show remarkable eco- logical differences between and within-islands (Bramwell and Bramwell, 1990). These geological and ecological characteristics, together with the presence of a high level of endemism, make the Canaries very attractive for the study of speciation and genetic differentiation. constitute the major group of endemic terrestrial vertebrates in the archipelago. This group provides the oppor- tunity to analyze different levels of genetic differentiation as well as the possibil- ity of comparing the patterns of evolution among the different families present in the islands (see review of Machado et al., 1985). All native reptiles from the Canaries belong to the Order Sauria and are included in three families: F. Lacertidae, F. Gekkonidae and F. Scincidae. Geckos are represented in the Canary Islands by two genera one of them native: Tarentola (Bischoff, 1985) and the other introduced by man: Hemidactylus, represented only by the species H. turcicus Linnt, 1758 and restricted to small populations on the islands of Tenerife and Gran Canaria (Baez, 1979). Classical based on morphological differentiation permits us to dis- tinguish four endemic species of genus Tarentolu in the Canaries (Joger, 1984a and 1984b): T. angustimentalis Steindachner, 1891 from Lanzarote, Fuerteventura and its surrounding islets (Steindachner, 1891), T. boettgeri from Gran Canaria (T. 6. boettgeri Steindachner, 1891) and El Hierro (T. b. hierrensis Joger and Bischoff, 1983), T. delalundii (Dumeril and Bibron, 1836) from Tenerife and La Palma, and T. gomerensis (Joger and Bischoff, 1983) from La Gomera Island. These species show a great deal of inter and intra-population morphological variation (Thorpe, 1991). Previous studies on the phylogenetic relationships of this genera based on classical morphological methods, scanning electron microscopy of scale surfaces, serum protein electrophoresis, and quantitative precipitin tests of serum albumin suggested that colonization of the Canaries occurred independently from Madeira (for the islands of El Hierro, La Palma, La Gomera, Tenerife and Gran Canaria) and from Africa (for the islands of Lanzarote and Fuerteventura) (Joger, 1984~). This contrasts with the colonization pattern observed in the Fam- ily Lacertidae, whose relative affinities among species follow an eastern-western geographic cline with a clear monophyletic African origin (Gonzalez et al., 1996). With the aim of gaining more insight into the process of genetic differentiation and in the colonization pattern of genus Turentola in the Canary Islands, we have sequenced two fragments of mitochondrial DNA (a 305 bp region of the cytochrome b gene and an approximately 390 bp region of the 12s rRNA gene) for populations from islands of the Canary, Selvages and Madeira archipelagos, and continental populations from Morocco and the Iberian Peninsula. Evolution Tarrnrolu Canary Islands 483 Materials and methods

Specimens unulyzrd

Figure 1 shows the geographic origin of the specimens examined in this study. This includes representatives from the four species of genus Turentolu present in the Canary Islands, as well as two individuals from Selvages Islands, and six individuals of T. mauritunicu from the Iberian Peninsula, Madeira and Morocco. One specimen of H. turcicus was included as an outgroup genus. Most samples were obtained from live captured in the field. A piece of the tail of about 0.5 g in weight was clipped off and kept in absolute ethanol until being processed for DNA extraction. Specimens were released alive in the same capture place. Some samples were obtained from collection material (Zoology Department of La Laguna Uni- versity) kept in 70% ethanol and processed in the same way as the living specimens. Taxonomic affiliation, source and locality of all specimens included in this study are summarized in Table 1.

DNA extraction, umplification and sequencing

DNA was extracted from a slice of tail using the procedure described by Walsh et al. (1991). A portion of the cytb gene was amplified using the primers L14841 and H15149 (Kocher et al., 1989). For samples whose amplification failed, primer Ll4841 was replaced by primer GLUDG-L (Palumbi, 1991). A second fragment of mtDNA corresponding to the 12s rRNA gene was amplified with primers L1091 and H1478 (Kocher et al., 1989). Amplification reactions were carried out in most cases at 95 “C 1 min, 50 “C 1 min and 72 “C 1 min for 35 cycles. PCR products were precipitated with 0.6 volumes of polyethylene glycol/NaCl (20%wt/vol PEG 6000, 2.5 M NaCl) and dissolved in water for direct cycle sequencing. Both complementary strands were sequenced with the same primers as for PCR amplifications using the jino/ DNA sequencing system from Promega. Cycle sequencing conditions were: 1 min at 94 “C, 1 min at 58 “C and 1 min at 72 “C for 35 cycles.

