Journal of Biogeography (J. Biogeogr.) (2011) 38, 272–284

ORIGINAL Seri Indian traditional knowledge and ARTICLE molecular biology agree: no express train for island-hopping spiny-tailed in the Sea of Corte´ s Christina M. Davy1,2*, Fausto R. Me´ndez de la Cruz3, Amy Lathrop2 and Robert W. Murphy1,2,4

1Department of Ecology and Evolutionary ABSTRACT Biology, University of Toronto, 25 Wilcocks Aim The role of human activities in biogeography can be difficult to Street, Toronto, ON M5S 3B2, Canada, 2Department of Natural History, Royal identify, but in some cases molecular techniques can be used to test hypotheses of Ontario Museum, 100 Queen’s Park, Toronto, human-mediated dispersal. A currently accepted hypothesis states that humans ON M5S 2C6, Canada, 3Laboratorio de mediated the divergence of two species of spiny-tailed iguanas in the Herpetologı´a, Instituto de Biologı´a, hemilopha species complex, namely C. conspicuosa and C. nolascensis, which Universidad Nacional Auto´noma de Me´xico, occupy islands in the Sea of Corte´s between the peninsula of Baja and AP 70-153, CP 04510, Me´xico, DF, , mainland Mexico. We test an alternative hypothesis that follows the traditional 4State Key Laboratory of Genetic Resources and knowledge of the Seri Indians and states that the divergence of these species was Evolution, Kunming Institute of Zoology, The not mediated by humans. Chinese Academy of Sciences, Kunming Location Mexico, including Baja California, Sonoran and Sinaloan coastal 650223, China regions, and Isla San Esteban and Isla San Pedro Nolasco in the Sea of Corte´s. Methods We analysed mitochondrial (cytochrome b and cytochrome c oxidase subunit III) DNA sequences from four species in the C. hemilopha species complex. Maximum parsimony and Bayesian inference were used to infer matriarchal genealogical relationships between the species and several outgroup taxa. Bayesian methods were used to estimate divergence times for the major nodes on the trees based on previously published, fossil-calibrated priors. Results Our analysis indicated that lineages within the C. hemilopha species complex diverged long before human colonization of the Americas. The divergence of C. nolascensis and C. conspicuosa could not be attributed to Seri translocations. The matriarchal genealogy of the species complex currently defies a simple biogeographical interpretation. Main conclusions We conclude that humans did not mediate the divergence of C. nolascensis and C. conspicuosa. This conclusion is consistent with the traditional knowledge of the Seri people. These results demonstrate the utility of molecular techniques in investigating potential cases of human-mediated dispersal of plants and , and reinforce the importance of considering traditional knowledge in the formation of scientific hypotheses and the interpretation of results. *Correspondence: Christina M. Davy, Keywords c/o Department of Natural History, Royal Baja California, Ctenosaura conspicuosa, Ctenosaura hemilopha, Ctenosaura Ontario Museum, 100 Queens Park, Toronto, ON M5S 2C6, Canada. nolascensis, genealogy, human-mediated dispersal, , island biogeo- E-mail: [email protected] graphy, molecular clock, .

272 www.blackwellpublishing.com/jbi ª 2010 Blackwell Publishing Ltd doi:10.1111/j.1365-2699.2010.02422.x Human translocation is not responsible for Ctenosaura hemilopha dispersal

Grismer (1999) elevated to full species level. Ctenosaura INTRODUCTION hemilopha occurs on the southern half of the peninsula of Untangling the historical causes of the geographic distributions Baja California, and C. h. insulana is found on Isla Cerralvo, of species or species complexes is central to the study of c. 8.73 km from the peninsula (Murphy et al., 2002). Cten- biogeography. However, the factors that are responsible for osaura macrolopha (Grismer, 1999) is found on the Mexican current species distributions cannot always be directly inferred mainland, from Hermosillo, , southwards to mid- from available data. For example, historical climatic conditions Sinaloa. Ctenosaura nolascensis (Grismer, 1999) is restricted to are often difficult to determine, although these may explain Isla San Pedro Nolasco, a small island c. 14.61 km off the coast current species ranges and distributions. Likewise, the geolog- near Guaymas, Sonora (Murphy et al., 2002). Finally, Cten- ical history of many areas is not completely understood; it may osaura conspicuosa (Grismer, 1999) occurs only on Isla San not be ‘written in stone’ (e.g. Murphy & Aguirre-Leo´n, 2002; Esteban and the neighbouring Isla Cholludo (also referred to as Riddle et al., 2008). Human impacts on species distributions Isla Lobos, e.g. Smith, 1972). Isla Cholludo is located near the must also be considered, because humans have both deliber- southernmost point of Isla Tiburo´n (Fig. 1), and it was ately and accidentally mediated the dispersal of many plants connected to this island and mainland Mexico at times of and animals (e.g. Austin, 1999; Nabhan, 2002; Carlton, 2003). maximum glaciation in the Pleistocene. The oceanic islands in In this study we use molecular techniques to investigate the Sea of Corte´s are estimated to have uplifted between 5 and whether or not human-mediated translocations played a role 2 Ma (Carren˜o & Helenes, 2002). Some of these islands have in the evolutionary history of an iguanid species complex in never been connected to the mainland, and Isla San Esteban the Sea of Corte´s. may be one of these (Carren˜o & Helenes, 2002). Thus, the The Cape spiny-tailed (Ctenosaura hemilopha Cope, occurrence of C. conspicuosa on Isla San Esteban, and the 1863) species complex (, Iguanidae) is found in the distribution of this species complex in general, is a biogeo- southern part of the Baja Californian peninsula, on mainland graphical conundrum. Mexico (Sonora and Sinaloa), and on several islands in the Sea Bailey (1928) suggested that C. conspicuosa on Isla San of Corte´s (Smith, 1935; Lowe & Norris, 1955; Fig. 1). Colour Esteban ‘were in all probability carried there by man’. When pattern variability and other morphological attributes among Smith (1972) later described the taxon, he suggested that the individuals from these isolated locations led Smith (1972) to insular populations were founded by individuals from the recognize five geographically isolated subspecies, four of which peninsula of Baja California, as ‘a few waif populations in a

