Evolution of Galapagos Island Lava Lizards (Iguania: Tropiduridae: Microlophus)

Home , Microlophus, Microlophus occipitalis, Tropidurus

MOLECULAR PHYLOGENETICS AND EVOLUTION Molecular Phylogenetics and Evolution 32 (2004) 761–769 www.elsevier.com/locate/ympev

David Kizirian,a,b,* Adrienne Trager,c Maureen A. Donnelly,b and John W. Wrightd

a Department of Organismic Biology, Ecology and Evolution, University of California–Los Angeles, Los Angeles, CA 90095-1606, USA b Department of Biological Sciences, Florida International University, Miami, FL 33199, USA c Moorpark College, 7075 Campus Road, Moorpark, CA 93021, USA d Natural History Museum of Los Angeles County, 900 Exposition Boulevard, Los Angeles, CA 90007, USA

Received 18 June 2003; revised 24 March 2004 Available online 2 June 2004

Abstract

Nucleotide sequences of mitochondrial genes (ND1, ND2, COI, and tRNAs) were determined for 38 samples representing 15 taxa of tropidurid lizards from the Galapagos Islands and mainland South America. Phylogenetically informative characters (759 of 1956) were analyzed under Bayesian, maximum likelihood, and parsimony frameworks. This study supports the hypothesis that tropidurid lizards dispersed to the Galapagos on at least two separate occasions. One dispersal event involved an eastern Galapagos clade (Microlophus habelii and M. bivittatus, on Marchena and San Cristobal islands, respectively) the sister taxon of which is M. occipitalis from coastal Ecuador and Peru; the closest mainland relative of the western Galapagos clade was not unambiguously identiﬁed. The wide-ranging M. albemarlensis is revealed to be a complex of weakly divergent lineages that is paraphyletic with respect to the insular species M. duncanensis, M. grayii, and M. paciﬁcus. Ó 2004 Elsevier Inc. All rights reserved.

Keywords: Biogeography; Galapagos; Microlophus; Phylogeography; Tropiduridae; Tropidurus

1. Introduction named and unnamed lineages but has not been widely accepted. Central to the disagreement about Lava Liz- Despite more than a century of intensive study there ard diversity is the status of the populations of M. al- are still many unanswered questions regarding the evo- bemarlensis, which occur on four major and at least six lutionary history of organisms inhabiting the Galapagos satellite islands. In addition to addressing species di- Islands (e.g., Grehan, 2001). A fundamental issue ad- versity we also address the biogeographic history of dressed herein is the number of species of the conspic- Lava Lizards. Previous biogeographic hypotheses in- uous and abundant Lava Lizards (Microlophus; auct. ferred from the electrophoretic migration rates of allo- Tropidurus) occurring on the islands. Based on an zymes (Wright, 1983, 1984) and micro-complement analysis of morphological variation, Van Denburgh and ﬁxation of albumins (Lopez et al., 1992) indicated that Slevin (1913) recognized seven species of Lava Lizards at least two independent dispersal events from the in the archipelago, an arrangement that has been fol- mainland to the archipelago were required to explain lowed by most (e.g., Lopez et al., 1992; Wright, 1983). Lava Lizards diversity. Herein, we revisit hypotheses An alternative arrangement (Lanza, 1974, 1980; Talurri about the diversity and biogeography of Galapagoan et al., 1982) based on geographic distribution and be- Microlophus in light of nucleotide sequence data ana- havioral data (Carpenter, 1966, 1970) recognized 17 lyzed in a phylogenetic context and broader taxonomic sampling than previous studies. At the same time, we evaluate hypotheses of monophyly for the M. occipitalis * Corresponding author. and M. peruvianus groups (Dixon and Wright, 1975) and E-mail address: [email protected] (D. Kizirian). the genus Microlophus (Frost et al., 2001; Harvey and