Sequrnce unulysis

Nucleotide sequences were aligned by eye using the program SeqEd from Applied Biosystems. As is common for multiple sequences of rRNA genes, the alignment has several indels (insertions and deletions). Sites including indels were conserva- tively omitted for the phylogenetic analysis. A combined analysis of 12s rRNA and cytb sequences was performed. UPGMA (Sneath and Sokal, 1973; Nei et al., 1985) and Neighbor Joining (Saitou and Nei, 1987; Felsenstein, 1985) phylogenies were constructed using Kimura (1980) genetic distances with the MEGA 1.01 program (Kumar et al., 1993). Parsimony analysis was performed using PAUP 3.1.1 (Swof- ford, 1993) with random stepwise addition of sequences and TBR branch swapping. H. turcicus was used as outgroup to root the trees. Ten separate searches were 484 Nogales et al. performed, each terminating after the accumulation of 1000 equally parsimonious trees. One of the most parsimonious trees was arbitrarily chosen for representation.

Results

After several attempts under different PCR conditions, none of the specimens from T. mauritanica and T. nngustimentalis amplified with primers L14841 and H 15 149 (Kocher et al., 1989). When primer L14841 was replaced by GLUDG-L (Palumbi, 1991) amplification products were obtained for these samples with the exception of individuals from Lobos and Graciosa. Individuals Tenerife-2, 4, 5, 6, and 7 did not amplify with either set of primers for the cytb fragment. Sequences of the cytb fragment provided a matrix with 305 nucleotides out of which 158 were variable among 28 different sequences. 41% of substitutions

Fig. 1. Distribution of the specimens (genus Turenrola) analyzed in the present study on the Canary Islands. Numbers of the localities correspond with numbers in Table I. Evolution Tarentola Canary Islands 485

Table I. Samples used in this study.

Taxon Abbreviation“ Sourceb Locality’

Tarmtola clelalandii Tenerife-I w El Palmar (Tenerife) [I] Tenerife-2* w Arafo (Tenerife) [2] Tenerife-3 w El Sauzal (Tenerife) [3] Tenerife-4” w Tejina (Tenerife) [4] Tenerife-5* c lcod de 10s Vinos (Tenerife) [5] Tenerife-6’ c Roque de Fuera de Anaga (Tenerife) [6] Tenerife-7* C Roque de Garachico (Tenerife) [7] Tenerife-8 w Punta de la Rasca (Tenerife) [8] La Palma-l w San And& (La Palma) [9] La Palma-? w San And& (La Palma) [IO] La Palma- C Los Sauces (La Palma) [ll] La Gomera- 1 w Valle de Gran Rey (La Gomera) [I21 La Comera- w San Sebastian (La Gomera) [I31 La Gomera-3 w San Sebastian (La Gomerd) [I41 T. boettgeri hoettpri Gran Canaria- I w Mogan (Gran Canaria) [IS] Gran Canaria- w Arucas (Gran Canaria) [l6] T. h. hierrrnsis El Hierro-1 C Roque Grande de Salmor (El Hierro) [17] El Hierro-2 w Tamaduste (El Hierro) [I81 T. h. bkhoffi Selvages- 1 w Selvagem Grande (Selvages Islands) [ 191 Selvages-2 w Selvagem Grande (Selvages Islands) [20] T. angustimmtnlis Fuerteventura- I w Barranco Esquinzo (Fuerteventura) [2l] Fuerteventurd-2 w Gran Tarajal (Fuerteventura) [22] Fuerteventura-3 w Gran Tarajal (Fuerteventura) [23] Lobos-I * w Lobos [24] Lobos-2* w Lobos [25] Lobos-3* w Lobos [26] Lanzarote- 1 w Playa Quemada (Lanzarote)[27] Lanzarote-2 w Playa Quemada (Lanzarotc)[28] Lanzarote-3 C Gudtizd (Ldnzarote) [29] Algranza- 1 w Alegranza [30] Graciosa- I * w Graciosa [3l] Graciosa-2* w Graciosa [32] M. Clara-l w Montana Clara [33] M. Clara-2 w Montana Clara [34] R. del Este-I w Roque del Este [35] R. de1 Este-2 w Roque del Este [36] T. maur’itanica Madeira- I w Funchal (Madeira) [37] Madeira-2 w Ponta do Gardjao (Madeira) [38] Sevilla w Sevilla (Iberian Peninsula) [39] Valencia w Valencia (Iberian Peninsula) [40] Morocco- I w Marrakech (Morocco) [ill] Morocco-2 w Essaouira (Morocco) [42] H. turcicus w Lorca (I berian Peninsula) [43]

I’ Samples with an asterisk were analyzed only for 12s rRNA. h w, wild: c, collection. ’ Numbers in brackets correspond with numbers in Figure I. 486 Nogales et al.