32o N

0 100 200 see inset km

30o

25-28 29-36

28o 7

Figure 1 Distribution of the Ctenosaura hemilopha species complex, with arrows 13,14 Isla Tiburon indicating Isla San Esteban and Isla San 20-24 18, 19 26o o 6 Pedro Nolasco. The location of Isla Cholludo 29.7 is indicated in the inset. Numbers indicate Isla Cholludo Isla Datil 4 samples included in the analysis (see Table 1 o 2, 3 29.6 0510 for sample details). Shaded areas indicate the Isla San ranges of C. hemilopha (dotted line) and Esteban km o o o 5 o 112.6 112.5 112.4 o C. macrolopha (dashed line). Samples 1–6, 24 112.3 1 C. hemilopha; 13, 14, 20–24, C. conspicuosa;7, 18, 19, 25–28, C. macrolopha; 29–36, o o o o o o C. nolascensis. 117 115 113 111 109 107

Journal of Biogeography 38, 272–284 273 ª 2010 Blackwell Publishing Ltd C. M. Davy et al. sweepstake pattern reached a number of the Gulf Islands’. had translocated Ctenosaura from Isla San Pedro Nolasco to Grismer (1994, 2002) further considered the hypothesis that Isla San Esteban, the response was that, although it was the indigenous culture in and around the Sea of Corte´s certainly possible, they had no history of such a translocation. mediated the dispersal of Ctenosaura sp. (presumably When translocating animals for live food, the Seri have a C. nolascensis) from Isla San Pedro Nolasco to Isla San Esteban. practice of breaking the legs of in order to prevent Nabhan (2003) documented in detail the complex cultural escape (Nabhan, 2003), which makes accidental introductions relationship between the Seri (Comca´ac) people indigenous to unlikely (although not impossible). the Sea of Corte´s and the native reptiles of the region. Many Recent translocations are unlikely to be detectable mor- Seri recognize snakes, lizards, tortoises and marine turtles by phologically. For example, translocated populations of chuc- species, and some species have more than one common name kwallas cannot be morphologically distinguished based on in the Comca´ac language. Each species has a cultural signif- their place of origin. In contrast, phenotypic distinctions icance to the Seri. Some may be included in feasts at important between C. conspicuosa on Isla San Esteban and C. nolascensis celebrations; for example, marine turtles are served during on Isla San Pedro Nolasco have been listed by Grismer coming-of-age ceremonies. Some are avoided; for example, the (1999). These include the presence of small black spots on Seri believe that looking at certain lizards can cause a pregnant the ventral surface of the hind limbs of adult C. nolascensis, woman to miscarry (Nabhan, 2003). while C. conspicuosa, C. macrolopha and C. hemilopha have Along with the cultural importance of the Seri’s relationship large circular blotches. In C. nolascensis, the dorsal hind limb with reptiles, Seri oral history contains information about pattern is mottled, while in C. conspicuosa it is banded. historical translocations of reptiles. Nabhan (2002, 2003) Hatchling coloration tends to differ between the populations documented Seri accounts of the deliberate translocation of as well, although less consistently than adult coloration (Sauromalus) between islands in the Sea of Corte´s. (Grismer, 1999). Consistent differences in coloration between Chuckwallas are an important source of food for the Seri these two populations (Smith, 1972; Grismer, 1999) imply (Nabhan, 2003). Both molecular evidence and Seri traditional prolonged reproductive isolation, which is inconsistent with knowledge suggest that the Seri were responsible for trans- ongoing human translocation of individuals between recently locating Sauromalus hispidus from Isla A´ ngel de la Guarda diverged populations. Overall, the evidence for human- southwards to Isla San Lorenzo Sur, and probably also to Isla mediated dispersal of Ctenosaura to Isla San Esteban is San Lorenzo Norte and Islote Granito (Petren & Case, 1997, currently equivocal. In the case of Sauromalus, molecular 2002; Murphy & Aguirre-Leo´n, 2002). Seri involvement is also evidence helped to confirm Seri translocations, but such implicated in the dispersal of several other reptilian species evidence is lacking for the C. hemilopha complex. throughout the Gulf islands, including side-blotched lizards, To date, the sole genetic analysis of the C. hemilopha species which probably dispersed as hitchhikers (Uta; Upton & complex, an MSc thesis (Cryder, 1999), suggested a genealogy Murphy, 1997), and giant chuckwallas (Sauromalus varius; based on 22 cytochrome b (cyt b) and cytochrome c oxidase Murphy & Aguirre-Leo´n, 2002), which were probably trans- subunit III (COIII) sequences (Fig. 2). Grismer (2002) cited located deliberately (Nabhan, 2003). Human translocations Cryder’s genealogy and the morphological variation between have also mediated the dispersal and subsequent divergence of species (Grismer, 1999) as evidence that the Seri people had lizards in other parts of the world. For example, molecular data ‘created’ C. conspicuosa by moving C. nolascensis from Isla San demonstrate how Lipinia noctua ‘took the express train’ to Pedro Nolasco to Isla San Esteban. Grismer (2002) also cited distant Polynesian islands by hitchhiking with humans Nabhan’s (2003) ethno-herpetological study of the Seri culture dispersing out of Melanesia (Austin, 1999). as evidence for Seri translocation of Ctenosaura from Isla San Could translocations by the Seri explain the peculiar Pedro Nolasco to Isla San Esteban. occurrence of C. conspicuosa on the islands of San Esteban The question of Seri involvement has not yet been and Cholludo, which are so far north of the other insular adequately addressed. The Seri elders cited by Grismer Ctenosaura and surrounded by islands on which Ctenosaura (2002) did not, in fact, claim an oral history of Ctenosaura are not found? The Seri people hunt spiny-tailed iguanas translocations between those two islands (Nabhan, 2003). The (Nabhan, 2003), so it would have benefited them to move practice of breaking the legs of lizards being transported for Ctenosaura species to islands on which they lived or hunted. food makes accidental translocations unlikely. Equally prob- They have successfully translocated and established new lematic, Cryder’s (1999) phylogenetic results (Fig. 2) do not populations of other iguanid lizards, as evidenced by their show the topology expected under the hypothesis of the translocations of Sauromalus and other reptiles, and they have translocation of Ctenosaura from Isla San Pedro Nolasco to Isla an oral history of the translocation of C. conspicuosa from Isla San Esteban. Recent divergence between young species or San Esteban to nearby Isla Cholludo. However, Isla San diverging populations typically manifests in genealogies as Esteban itself is located far to the north of the other insular incomplete lineage sorting (e.g. Murphy & Aguirre-Leo´n, 2002; species of Ctenosaura (Fig. 1) and is isolated from other Morando et al., 2004; Heckman et al., 2007). In contrast to populations of Ctenosaura. The occurrence of C. conspicuosa this prediction, Cryder’s (1999) genealogy showed that the on Isla San Esteban is, therefore, more difficult to explain. four species and the one subspecies form distinct lineages. The When Nabhan (2003) directly asked a Seri elder if his people exception was C. nolascensis, which had two independent