Gutberlet, 2000). Lastly, we employ the principles of NADH 1 and CO1, and the intervening tRNAs were phylogenetic classification (de Queiroz and Gauthier, amplified using the primers in Macey et al. (1997a,b) and 1990) to represent nested diversity at the species level. Jackman et al. (1999) as well as [50] taarataagtcattttggg [30], which was designed for use with some light strand primers from those studies. 2. Materials and methods Some fragments were amplified using a capillary thermal-cycler (Rapidcycler) and DNA amplification 2.1. Taxon sampling and data kits available from Idaho Technology (Salt Lake City, Utah). PCR cocktails comprised water (25 ll), 10 X Exemplars for this study include some of the tissue buffer (20, 30, or 40 mM MgCl2, 5.0 ll), dNTPs (2 mM samples used by Wright (1983) and Lopez et al. (1992) each; 5.0 ll), bovine serum albumin (2.5 mg/ml; 5.0 ll), as well as additional samples of mainland species not light and heavy strand primers (10 mM; 2.5 ll each), Taq previously examined (Table 1). Genomic DNA was ex- polymerase (ProMega; 0.3 ll), and template DNA (0.7– tracted from frozen tissues (1–25 mg) using the DNEasy 1.0 ll). Reactions were subjected to an initial heating Tissue Kits (Qiagen-Operon Catalog # 69504, 69506). (15 s, 94 °C), then 40–42 cycles of denaturation (15 s, Nucleotide sequence data were collected using poly- 94 °C), annealing (15 s, 53 °C), and extension (35 s, merase chain reaction (PCR) and automated DNA se- 71 °C), and then a final extension (60 s, 71 °C). quencing technology. Approximately 2 kb of the Most fragments were amplified using a block ther- mitochondrial genome, including NADH 2, portions of mal-cycler (Perkin–Elmer 9700) and AmpliTaq Gold

Table 1 Samples of Microlophus and Tropidurus used in this study Species Voucher # (Tissue #) Locality M. albemarlensis LACM 106273 (G302) Galapagos: Baltra M. albemarlensis LACM 106267 (G216) Galapagos: Bartolome M. albemarlensis LACM 106268 (G217) Galapagos: Bartolome M. albemarlensis LACM 106288 (G327) Galapagos: Daphne M. albemarlensis LACM 106282 (G321) Galapagos: Daphne M. albemarlensis LACM 106254 (G150B) Galapagos: Fernandina: Punta Espinosa M. albemarlensis LACM 106244 (G142) Galapagos: Fernandina: Punta Espinosa M. albemarlensis LACM 106193 (G170) Galapagos: Isabela: Black Cove M. albemarlensis LACM 106185 (G162) Galapagos: Isabela: Black Cove M. albemarlensis LACM 106205 (G280B) Galapagos: Isabela: Cartago Bay M. albemarlensis LACM 106217 (G341) Galapagos: Santiago: James Bay M. albemarlensis LACM 106207 (G232) Galapagos: Santiago: Sullivan Bay M. albemarlensis LACM 106206 (G231) Galapagos: Santiago: Sullivan Bay M. albemarlensis LACM 106181 (G290) Galapagos: Santa Cruz: Conway Bay M. albemarlensis LACM 106162 (G11) Galapagos: Santa Cruz: Academy Bay M. albemarlensis LACM 106168 (G17) Galapagos: Santa Cruz: Academy Bay M. bivitattus LACM 106298 (G43) Galapagos: San Cristobal: Wreck Bay M. bivitattus LACM 106303 (G49) Galapagos: San Cristobal: Wreck Bay M. delanonis LACM 106327 (G104) Galapagos: Espanola: Gardner Bay M. delanonis LACM 106314 (G92) Galapagos: Gardner: near Espanola M. delanonis LACM 106315 (G93) Galapagos: Gardner: near Espanola M. duncanensis LACM 106340 (G261) Galapagos: Duncan (Pinzon): east side M. grayii LACM 106363 (G132) Galapagos: Floreana: Black Beach M. habelii LACM 106384 (G205) Galapagos: Marchena: south side M. habelii LACM 106389 (G210) Galapagos: Marchena: south side M. koepckeorum LACM 122590 (P6-193) Peru: Lambayeque: Cerro de la Vieja: ca. 7 km S (by rd) Motupe M. occipitalis LACM 154351 (G4-78) Ecuador: Ancon at Basurera M. paciﬁcus LACM 106398 (G192) Galapagos: Pinta: southwest side M. paciﬁcus LACM 106399 (G193) Galapagos: Pinta: southwest side M. peruvianus LACM 122684 (P6-381) Peru: Lima: El Paraiso Peninsula: ca 6.2 km W (by rd) jct Pan Am Hwy M. peruvianus LACM 154394 (G4-91) Ecuador: Santa Elena Peninsula, Punto Carnero M. stolzmanni LACM 122648 (P6-290) Peru: Cajamarca: ca 13 km SSE (by rd) Hacienda Molino Viejo (Ochentiuno) M. stolzmanni LACM 122667 (P6-258) Peru: Cajamarca: Bellavista M. theresiae LACM 122695 (P6-389) Peru: Lima: El Paraiso Peninsula: ca 6.2 km W (by rd) jct Pan Am Hwy M. tigris LACM 122720 (P6-84) Peru: Lima: ca 3 km SE Asia Vieja T. etheridgei [at LACM] (TC-921) Bolivia: 0.5 km SW Parotania RR station T. hispidus [at LACM] (4-94) Venezuela: Isla Margarita T. hispidus [at LACM] (4-76) Venezuela: Playa Guiria D. Kizirian et al. / Molecular Phylogenetics and Evolution 32 (2004) 761–769 763