Fig. 2. Cyth nucleotide differences among the 33 specimens studied. Site I corresponds with position 14 842 in the published human reference sequence (Anderson et al., 1981). The following individuals had identical sequences and only one of each group was represented: Tenerife-3 = Tenerife-8; Selvages-I = Selvages-2; Alegranza-I = M. Clara-l = M. Clara-2 = R. del Este-2. Evolution Trrren/oh Canary Islands 487 occurred in third base codon (Fig. 2). The 12s rRNA fragment amplified ranged in size from 376 to 390 nucleotides. A number of gaps had to be introduced to maximize the similarity among sequences. After removal of sites with indels the matrix included 373 sites, 143 of them informative. Variable sites (including indels) are represented in Figure 3. The combined matrix with cyth and 12s rRNA sequences generated for phyloge- netic analysis included 678 sites, 301 of which were informative. The average transition/transversion ratio was 1.56 ranging from 9.10 for some within subspecies comparisons to 1.05 between Hemiductylus and Turentolu. The most parsimonious trees obtained were 646 steps(C. I. = 0.633) (Fig. 4). The topology of UPGMA and Neighbor Joining trees (not shown) was essentially identical to that of the PAUP trees. Important levels of molecular differentiation were found between the two individ- uals of T. h. boettgeri from north and south of Gran Canaria (Dxy = 0.110 for cytb; Dxy = 0.064 for 12s rRNA) and between north and south populations of T. deldundii in Tenerife (Dxy = 0.129 k 0.000 for cytb; Dxy = 0.015 k 0.002 for 12s rRNA). T. boettgeri shows the expected intraspecific differentiation in three subspe- cies, with T. b. hierrensis being closer to T. b. bischoj,, than to T. b. boettgeri (Fig. 4). Interestingly, T. muuritanicu and T. angustimentalis also show significant in- traspecific differentiation between individuals from the Iberian Peninsula and Morocco (Dxy = 0.154 f 0.006 for cyth; Dxy = 0.040 f 0.002 for 12s rRNA) in the first case and between Fuerteventura and Lanzarote, plus their surrounding i lets (Dxy = 0.083 f 0.003 for cytb; Dxy = 0.045 + 0.003 for 12s rRNA), in the seco Genetic distances between species are relatively high (Tab. 2) and permit us% t recognize the close relationship between T. angustimentulis and T. mauritanica.‘ (Dxy = 0.173 f 0.030 for cytb; Dxy = 0.048 k 0.007 for 12s rRNA), and to estab- lish that T. gomerensis and T. delulandii (Dxy = 0.249 k 0.028 for cytb; Dxy = 0.059 f 0.005 for 12s rRNA) are more closely related to one another than either is to T. boettgeri (Dxy = 0.220 f 0.036 for cytb; Dxy = 0.132 k 0.017 for 12s rRNA).

Discussion

Intraspecijic dfjtirentiution

Although the morphological variation for Tarentob specieswithin the Island of Tenerife (Thorpe, 1991) and Gran Canaria (Nogales, pers. obs.) already pointed towards some degree of isolation between the northern and southern populations, the average molecular distances between individuals from the north and south of Gran Canaria in T. boettgeri and between the north and south of Tenerife in T. de/ulundii are surprisingly high (Tab. 2). These distances are also more than one order of magnitude higher than those found between the northern and southern populations of lacertids of the genus Gullotia in Tenerife (Dxy = 0.013 for cytb; Dxy = 0.003 for 12s rRNA) (GonzBlez et al., 1996) which already showed an significant level of morphological differentiation (Bischoff, 1982; Thorpe and BBez, 488 No&ales et al

Fig. 3. 12s rRNA nucleotide differences among the 43 specimens studied. Site 8 corresponds to position 1099 in the published human reference sequence (Anderson et al., 1981). The following individuals had identical sequences and only one of each group was represented: La Palma- = La Palma-3; Selvages- I = Selvages-2; Fuerteventura-2 = Fuerteventura-3; Lobos ~ I = Lobos-2 = Lobos-3; Lanzarote-2 = Lanzarote-3; Graciosa-I = M. Clara-l = M. Clara-2 = R. del Este-1 = R. del Este-2; Madeira-l = Madeira-2; Morocco-l = Morocco-2. Evolution Turenroh Canary Islands 489

Fig. 4. Phylogeny of the 43 DNA sequences analyzed. Values in branches are bootstrap indices of support (percentage of 100 replications). Asterisks indicate specimens analyzed for the 12s rRNA fragment only. 490 Nogales et al.