274 Journal of Biogeography 38, 272–284 ª 2010 Blackwell Publishing Ltd Human translocation is not responsible for Ctenosaura hemilopha dispersal

C. nolascensis Isla San Pedro Nolasco

C. conspicuosa Isla San Esteban

C. conspicuosa Isla Cholludo

C. macrolopha Sonora

C. nolascensis Isla San Pedro Nolasco

C. hemilopha Baja California Figure 2 Genealogy of the matrilines of the Ctenosaura hemilopha species complex inferred by Cryder (1999) from cytochrome b and cytochrome c oxidase subunit III C. hemilopha Isla Cerralvo sequences using maximum parsimony methods (redrawn from Grismer, 2002). maternal lineages, both of which were resolved and neither of sample from a number of locations within the range of each which nested within another species. species in order to avoid a geographic sampling bias. The We used mtDNA sequences to test the hypothesis suggested subspecies C. h. insulana was not included owing to sampling by Bailey (1928) and Grismer (2002) that the Seri people restrictions. We also collected tissues from the Mexican spiny- founded the population of C. conspicuosa on Isla San Esteban tailed iguana (C. pectinata Wiegmann) in Sinaloa (near by translocating C. nolascensis from Isla San Pedro Nolasco. Chametla, Culiaca´n and Mazatla´n) as an outgroup taxon. Acceptance of this hypothesis requires that the divergence of Previously, this iguanid was shown to be closely related to the the separate species lineages occurred after the first known C. hemilopha complex (Ko¨hler et al., 2000). As more distant human colonization of the Americas (c. 16,500 years ago; outgroups we included sequences from Petrosaurus thalassinus Goebel et al., 2008). We inferred the relationships between Cope collected in Baja California, Iguana iguana Linnaeus taken maternal lineages of the C. hemilopha species complex using from GenBank, and Dume´ril collected from standard phylogenetic methods, and used Bayesian inference Sonora (near Sonoyta and Caborca). Petrosaurus thalassinus (BI) to estimate divergence times between the species. Rejec- was specified as the most distant outgroup whenever required. tion of the hypothesis requires that estimated divergence times GenBank accession numbers and voucher specimen informa- between the species occurred before human colonization of the tion for all individuals are listed in Table 1. Americas, and that there is no evidence of incomplete lineage sorting between matrilines sampled within the ranges of DNA extraction, amplification and sequencing C. conspicuosa and C. nolascensis. We isolated total genomic DNA from frozen or 95% ethanol- preserved tissues using standard proteinase K digestion MATERIALS AND METHODS followed by phenol-chloroform extraction (Sambrook et al., Because the sequences obtained by Cryder (1999) were not 1989). Cyt b and COIII were amplified using polymerase chain available in GenBank, we resampled the four species. Phyloge- reaction (PCR) (Saiki et al., 1988). DNA amplification and netic analysis of mtDNA was assumed to produce a genealogy purification followed the methods of Blair et al. (2009), using of maternal lineages that closely reflects the evolutionary the primers and primer-specific annealing temperatures listed history of the species in question (e.g. Upton & Murphy, 1997). in Table 2. Sequencing reactions were performed on a Gene- To produce a mtDNA genealogy for the C. hemilopha complex, Amp 9700 thermal cycler (Applied Biosystems, Foster City, CA, we examined mitochondrial cyt b and COIII sequences from 31 USA), using the BigDye Terminator v 3.1 Cycle Sequencing kit individual Ctenosaura representing the four recognized species (Applied Biosystems). Sequences were visualized on an ABI 377 in the C. hemilopha complex. Where possible, we attempted to automated sequencer (Applied Biosystems).