DNA polymerase (Perkin–Elmer) kits. PCR cocktails Decay indices (Bremer, 1988) were calculated using comprised water (17–35 ll), 10 X buffer (5.0 ll), MgCl2 TreeRot (version 2.0) with the number of heuristic (4.0–7.0 ll), dNTPs (5.0 ll), heavy and light strand searches increased to 1000 to increase accuracy of indi- primers (10 mM; 1.0 or 2.5 ll), Taq polymerase (0.3 ll), ces (Sorenson, 1999). Bootstrap statistics were calcu- and template DNA (0.7–1.0 ll). Reactions were sub- lated using PAUP* (fast-heuristic search, nreps ¼ 1002) jected to a variety of thermal-cycling profiles including and assumed model parameters calculated with Model an initial heating (1–5 min, 95 °C) then 32–43 cycles of test (i.e., GTR + I + C). denaturation (35 s, 94 °C), annealing (35–45 s, 48–58 °C), MrBayes (version 3.0; Huelsenbeck and Ronquist, extension (60 s; or 150 s plus 4 s added to each sub- 2001) was used to estimate posterior probabilities of sequent cycle; 70 °C, or 72 °C), and a final extension clades under a likelihood model. Substitution rates were (7 min, 72 °C). allowed to be different, subject to the constraints of time- To reduce the risk of contamination, all reagents, reversibility (GTR; n ¼ 6). Among-site rate variation was primer stocks, DNA extractions, and PCR cocktails drawn from a gamma distribution with some proportion were prepared in a facility that was physically isolated being invariant (rates ¼ invgamma). Model parameters from areas where post-PCR procedures were performed. for structural and protein-coding partitions were treated Kim Wipes were used to shield aerosol created when as unlinked [unlink revmat ¼ (all) shape ¼ (all) pinvar ¼ tubes were opened. In addition, pre-PCR tasks were (all) statefreq ¼ (all)]. Four (nchains ¼ 4) heated (temp ¼ performed earlier in the day than less sensitive proce- 0.5) Markov chains were calculated simultaneously and dures to reduce risk of contamination due to accumu- sampled every 100 generations (samplefreq ¼ 100) for lated contaminant on skin and clothing (Wayne et al., 2,000,000 generations (mcmc ngen ¼ 2,000,000). Sta- 1999). tionarity was evaluated graphically (plot) and the first Amplified fragments were purified using agarose gel 2001 (of 20,001) trees were discarded (burnin ¼ 2001). electrophoresis and QIAquick Gel Extraction kits (Qia- Best trees were compared to those incorporating to- gen-Operon Catalog #28706). In a few cases, fragments pological constraints (i.e., monophyly of Galapagoan were re-amplified using nested primer pairs. Fragments Microlophus and monophyly of M. albemarlensis) using were labeled using Big Dye (2.0) florescent dye termina- Kishino–Hasegawa (for parsimony trees) and Shimoda- tors and cycle-sequencing protocols for the ABI PRISM ira–Hasegawa (for likelihood trees) tests in PAUP*. The 377 Automated