Table 2. Mean distances within and between subspecies and bctwccn species and genera compared with genus Gallotia.

Cyth 12s rRNA

N Dxy k SE N Dxy + SE

Within subspecies Tuwntola ( I ) 91 0.055 * 0.05 I 196 0.022 + 0.0 I7 Taren toh (2 ) 50 0.018 + 0.022 109 0.010 * 0.008 Gall0 tia ( 3) 40 0.006 i 0.004 58 0.002 * 0.003 Between subspecies Ttrrentoh (1) 12 0.108 * 0.053 12 0.073 i 0.025 Tarentola (2) 71 0.106 & 0.038 98 0.043 f 0.018 Gullotiu (3) 60 0.055 + 0.018 73 0.023 f 0.015 Between species Tarenioltr 393 0.236 i 0.042 346 0. I I3 * 0.033 GaIlotIa (3) 252 0. I57 f 0.024 305 0.079 * 0.0 18 Between genera Tarm toh 32 0.312kO.027 42 0.253 f 0.013 Gall0 t ia ( 3 ) 83 0.244 k 0.025 92 0.193 f 0.010

(I) Standard taxonomy. (2) Two subspecies in T. cmgu.stinwntcrli.s, T. mcruritanrc~cr, T. d~~luhtrdii and T. h. hoettgrri. (3) From Gonziilez et al. (1996).

1987; Thorpe and Brown, 1989). It can be claimed that this molecular divergence resulted from geographical or ecological isolation. In the case of Tenerife geograph- ical isolation could result from the geological origin of the Island by the union, about one million years ago of three smaller islands 6 to 11.6 million years old (Ancochea et al., 1990). However, in Gran Canaria the level of differentiation is not lower than in Tenerife and this Island is believed to have been a single geological unit since its origin 9- 16 million years ago (Abdel-Monem et al., 1971). A different line of explanation for the strong molecular differentiation in Tenerife and Cran Canaria comes from the marked ecological differences between the northern and southern regions of the islands (Bramwell and Bramwell, 1990; Thorpe, 1991). It remains to be explained, however, why differentiation within these islands is so much higher in geckos than in the genus Gulfotiu. A possible explanation could be an earlier colonization of the emerging islands by geckos which could require less restrictive conditions for their establishment. Vagility differences between Lacer- tidae and Gekkonidae seemsto be also a plausible explanation. Populations of T. angustimrntalis from Fuerteventura and Lobos should be considered a different subspeciesfrom those of Lanzarote and its surrounding islets on the basis of the noticeable level of molecular differentiation between them and the remarkable lack of molecular variation within both groups (Fig. 4). The ecological similarity between the north and south of these islands and the absence of geographical barriers may be responsible for this homogeneity at molecular level, in contrast to the differentiation found in Gran Canaria and Tenerife. Evolution Tarcwtoh Canary Islands 491 The molecular differences between the populations of T. mauritanica from Morocco and the Iberian Peninsula also indicate an important degree of isolation between these two populations (Fig. 4) which should be considered as different subspecies.