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Table 1 Sample details for iguanid lizards Voucher GenBank GenBank included in this study. Sample ID numbers number/Field accession no. accession no. correspond to the ID numbers on the trees Species Sample ID number (cyt b) (COIII) and in Fig. 1. Where vouchers were taken, ROM accession numbers are designated (i.e. Ctenosaura hemilopha CH1 ROM 26795 HQ141246 HQ141198 ‘ROM xxxx’). If vouchers were not taken, the CH2 RWM 2280 HQ141251 HQ141203 field number corresponding to the tissue CH3 RWM 2282 HQ141247 HQ141199 sample is indicated. CH4 RWM 623 HQ141248 HQ141200 CH5 RWM 631 HQ141249 HQ141201 CH6 RWM 879 HQ141250 HQ141202 C. macrolopha CH7 JRO 645 HQ141235 HQ141187 CH18 ROM 38003 HQ141268 HQ141220 CH19 ROM 38004 HQ141267 HQ141219 CH25 ROM 38021 HQ141236 HQ141188 CH26 ROM 38022 HQ141237 HQ141189 CH27 ROM 38023 HQ141238 HQ141190 CH28 ROM 38024 HQ141239 HQ141191 CH37 ROM 38037 HQ141231 HQ141183 CH38 ROM 38038 HQ141232 HQ141184 CH39 ROM 38039 HQ141233 HQ141185 C. conspicuosa CH13 KP-EC102 HQ141252 HQ141204 CH14 KP-EC103 HQ141253 HQ141205 CH 20 ROM 38011 HQ141254 HQ141206 CH21 ROM 38012 HQ141255 HQ141207 CH22 ROM 38013 HQ141256 HQ141208 CH23 ROM 38014 HQ141257 HQ141209 CH24 ROM 38015 HQ141258 HQ141210 C. nolascensis CH29 ROM 38028 HQ141240 HQ141192 CH30 ROM 38029 HQ141271 HQ141223 CH31 ROM 38030 HQ141272 HQ141224 CH32 ROM 38031 HQ141273 HQ141225 CH33 ROM 38032 HQ141274 HQ141226 CH34 ROM 38033 HQ141275 HQ141227 CH35 ROM 38034 HQ141276 HQ141228 CH36 ROM 38035 HQ141277 HQ141229 Petrosaurus thalassinus Petrosaurus RWM 2263 HQ141230 HQ141182 Iguana iguana Iguana GenBank AJ278511.2 AJ278511.2 Sauromalus ater S1 KP-20 HQ141242 HQ141194 S2 KP-22a HQ141243 HQ141195 S3 KP-22b HQ141244 HQ141196 S4 KP-19 HQ141241 HQ141193 S5 KP-21 HQ141245 HQ141197 C. pectinata CP1 ROM 38001 HQ141269 HQ141221 CP2 ROM 38002 HQ141270 HQ141222 CP3 ROM 38040 HQ141234 HQ141186 CP4 ROM 38041 HQ141259 HQ141211 CP5 ROM 38042 HQ141260 HQ141212 CP6 ROM 38043 HQ141261 HQ141213 CP7 ROM 38044 HQ141262 HQ141214 CP8 ROM 38045 HQ141263 HQ141215 CP9 ROM 38046 HQ141264 HQ141216 CP10 ROM 38047 HQ141265 HQ141217 CP11 ROM 38050 HQ141266 HQ141218

Cyt b, cytochrome b; COIII, cytochrome c oxidase subunit III.

ClustalW (Larkin et al., 2007) and subsequently checked Alignment and sequence analysis by eye. Conversion to amino acids confirmed the align- We sequenced 1632 base pairs combined from COIII and ment. We calculated the percentage sequence divergence cyt b for 49 individuals. Sequences were aligned with (uncorrected p-distances) for all ingroup taxa based on

276 Journal of Biogeography 38, 272–284 ª 2010 Blackwell Publishing Ltd Human translocation is not responsible for Ctenosaura hemilopha dispersal

Table 2 Primers used to amplify and sequence cytochrome b (cyt b) and cytochrome c oxidase subunit III (COIII) from the Ctenosaura hemilopha species complex, C. pectinata, Sauromalus ater and Petrosaurus thalassinus.

Primer/annealing Target gene temperature Sequence (5¢–3¢) Source

Cyt b GLUDG-L TGACTTGAARAACCAYCGTTG Palumbi et al. (1991) 50 C CTEN-8H TTACTGTGGCGCCTCGGAAGGATATTTGGCCTCA Cryder (1999) 50 C COIII L8618CO3 CATGATAACACATAATGACCCACCAA Cryder (1999) 46 C H9323CO3 ACTACGTCTACGAAATGTCAGTATCA Cryder (1999) 46 C Petrosaurus and Sauromalus cyt b Cyt b B1L CCATCCAACATCTCAGCATGATGAAA Kocher et al. (1989) Cyt b B6H GTCTTCAGTTTTTGGTTTACAAGAC Tim Birt (Queens University, pers. comm.) 50 C uncorrected p-distances as implemented in mega 4 (Tamura they had posterior probability values ‡ 0.95 (Felsenstein, et al., 2007). 2004). Because direct comparison of our sampled sequences with those of Cryder (1999) was not possible, we initially followed Estimates of divergence time his methods in order to determine if any inherent differences between the two data sets might have caused our genealogy to It is not advisable to estimate divergence times for nodes in a differ in topology from his (Fig. 2). Thus, maximum parsi- tree without at least one point of geological or palaeonto- mony (MP) analysis was implemented in paup* 4.0b10 logical reference, and preferably several (Benton & Ayala, (Swofford, 2002), employing a heuristic search with 50 2003; Reisz & Mu¨ller, 2004). Robust estimations of diver- random addition sequences (RAS) and tree bisection–recon- gence dates require several points of reference, preferably nection branch swapping. We then assessed nodal confidence from fossil evidence. Unfortunately, the fossil record for for the MP strict consensus tree by nonparametric bootstrap- iguanid lizards in western Mexico is scarce, and it is therefore ping (Felsenstein, 1985) using a heuristic search with 1000 not possible to date the evolution of the C. hemilopha species pseudoreplicates, 50 RAS per pseudoreplicate, and nearest- complex by dating fossils of these species. Therefore, we base neighbour interchange branch swapping (Nei & Kumar, 2000). our estimates of divergence time within the C. hemilopha We considered bootstrap values > 70 to indicate strong nodal complex on the divergence time (most recent common support (Hillis & Bull, 1993). ancestor, MRCA) of C. pectinata and C. hemilopha estimated We used MrBayes 3.1.2 (Huelsenbeck & Ronquist, 2001; by Zarza et al. (2008). Using a Bayesian approach and Ronquist & Huelsenbeck, 2003) to determine the most calibration points based on fossil evidence, they estimate that probable evolutionary history for the matrilines based on the the divergence of C. pectinata and C. hemilopha occurred available sequences. MrModeltest 2.3 (Nylander, 2004) 9.24 Ma (SD = 2.9), and that divergence within C. pectinata indicated that the best-fit evolutionary model for our data began between 2.3 and 6.5 Ma (Zarza et al., 2008). We use was (GTR+I+C), which was selected for each gene using the these priors in our analysis. Akaike information criterion (Akaike, 1974, 1979). To We used the program beast 1.4.2 (Drummond et al., 2007) account for potential rate variation and differing rates of to infer divergence times for species of the C. hemilopha substitution between the two genes (Nylander, 2004; Brandley complex under an uncorrelated lognormal relaxed molecular et al., 2005), we partitioned our data set by gene, and set the clock model (Drummond et al., 2006). Input files were created analysis to account for variable rates between partitions using with BEAUti 1.4.2 (Drummond et al., 2007), and manually the command prset applyto = (all) ratepr = variable in edited to partition the data by gene and to specify substitution MrBayes. rates, gamma shape parameters and proportion of invariable The Bayesian analysis was run for 1 · 105 generations, with sites for each partition based on the estimates made in two simultaneous runs of six chains sampled at 100-generation MrModeltest. We specified monophyly of the lineages intervals. The first 2500 trees (25%) were discarded as burn-in, identified by our BI analysis but did not specify an input tree. and the inferred genealogy was based on 7500 data points. beast analyses considered the Yule process tree prior, as Examination of the raw trace, the log-likelihood plot and the recommended for analyses of speciation (Drummond et al., standard deviations of the split frequencies all indicated that 2007). Three Markov chain Monte Carlo (MCMC) runs were convergence had occurred, and that the burn-in period was made, each with 3 · 107 generations, sampling every 100 sufficient. We considered lineages to have significant support if generations with a burn-in period of 10% of the samples.