Sequencer. Sequencher software (version Shimodaira–Hasegawa test results are based on model 4.1, Gene Codes) was used to edit the raw data and to parameters calculated with Modeltest (i.e., GTR + I + C), produce a preliminary alignment of fragments. Ulti- a RELL test distribution, and 10,000 bootstrap replicates. mately, protein-coding genes were aligned assuming translation into a functional protein and transfer RNAs were aligned assuming secondary structure models for 3. Results closely related taxa (Macey et al., 1997b). Nucleotide sequences were deposited at GenBank (AY625152-89). Low guanine content (12%) suggests that nucleotide sequences represent mitochondrial genes rather than nu- 2.2. Data analysis clear pseudogenes (Zhang and Hewitt, 1996). Parsimony analysis yielded three cladograms (Figs. 1 and 4; Of 1956 nucleotide positions, seven (characters 1603– L ¼ 2558; CI ¼ 0.5625; CI excluding uninformative 09) in the TwC loop of tRNATrp were excluded from characters ¼ 0.5202; RI ¼ 0.7785; RC ¼ 0.4379) among phylogenetic analyses because they could not be un- which topological variation is due to ambiguous rela- ambiguously aligned, 980 were invariant, 210 were tionships within island groups (i.e., Santa Cruz + Bal- parsimony-uninformative, and 759 were parsimony-intra + Daphne, Santiago + Bartolome, and Fernandina + formative. A molecular clock was not assumed in any of Isabela). Maximum likelihood analysis under the best- the analyses. Trees were rooted with Iguana iguana fitting model (GTR + I + C) yielded a single tree (Fig. 2; (GenBank llG278511). Parsimony analysis was per- )ln L ¼ 13578:23698). The likelihood of the best state for formed assuming equal weights for all transformation the ‘‘cold chain’’ in the Bayesian analysis was )13192.57 series and gaps were treated as a fifth character state. (Fig. 3). Topologies from likelihood and parsimony Heuristic tree search settings in PAUP* (version 4.0b10; analyses are largely congruent and possess comparable Swofford, 2002) included addseq ¼ random, nreps ¼ levels of support for most major clades. Likelihood and 50,000, swap ¼ tbr, steepest ¼ yes, hold ¼ 5 or 10. Mod- parsimony trees differ in the position of the M. koe- eltest (version 3.06; Posada and Crandall, 1998) was used pckeorum, which may be the result of missing data (418 of to identify the likelihood model that best fits the data 1956 positions were coded as missing for this taxon). (i.e., GTR + I + C), which was assumed in two subsequent Nevertheless, this study places this enigmatic taxon tree searches using PAUP* (addseq ¼ random, nreps ¼ (Plesiomicrolophus of Frost, 1992; Microlophus of 825; start ¼ nj, nreps ¼ 1000, hold ¼ 1). Frost et al., 2001; Harvey and Gutberlet, 2000) in the 764 D. Kizirian et al. / Molecular Phylogenetics and Evolution 32 (2004) 761–769