Phylogenetic rrlutionships and biogeography

Phylogenetic relationships found in this study (Fig. 4) are basically concordant with the phylogeny proposed by Joger (1984~). The only topological difference between both phylogenies affects the subspecies of T. boettgeri. According to Joger (1984~) T. 6. hirrrensis is closer to T. b. boettgrri than to T. b. bischofj, while according to our own data T. b. hierrrnsis is more closely related to T. b. hischoffi. Since the island of El Hierro is much more recent than Gran Canaria and Selvages (Carracedo et al., 1984; Jeremie, 1950) the more likely explanation is that El Hierro was colonized from the Selvages Islands. According to Joger’s correction (1985) in relation to the presence of T. b. boettgeri in Madeira Island, we could not find any specimen of this subspecies either in museum material or in the wild, nor could we verify any indication that could suggest the actual or past presence of T. boettgeri in that island. Both specimens of Turentola captured in Madeira were unequivocally identified as T. mmuritunicu and the analysis of mtDNA sequences revealed a recent origin from the Iberian Peninsula consistent with the human introduction during the Portuguese colonization. The separation between T. gomerensis and T. delulundii can be explained as the result of the formation of the islands of La Gomera and Tenerife which is estimated to have happened about 14-19 million years ago (Cantagrel et al., 1984). It is more difficult, however, to find an earlier geological event that could be associated with the separation between T. boettgrri and the complex T. gomerensis-del&ndii, making difficult to postulate reasonable hypothesis to explain the actual distribu- tion of these species in the Canaries. Although the general radiation hypothesis of Joger (1984~) could be supported by our data with the simple modifications that the insular origin of radiation were Selvages instead of Madeira, it still remains how to explain the fact that T. delalundii and T. gomerensis in geologically younger islands are phylogenetically so divergent from T. boettgrri from the older islands of Selvages and Gran Canaria. One explanation could be that Gran Canaria underwent several substitutive colo- nizations from Selvages but La Gomera and Tenerife were colonized only once. Against this supposition is the fact that T. borttgeri from the south of Gran Canaria is more similar to T. h. bischof$ from Selvages than the nearest northern specimen (Fig. 2). An alternative hypothesis can be formulated with Gran Canaria as the insular radiation center. Under this supposition an ancestor of T. boettgeri arrived on Gran Canaria and radiated first to the geological younger islands of Tenerife and La Gomera giving rise to T. d&lundii and T. gomerensis and more recently to the Selvages Islands. Arguing against this alternative is the low probabil- 492 Nogales et al. ity of South-North migrations against the dominant winds and marine cur- rents. In fact, the Selvages Islands have not been colonized by Gullotiu from the Canaries but by Podurcis from Madeira. It is particularly interesting, however, to observe that the recent colonization of El Hierro did not occur from the closest Island of La Gomera, but from the more distant Selvages indicating that colonization pathways may be determined by other kinds of factors different from geographical proximity and the direction of the marine currents. According to the molecular data, separation between T. angustimentalis and T. mnuvitanicu occurred after the divergence of T. boettgeri, T. delulundii and T. gomerensis (Fig. 4). This indicates that the colonization of Fuerteventura and Lanzarote by the ancestors of T. ungustimentulis, resulted from a later colonization event different from the one responsible for the presence of the other three species in the Canaries. The lack of fossil records makes it impos- sible to determine if geckos different from the actual forms were once present in these islands. However, the ability to colonize new islands shown by this group makes it difficult to believe that Fuerteventura and Lanzarote were not colonized since their origin (about 30 million years ago) until the colonization by the ancestor of T. angustimentulis. Fuerteventura and Lanzarote are the closest islands to the African coast and the ones that at the present time show greater ecological similarities with the neighboring continental region. It is be- lieved that important ecological changes occurred in the history of these is- lands as well as in the northern African region (Bramwell, 1974; Santos, 1983). These changes could be responsible for the possible replacement of geckos bet- ter adapted to the old ecological conditions of the islands, by the ancestor of T. ungustimentulis, which may then have evolved under ecological conditions more similar to the present ones. The average sequence divergence among the species of Tuwntolu in the Ca- nary Islands is higher than that previously found in the lacertids of genus Gullotiu (Tab. 2). Assuming constancy in the rate of evolution of mtDNA in both groups, differentiation among the Turcntollz species started earlier than among species of Gullotiu, pointing to an earlier colonization of the Canaries by geckos as mentioned before. The actual distribution and molecular differentiation of geckos in the Canary Islands indicates that colonization did not follow a simple pattern like in the lacertids of genus Gullotiu (Gonzalez et al., 1996). The origin of one island’s population is not necessarily found in the island that, because of its geological age, ecological characteristics, geographical situation and the direction of the marine currents, initially would appear as the most obvious candidate. More complicated ecological and historical factors have influenced the evolutionary history of genus Turentolu in the Canary Islands leading to separate coloniza- tion events and probably replacements of some species for others from differ- ent origins. Evolution Turmtoh Canary Islands 493

Sequence availability

The nucleotide sequences in this paper are available from the GenBank/EMBL databases and are as follows: T. angustimentalis 293325 and 293326; T. boettgeri boettgeri 293327 and 293328; T. gomrrensis 293329 and 293330; H. turcicus 293332 and 293331; T. delalandii 293333 and 293334; and T. mauritanica 293335 and 293336 for 12s rRNA and cytb respectively.

Acknowledgements

We are grateful to A. Martin and J. M. Mateo for their helpful suggestions which improved the manuscript, to F. M. Medina, A. Brehm and P. Oliveira for collecting some of the specimens analyzed in this study, to Museu Municipal do Funchal for permitting us to study its material, to R. Hutterer for providing important information on the genus Turentola in Madeira, and to P. H. Carpio for technical assistance. This work has been supported by Grant 29/95 from the Gobierno de Canarias.

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Received 2 April 1997; revised I I June 1997; accepted 12 June 1997.