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We examined output files for each of the three runs in The topologies of the MP strict consensus tree and the BI Tracer (Rambaut & Drummond, 2007) to assess whether or majority-rule consensus tree differed slightly at the tips. The not they had converged on similar estimates of divergence trees also differed in their placement of Ctenosaura in relation times. Next, we combined the results of the runs using to the three outgroup genera, but both methods inferred the LogCombiner (Drummond et al., 2007) for further analysis, same relationships between the five species of Ctenosaura, and sampling the combined runs every 300 generations for a final resolved the same major lineages within Ctenosaura, without sample of 89,991,300 states with a burn-in of 10% of the exception (Fig. 3). Six major mtDNA lineages were recovered. samples. We considered effective sample size (ESS) values formed a distinct lineage distantly related > 200 to indicate good mixing and a valid estimate of to the C. hemilopha complex. Ctenosaura nolascensis was continuous parameters and likelihoods given the specified resolved into two distinct and distantly related lineages. The priors (Drummond et al., 2007). We checked the distribution first of these lineages (C. nolascensis-1) was resolved as sister to of the standard deviation of the uncorrelated lognormal relaxed a single sample of C. macrolopha from Culiaca´n, Sinaloa, and clock model and its coefficient of variation in Tracer during this group was resolved as sister to C. conspicuosa from Isla San examination of the output files. Neither parameter approached Esteban. The remaining samples of C. nolascensis formed a zero, indicating rate variation between branches and suggesting separate lineage (C. nolascensis-2), sister to a lineage containing that a strict clock would have been an inappropriate model for both C. hemilopha and C. macrolopha. There was high our data. The maximum clade credibility tree for the combined bootstrap and posterior probability support for most nodes runs was computed using TreeAnnotator 1.4.7 (Drummond between species (Fig. 3). et al., 2007). We used the estimated lower bound of the 95% Our genealogy recovered the same mtDNA groups as Cryder highest posterior density (HPD) region of the MRCA param- (1999), but differed slightly in the relationships between eters to test the hypothesis that C. conspicuosa and C. nolascensis C. nolascensis-2, C. hemilopha and C. macrolopha. Otherwise, could have diverged as a result of human-mediated dispersal, our genealogies agreed on the relationship between the that is, after human colonization of the Americas matrilines. Interestingly, the proportion of the two C. nolasc- (c. 16,500 years ago; Goebel et al., 2008). ensis haplotypes in our sample (5:3) approximates that shown in Fig. 2 (3:2; Cryder, 1999). Based on the sample from Culiaca´n (CH19), which was resolved as sister to the RESULTS C. nolascensis-1 lineage, C. macrolopha did not consist of a single matrilineal lineage but contained at least two distinct Sequence analysis matrilines. Finally, the tree topology and p-distances indicated Average percentage sequence divergences (p-distances) within that the degree of divergence between C. conspicuosa and and between C. pectinata and species in the C. hemilopha C. nolascensis-1 was comparable to the divergence between complex are summarized in Table 3. Sequence divergence C. macrolopha and C. hemilopha (Table 4). between C. pectinata and the C. hemilopha species complex averaged 11.2%. Divergence between lineages within the Estimates of divergence time C. hemilopha species complex ranged from 0.8% to 4.5%, with the highest percentage divergence occurring between two Estimated divergence times within the major lineages and the lineages in C. nolascensis. 95% HPD of the estimates are summarized in Table 4. Of 1632 nucleotide sites, 547 were variable and 378 were Estimated divergence times for all major nodes are also shown potentially phylogenetically informative. Within the sequences on the maximum clade credibility tree generated by from C. hemilopha and C. pectinata (excluding other out- TreeAnnotator from the three combined MCMC runs groups), 241 sites were potentially phylogenetically informative. (Fig. 4). The estimated divergence time for the matrilines The MP analysis recovered 2111 most-parsimonious trees of within C. conspicuosa was 1.73 Ma, with a lower 95% HPD 925 steps (consistency index = 0.765, retention index = 0.943). of 326,200 years ago. Divergence between the C. nolascensis-1

Table 3 Average pairwise genetic divergence (percentage uncorrected p-distances) within and between Ctenosaura pectinata, C. hemilopha, C. macrolopha, C. conspicuosa and C. nolascensis. The two matrilines within C. nolascensis are presented separately. The standard error of percentage divergence is indicated in parentheses.