Fig. 1. Strict consensus of three shortest cladograms (L ¼ 2558). Numbers on branches are decay indices.

M. occipitalis group. Trees incorporating topological This study finds M. albemarlensis to be paraphyletic constraints (monophyly of Galapagoan Microlophus and with respect to M. duncanensis, M. grayii, and M. pac- monophyly of M. albemarlensis) are significantly different ificus, corroborating an analysis based on allozyme from best trees (Table 2). variation (Wright, 1983) and a remark in Frost (1992, p. Although our taxonomic sampling was not exhaus- 49) that M. albemarlensis might represent ‘‘grouping by tive, the monophyly of Microlophus, Tropidurus, the M. plesiomorphy rather than on an understanding of the peruvianus group, and the M. occipitalis group is sup- historical relationships of these island forms.’’ ported here. Our results are congruent with those of Lopez et al. (1992), except that this study finds unam- biguous support for the placement of M. peruvianus 4. Discussion outside the smallest clade including all Galapagos island tropidurids. Phylogenetic analyses herein agree with 4.1. Classification WrightÕs (1983) phenetic clustering of island groups (i.e., Santa Cruz + Baltra + Daphne, Santiago + Bartolome) The existence of geographically restricted haplotype except for Isabela and Fernandina, which we find to be clades suggests that there is greater species diversity than sister lineages and Wright (1983) identified as neighbors. implied by the classifications in Lopez et al. (1992) and D. Kizirian et al. / Molecular Phylogenetics and Evolution 32 (2004) 761–769 765

Fig. 2. Unrooted phylogram resulting from maximum likelihood analysis ()ln L ¼ 13578:23698) with proposed classiﬁcation.

Frost (1992), particularly with respect to the Microlo- than 2000 specimens from 24 islands, Van Denburgh phus albemarlensis complex. Greater inter-island diver- and Slevin (1913, p. 188) stated that the populations of sity in Galapagos Microlophus is also suggested by M. albemarlensis are ‘‘so similar that we have been able variation in behavior (Carpenter, 1966, 1970), allozymes to find no characters which will distinguish them.’’ The (Wright, 1983), and microsatellite DNA (Jordan et al., weak divergence within the M. albemarlensis complex 2002). Intra-island variation in sprint speed (Snell et al., could reflect slower rates of evolution or relatively recent 1988) and endurance capacity (Miles et al., 2001) in M. isolation; a mechanism consistent with the latter is dis- albemarlensis has also been reported. Much of the di- cussed below (see Section 4.2). vergence among M. albemarlensis, however, is weak. This study corroborates previous work based on For example, sequence divergence among M. albemarl- morphology (Van Denburgh and Slevin, 1913), behavior ensis (excluding M. duncanensis, M. grayii, and M. (Carpenter, 1966), and allozymes (Wright, 1983) that M. pacificus) varies from 0 to 6% and morphological vari- duncanensis, M. grayii,andM. pacificus represent di- ation is nearly non-existent (Lanza, 1974, 1980; Van agnosable entities, therefore, we continue to recognize Denburgh and Slevin, 1913); after examination of more them. Because the relatively plesiomorphic lineages of 766 D. Kizirian et al. / Molecular Phylogenetics and Evolution 32 (2004) 761–769

Fig. 3. Majority rule consensus tree resulting from Bayesian analysis. Numbers above branches are posterior probabilities of clade support and those below are bootstrap values.

M. albemarlensis (i.e., those on Fernandina, Isabela, because it is paraphyletic. Should it become desirable to Santa Cruz, Santiago, and satellites) by themselves do identify lizards of the M. albemarlensis complex from, for not form a natural entity, we do not associate an ex- example, Isabela, we suggest ‘‘M. albemarlensis complex clusive binominal with it. Instead, we recognize the more from Isabela.’’ Alternatively, there are numerous inclusive ‘‘M. albemarlensis complex,’’ which includes available names (e.g., Lanza, 1974, 1980; Talurri et al., the historically nested lineages M. duncanensis, M. 1982) that could be resurrected to recognize formally grayii, and M. pacificus, as well as the lineages previ- additional insular diversity within M. albemarlensis ously recognized as ‘‘M. albemarlensis.’’ It should be complex. emphasized that the unappended binominal ‘‘M. albe- The indented classification proposed here (Table 3) marlensis’’ does not appear in our classification (Table 3) employs the principles of classification in de Queiroz D. Kizirian et al. / Molecular Phylogenetics and Evolution 32 (2004) 761–769 767

Table 2 Results of topological constraints tests Tree Shimodaira–Hasegawa Kishino–Hasegawa )ln L P Length P 1 13699.50030 0.0000 2606 <0.0001 2 13672.70332 0.0004 2618 <0.0001 3 13578.23698 2558 In Tree 1, M. albemarlensis was constrained to be monophyletic. In Tree 2, the monophyly of Galapagoan Microlophus was constrained, consistent with a single dispersal event to Galapagos. Tree 3 represents the best tree(s) for likelihood and parsimony analyses, respectively. Asterisks indicate signiﬁcant diﬀerence at P < 0:05.