C. pectinata C. conspicuosa C. nolascensis-1 C. nolascensis-2 C. hemilopha C. macrolopha

C. pectinata 0.9 (± 0.2) C. conspicuosa 11.1 (± 1.2) 0.1 (± 0.1) C. nolascensis-1 11.2 (± 1.2) 0.8 (± 0.2) 0.0 (± 0.1) C. nolascensis-2 11.2 (± 0.012) 4.0 (± 0.6) 4.5 (± 0.2) 0.0 (± 0.0) C. hemilopha 11.3 (± 1.2) 3.9 (± 0.6) 4.0 (± 0.6) 1.1 (± 0.3) 0.0 (± 0.0) C. macrolopha 11.3 (± 1.2) 4.3 (± 0.6) 1.5 (± 0.6) 1.5 (± 0.3) 0.9(± 0.2) 0.1 (± 0.0)

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P. thalassinus C.h. 19 C. macrolopha 1.0/99 C.h. 30 C.h. 31 C.h. 32 C. nolascensis-1 1.0/93 C.h. 35 * C.h. 36 0.93/63 C.h. 13 C.h. 20 0.97/64 C.h. 14 0.75/86 C.h. 21 C. conspicuosa C.h. 24 C.h. 22 C.h. 23 C.h. 1 * C.h. 3 0.93/98 C.h. 4 C. hemilopha C.h. 5 C.h. 6 C.h. 2 0.92/77 C.h. 37 C.h. 38 C.h. 39 1.0/91 C.h. 26 C. macrolopha 0.93/100 C.h. 7 C.h. 25 C.h. 27 1.0/97 C.h. 28 C.h. 18 1.0/99 C.h. 29 C.h. 33 C. nolascensis-2 0.64/58 C.h. 34 0.98/85 C.p. 3 0.55 C.p. 4 Figure 3 Evolutionary history of the matri- C.p. 1 lines of the Ctenosaura hemilopha complex * 0.65 C.p. 2 inferred using Bayesian inference analysis of C.p. 11 C. pectinata C.p. 5 cytochrome b and cytochrome c oxidase 0.59/61 C.p. 6 subunit III sequence data. C.h., Ctenosaura * C.p. 7 hemilopha complex; C.p., C. pectinata; C.p. 10 C.p. 8 S, Sauromalus ater. Numbers at nodes indi- C.p. 9 cate Bayesian posterior probability values 1.0/91 S4 0.96 followed by the percentage of replicate trees S2 * S3 in which the associated taxa clustered 0.96/51 S5 together in the nonparametric bootstrap 0.68 S1 analysis; * indicates a posterior probability/ I. iguana bootstrap proportion = 1.0/100.

Table 4 Estimated dates of divergence from the most recent common ancestor (MRCA) MRCA Mean (Ma) 95% HPD upper 95% HPD lower between the major matrilines within the Between lineages Ctenosaura hemilopha species complex. Dates (hemilopha/macrolopha) 3.6723 6.7291 1.2188 (Ma) were estimated under an uncorrelated + nolascensis-2 relaxed clock model in beast, with the hemilopha + macrolopha 2.7318 5.162 0.7606 analysis partitioned by gene and priors as conspicuosa + nolascensis-1 2.9032 5.5111 0.8421 described in the Materials and Methods. nolascensis-1 + CH19 1.5714 3.3261 0.2287 Mean estimated divergence dates are listed, (C. macrolopha) with the upper and lower bounds of the 95% Within lineages highest posterior density (HPD) of each pectinata 3.5842 5.6412 2.3001 estimate. hemilopha 1.3676 3.0473 0.1667 macrolopha 1.6646 3.4142 0.3553 conspicuosa 1.7283 3.6096 0.3367 nolascensis-1* 0.7433 1.807 0.0447 nolascensis-2 1.2582 3.1166 0.504

*Excluding the sample of C. macrolopha from Culiaca´n (CH19), which was resolved as sister to this lineage.

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C.h. 19 C. macrolopha 1.57 C.h. 32 * C.h. 35 C. nolascensis-1 0.74 C.h. 30 2.90 * C.h. 31 * C.h. 36 C.h. 14 1.73 0.41 C.h. 20 * * C.h. 13 C. conspicuosa C.h. 23 0.26 C.h. 24 * C.h. 21 C.h. 22 C.h. 2 5.17 C.h. 4 * 1.37 C.h. 1 C. hemilopha * C.h. 5 C.h. 6 2.73 C.h. 3 C.h. 7 C.h. 38 1.66 C.h. 28 * C.h. 25 C. macrolopha 3.67 C.h. 27 C.h. 37 Figure 4 Chronogram of the Ctenosaura 1.29 C.h. 18 * 7.93 * hemilopha species complex: maximum clade * C.h. 26 0.39 C.h. 39 credibility tree from three combined Markov 0.45 C.h. 33 chain Monte Carlo analyses performed in 1.26 C. nolascensis-2 C.h. 34 beast. Ctenosaura pectinata and Iguana ig- * C.h. 29 0.52 C.p. 5 uana are included as outgroups. Posterior 1.56 * C.p. 6 estimates of divergence times were inferred 0.7 C.p. 10 9.27 by partitioning analyses by gene, and placing * C.p. 7 constraints on the divergence dates of two * 0.25 C.p. 2 C. pectinata 1.14 C.p. 1 nodes (see Materials and Methods). Values at nodes indicate posterior mean ages (Ma), and 3.58 * C.p. 4 * C.p. 3 node bars represent the 95% highest proba- C.p. 11 bility density (HPD). * indicates Bayesian 1.01 C.p. 8 C.p. 9 posterior probabilities > 95%. The full extent I. iguana of the upper 95% HPD for the two most basal nodes is not shown. The scale bar shows 10.0 9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0 time in millions of years ago. and C. conspicuosa matrilines was estimated at 2.9 Ma is especially intriguing and suggests historical dispersal (5.5–0.8421 Ma). The most recent divergence occurred bet- between Isla San Pedro Nolasco and the mainland. Our ween the matrilines within C. nolascensis-1 (1.8–0.0447 Ma). conclusion leaves us in need of a new biogeographical explanation for the distribution of the C. hemilopha species complex. DISCUSSION