Table 3 Proposed phylogenetic (indented) classification and geographic distri- butions of Microlophus occipitalis group species Microlophus occipitalis group M. albemarlensis complex Western Galapagos M. pacificus Pinta (Galapagos) M. duncanensis Pinzon (Galapagos) M. grayii Floreana (Galapagos) M. bivittatus San Cristobal (Galapagos) Fig. 4. Map of Galapagos including adapted topology from the par- M. delanonis Espanola, Gardner (Galapagos) simony analysis, island names used in this study, direction of the M. habelii Marchena (Galapagos) Humboldt Current, movement of the Nazca Plate, and proposed M. koepckeorum Coastal Peru classification. Roman numerals represent independent dispersal events M. occipitalis Coastal Ecuador and Peru from mainland. M. stolzmanni Intra-Andean valleys of Ecuador and Peru Previous studies based on allozyme variation (Wright, 1983; unpublished), immunological distances and Gauthier (1990), despite the lack of consensus re- (Lopez et al., 1992), and morphology (Wright, unpub- garding how species should be treated (Cantino et al., lished) found M. bivittatus (Isla San Cristobal) and 1999). Until such a consensus is reached, the classifica- Microlophus habelii (Isla Marchena) to be more closely tion used here is sufficiently informative and unambig- related to the mainland species M. occipitalis than to uous regarding the diversity of Lava Lizards, it other Galapagos species, a conclusion that is unambig- faithfully reflects the nested historical groupings dis- uously supported by this study. Given that the Gala- covered by phylogenetic analysis, and all recognized pagos Islands have never been attached to the mainland, taxa have meaning in an evolutionary context (i.e., no the most parsimonious explanation of the available data paraphyletic taxa are included). is that tropidurids have dispersed to the archipelago on at least two independent occasions. 4.2. Biogeography Isabela is the largest island in the Galapagos and is geologically complex with five volcanic peaks and recent Our biogeographic inferences assume geological hy- vulcanism (Cox, 1983). Studies of Lava Lizards (Wright, potheses summarized in Cox (1983), particularly the 1983) and tortoises (Beheregaray et al., 2003; Caccone plate tectonics model that explains the greater portion of et al., 1999, 2002; Ciofi et al., 2002) suggest that the the history of the archipelago. In addition, we consid- biota on this island also have complex histories. For ered the phylogeny of Lava Lizards (this study), eco- example, phylogenetic analysis of nucleotide variation logical data on extant tropidurids (Dixon and Wright, allies lizards from western Isabela with those from 1975), and data on the Humboldt Current (Wright, Fernandina, rather than samples from eastern Isabela. A 1984; Wyrtki, 1967; Wyrtki et al., 1976). A phylogenetic close relationship between western Isabela and Fernan- tree superimposed on a map of the emergent portions of dina Lava Lizards could be explained by relatively re- the Galapagos plate (Fig. 4) reveals concordance among cent (and not improbable) dispersal across the 5 km geographic, geological, oceanographic, and phylogenetic channel between the islands. In addition, although Lava data suggesting that we have recovered historical signal. Lizards from eastern Isabela are represented here by For example, younger (western) islands harbor younger only a single sample, divergence values (1.5%) and lineages, which were likely carried there by ocean cur- parsimony analyses (Figs. 1 and 4) suggest that there has rents from older (eastern) islands. been independent evolution of Lava Lizards on Isabela. 768 D. Kizirian et al. / Molecular Phylogenetics and Evolution 32 (2004) 761–769

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