Genealogies for the C. hemilopha complex and estimates of No ‘express train’ for C. conspicuosa divergence time between the species in the complex suggest that historical human involvement in their divergence is highly The hypothesis that Seri translocations caused the initial unlikely. We cannot refute the null hypothesis that C. conspic- divergence between the insular species of Ctenosaura requires uosa diverged from the other taxa in the C. hemilopha complex the divergence of the matrilines found on the two islands to before humans arrived in North America, and we cannot occur after c. 16,500 years ago (Goebel et al., 2008), but the accept Seri translocation as the explanation for the presence estimated divergence time between the insular matrilines of of C. conspicuosa on Isla San Esteban. Although this conclusion C. conspicuosa and C. nolascensis-1 is, at a minimum, 0.84 Ma differs from some previous interpretations, it is in agreement (Table 4). Consequently, the molecular data do not support with Seri traditional knowledge (Nabhan, 2002, 2003). the theory that the Seri (or any other human culture) mediated Ctenosaura hemilopha, C. macrolopha, C. nolascensis and the initial divergence of C. nolascensis and C. conspicuosa. C. conspicuosa show no evidence of recent female dispersal This finding is unlikely to surprise the Seri, whose oral and gene flow between them, although there are interesting histories do not include such a translocation (Nabhan, 2003). patterns present in the genealogy, as we discuss below. The As discussed earlier, the Seri’s reptilian translocations show occurrence of a C. nolascensis-like haplotype on the mainland deliberation and planning. Details of other translocations

280 Journal of Biogeography 38, 272–284 ª 2010 Blackwell Publishing Ltd Human translocation is not responsible for Ctenosaura hemilopha dispersal

(including those of C. conspicuosa between Isla San Esteban members of the scientific community can damage that trust. and Isla Cholludo) recorded by Nabhan suggest that such We acknowledge that the occurrence of C. conspicuosa on a events have been well documented in their oral history. small, oceanic island far from any obvious founder popula- Furthermore, the Seri practice of breaking the legs of lizards tions is a biogeographical conundrum. Human (specifically being transported for food (Nabhan, 2002) makes accidental Seri Indian) activities are known to have strongly shaped the translocations unlikely. Iguanas with broken or dislocated legs biogeography of the Sea of Corte´s, and human-mediated would be unlikely to escape successfully. Even if they did dispersal of C. hemilopha between the islands was first escape, an injured male would have difficulty mating, and suggested to explain this puzzle nearly a century ago (Bailey, crippled females might be unable to dig suitable nests. Thus, 1928). There is no doubt that the Seri are capable of they would be unlikely to contribute to the gene pool. Leg- successfully founding insular populations of iguanid lizards breaking was not used during deliberate translocations of (Petren & Case, 1997, 2002; Murphy & Aguirre-Leo´n, 2002; animals because the desired outcome (establishment of a new Nabhan, 2002, 2003), so Bailey’s original hypothesis of human- population that subsequently could be harvested) would be mediated dispersal was a reasonable one. However, the current thwarted (Nabhan, 2002). question is not one of ability, but simply of whether or not Other incidental evidence also suggests pre-Seri divergence humans actually translocated C. hemilopha from Isla San Pedro of C. conspicuosa and C. nolascensis. The population of Nolasco to Isla San Esteban. At this time, traditional knowl- C. conspicuosa on Isla Cholludo founded by the Seri (Grismer, edge and molecular biology both reject the hypothesis of 2002; Nabhan, 2002) can be used (albeit cautiously) as a null human-mediated dispersal of C. hemilopha between these two model for the expected genealogical pattern (i.e. incomplete islands, and a new interpretation of the biogeography of the lineage sorting) caused by Seri translocation. Although we C. hemilopha complex is needed. could not include these sequences in our analysis, Cryder’s (1999) analysis included two individuals from this population, Biogeography of the C. hemilopha species complex both of which fall undifferentiated into the lineage containing samples from Isla San Esteban (Fig. 2). In contrast to these Our tree topology differs slightly from that shown in Fig. 2 patterns, the genealogical distinctiveness of populations on Isla (Cryder, 1999), but both genealogies indicate that at least two San Pedro Nolasco and Isla San Esteban provide further independent colonization events were involved in the history evidence that these populations are not the result of human of C. nolascensis on Isla San Pedro Nolasco: one by an ancestor translocations. shared with C. conspicuosa, and the other by an ancestor shared The oral history of the Seri is a valuable cultural resource, with the mainland and peninsular species. Cryder’s tree places not only for the Seri themselves, but also for the rest of C. nolascensis-2 as the immediate sister of C. macrolopha, while humanity. As such, Nabhan’s (2002, 2003) documentation of our analyses resolves C. macrolopha and C. hemilopha in a the ethno-herpetology of this culture provides an important lineage sister to C. nolascensis-2. It is possible that we sampled record of Seri traditional knowledge, and a relatively unique different haplotypes within C. nolascensis, which would suggest anthropological work. Unfortunately, several species of reptiles the potential presence of three or more matrilines on Isla San pictured in the book are misidentified. For example, Derm- Pedro Nolasco. However, because the proportions of samples achelys coriacea is labelled as Caretta caretta; Pituophis is falling into each C. nolascensis lineage are roughly equal in the labelled as Lampropeltis; a desert tortoise (Gopherus agassizii)is two studies, it is more likely that we sampled the same two described as a ‘turtle’; a Sauromalus is labelled as Ctenosaura, matrilines of C. nolascensis as Cryder. Although sampling was with the location of the photo incorrectly listed as Isla San limited in both studies, the frequencies of the two haplotypes Esteban; and a Seri carving of a Ctenosaura is misidentified as are about 60% C. nolascensis-1:40% C. nolascensis-2. These two Sauromalus (Nabhan, 2003). These misidentifications raise the haplogroups had 4.5% divergence between them, a higher question of whether other species described by Seri elders divergence than occurred between any two recognized species could also be misidentified (i.e. assigned an incorrect scientific within the complex. Such genealogical patterns are often name). This is not the case. The names associated with the interpreted as evidence for cryptic speciation (e.g. Tavares & images are those in the photo archives of the -Sonora Baker, 2008). However, C. nolascensis is morphologically Desert Museum, and the errors are editorial in nature (G.P. distinct from the other species of Ctenosaura (Grismer, Nabhan, University of Arizona, pers. comm. to R.W.M., 14 1999), and we know of no previous mention of distinct June 2010). The discrepancy in names does not invalidate the morphotypes within C. nolascensis on Isla San Pedro Nolasco. traditional knowledge that Nabhan (2002, 2003) has so Because of the morphological distinctness of C. nolascensis and carefully collected. the lack of obvious mechanisms of reproductive isolation on Along with its inherent value, traditional knowledge can be the island, we find the hypothesis of sympatric cryptic species an important source of scientific inspiration, and can inform within C. nolascensis to be improbable. the development of scientific hypotheses and management Differences between Cryder’s and our trees probably result plans (e.g. Kimmins, 2008). However, the sharing of tradi- from different sampling strategies. Cryder’s analysis included tional knowledge by indigenous communities is a gesture of the subspecies C. h. insulana from Isla Cerralvo, which may trust, and selective interpretations of traditional knowledge by have affected the inferred relationships among the species.

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Furthermore, Cryder’s (1999) analysis of C. macrolopha was cladogenic event, probably during the Pleistocene. Evidence based on individuals collected at a single locality within the from nuclear genes would allow the construction of a species’ wide range. Because this strategy was unlikely to more informative species phylogeny, which may clarify the sample genetic diversity in mainland species, we collected evolutionary history of these species. The detailed biogeo- samples throughout the range of C. macrolopha along the coast graphy of this species complex continues to defy detailed of Sonora and Sinaloa from the north to south extremes, as interpretation, but we now know that humans were not well as for about half of the range for C. hemilopha on the involved. peninsula of Baja California (Fig. 1). However, our sampling Human activities have played a pivotal role in the did not fully cover these species’ ranges, and sampling further biogeographical history of many species, but testing hypo- inland in Sonora and Sinaloa and further north on the theses of human translocations is often challenging, and peninsula might have discovered additional lineages. limited by the available evidence. We successfully used Further sampling of C. macrolopha could be extremely traditional mtDNA analysis, together with Bayesian methods, informative, especially because the matrilines within this to refute the hypothesis of human-mediated dispersal in the species do not form a single lineage, and the species may case of the C. hemilopha complex, and reached an alternative contain several more divergent lineages. One sample of conclusion that is in keeping with the traditional knowledge of C. macrolopha (CH19, from Culiaca´n, Sinaloa) was resolved the Seri people. In our case, the traditional knowledge of the as sister to C. nolascensis-1, and this is particularly intriguing. Seri was carefully documented (Nabhan, 2002, 2003), but this The sample site is not near Isla San Pedro Nolasco (Fig. 1), and is often not the case. The teachings of many cultures are the haplotype occurred alongside the more common haplotype rapidly being lost. It is our hope that the information in C. macrolopha. Thus, there is no reason to suspect that contained in traditional teachings can be preserved, and that C. macrolopha haplotypes segregate geographically. There are such knowledge will continue to inform scientific studies. several potential explanations for the placement of this sample in the genealogy. First, it could be the result of incomplete ACKNOWLEDGEMENTS lineage sorting. Second, the pattern could be caused by Seri (or other human) transport of C. nolascensis to the mainland, We especially thank the Seri people of Tiburo´n and Isla San followed by deliberate or accidental introduction into the wild. Esteban for permission to enter and sample lizards on their Given the estimated average divergence date of 1.57 Ma for traditional lands. Sample collection in Mexico was approved this haplotype (95% HPD = 3.32–0.23 Ma; Fig. 4), we find by the Mexican government (SEMARNAT SGPA/ DGVS/ these two explanations unlikely. Third, this pattern could 03489/07). Access to an MSc thesis from Loma Linda indicate historical dispersal from San Pedro Nolasco to the University was kindly granted by W. Hays; R.L. Carter made mainland, followed by cytoplasmic capture and incorporation valiant attempts to retrieve M. Cryder’s sequence data; and of the C. nolascensis-like haplotype into C. macrolopha. The G.P. Nabhan graciously provided important information. We presence of iguanids on many deep-water oceanic islands thank C. Blair and two anonymous referees for valuable testifies to their ability to disperse across oceans, and Isla San comments on earlier versions of the manuscript. C.M.D. was Pedro Nolasco is only 14.6 km offshore. More rigorous supported by a Canada Graduate Scholarship from the sampling of the matrilines present throughout Sonora and National Research Council of Canada. This research was Sinaloa may shed further light on the question. supported by grants from the Natural Sciences and Engineer- The most perplexing aspect of the data is the counterin- ing Research Council of Canada, Discovery Grant A3148, the tuitive pattern of divergence, whereby the mainland and Royal Ontario Museum (ROM) Foundation, and the ROM peninsular species diverged after their origin from the two Members Volunteer Committee to R.W.M. insular species. 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