Abstract The taxonomy of giant Galapagos tortoises (Chelonoidis spp.) is currently based primarily on morphological characters and island of origin. Over the last decade, compelling genetic evidence has accumulated for multiple independent evolutionary lineages, spurring the need for taxonomic revision. On the island of Santa Cruz there is currently a single named species, C. porteri. Recent genetic and morphological studies have shown that, within this taxon, there are two evolutionarily and spatially distinct lineages on the western and eastern sectors of the island, known as the Reserva and Cerro Fatal populations, respectively. Analyses of DNA from natural populations and museum specimens, including the type specimen for C. porteri, confirm the genetic distinctiveness of these two lineages and support elevation of the Cerro Fatal tortoises to the rank of species. In this paper, we identify DNA characters that define this new species, and infer evolutionary relationships relative to other species of Galapagos tortoises.

Citation: Poulakakis N, Edwards DL, Chiari Y, Garrick RC, Russello MA, Benavides E, et al. (2015) Description of a New Galapagos Giant Tortoise Species (Chelonoidis; Testudines: Testudinidae) from Cerro Fatal on Santa Cruz Island. PLoS ONE 10(10): e0138779. https://doi.org/10.1371/journal.pone.0138779 Editor: Sergios-Orestis Kolokotronis, Fordham University, UNITED STATES Received: May 7, 2015; Accepted: September 3, 2015; Published: October 21, 2015 Copyright: © 2015 Poulakakis et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited Data Availability: All relevant data are within the paper and its Supporting Information files. All new D-loop sequences files are available from the GenBankdatabase (accession number(s) KT192434, KT192435, KT192436). Funding: This work was supported by funding from the Yale Institute for Biospheric Studies, the Turtle Conservation Society, Galapagos Conservancy, and the Paul and Bay and Eppley foundation to AC. Competing interests: The authors have declared that no competing interests exist.

Introduction Giant Galapagos tortoises are icons of the Galapagos archipelago. They represent a classic example of an island adaptive radiation [1, 2], and are keystone herbivores [3]. Despite their prominence, the taxonomy of Galapagos tortoises has long been debated. Van Denburgh [4] originally recognized 14 species (13 of them named) within the genus Testudo based on island of origin and differences in carapace morphology. Since then, the taxonomy of the group has undergone recurring changes. First, Mertens and Wermuth [5] demoted described groups to the subspecies level, under the name Testudo elephantopus. Next, on the basis of morphological data, Loveridge and Williams [6] established Geochelone (Fitzinger, 1835) as the most appropriate genus for Galapagos (and many other) tortoises, and placed all the Galapagos forms in one species (G. elephantopus) within the subgenus Chelonoidis (Fitzinger, 1856; also containing mainland South American species). More recently, Bour [7] promoted Chelonoidis to generic status and elevated the subspecies to species. Despite a nomenclatural review by Pritchard [8] arguing for Geochelone and Chelonoidis as the appropriate genus and subgenus respectively, genetic data presented by Le, Raxworthy [9] indicated that Geochelone is polyphyletic and thus the generic status of Chelonoidis is supported. Within his monograph, Van Denburgh [4] identified four general groups based on carapace shape: “saddle-back” (high anterior opening), “dome” (rounded cupola-like form), “intermediate” (between saddle-back and domed forms), and “unknown” (museum remains for which shape information is lacking). Tortoises from the islands of Española, San Cristóbal, Pinzón, Pinta, Floreana, Santa Fe (undescribed) and Fernandina are considered to be saddlebacks (the latter four taxa are now extinct). Tortoises from San Cristóbal Island, and Santiago Island have a carapace with an intermediate shape. Those from Isabela Island and Santa Cruz Island are domed [4, 10]. Saddleback tortoises have also been reported from northern Isabela, likely the result of human-mediated translocations [11]. Although useful for morphologically classifying tortoises, variation does exist within these three broadly defined carapace shapes [10]. Some authors have argued that Galapagos giant tortoise taxa should be considered subspecies [8], advocating for the synonymy of species described by Van Denburgh [4]; others accept the species status of all taxa except for four of the five named species on Isabela Island (the fifth being C. becki), which are lumped in a single species, C. vicina [12]. Genetic studies based broadly on mitochondrial DNA (mtDNA) sequence data support the evolutionary distinctiveness of described taxa [1, 2, 11, 13–16] with the clear exception of tortoises once found on Rábida Island, which are also likely human-mediated transplants [16]. Collectively, these studies further revealed that most populations from different islands represent clades and therefore independent evolutionary (and conservation) units. Moreover, geographically isolated populations within islands (e.g., those on separate volcanoes on Isabela Island) are readily distinguishable on the basis of nuclear microsatellite data, which indicates little or no gene flow among them [11, 15, 17–21]. Tortoises on Santa Cruz Island are currently considered members of a single named species, C. porteri (formerly Testudo porteri) [22] associated with the large population (“Reserva”) occurring on the island’s southwestern slopes in a mesic region of the island. This population occupies an area of ~156 km2 and includes 2,000–4,000 individuals [8, 23, 24]. A second tortoise population (“Cerro Fatal”) on the eastern side of Santa Cruz Island has long been recognized but considered a member of C. porteri (Fig 1). This population comprises vastly fewer tortoises (several hundred individuals) and occupies a smaller and dryer area (~40 km2) than the Reserva population from which it is separated by approximately 20 km (Fig 1) [25]. Although individuals of both populations exhibit a domed carapace morphology, morphological analyses indicated that tortoises from the two populations differ in size and shape [20, 26, 27]. Genetically, Reserva and Cerro Fatal tortoises are among the most divergent taxa within the archipelago: they belong to different major mtDNA clades [11, 16, 20] and were likely derived from separate colonizations of Santa Cruz Island from different source islands. Reserva tortoises are part of the oldest lineage in the archipelago (diverged ~1.74 million years ago, Mya), nested in a sub-clade including Isabela, Floreana and Pinzón Island tortoises. Cerro Fatal tortoises are much younger (~0.43 Mya), being most closely related to the tortoises from San Cristóbal, Pinta, and Española Islands [16]. Patterns and levels of genetic divergence based on nuclear microsatellite data support the relationships identified by mtDNA data. Each of the two taxa have numerous private alleles, implying very little recent gene flow, and they are as genetically divergent from each other as the other named species are from one another [11, 13, 20]. Previous studies have also revealed the existence of a limited amount of introgression between the two taxa [11, 13, 20], which is not unexpected given their geographical proximity. Over the last century, portions of the ranges of both species have been converted to farmland; the agricultural zone, a band stretching across the southern slope of the island from west to east, now provides a uniform habitat connection between the two species’ ranges. Moreover, the zone currently has many human residents, thus increasing the potential for human-mediated transport of tortoises. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 1. Geographic distribution of the two known lineages of giant tortoises on Santa Cruz Island: Chelonoidis porteri (Reserva) and Chelonoidis sp. nov. (Cerro Fatal) (indicated in dark gray). Light gray area connecting the distribution areas of the two species indicates agricultural land. Modified from Russello et al. [11]. https://doi.org/10.1371/journal.pone.0138779.g001 Given the genetic distinctiveness of the Reserva and Cerro Fatal populations and the current application of a single name (C. porteri) to all Santa Cruz Island tortoises, we sought to clarify their taxonomy by integrating data from extant populations with those from museum specimens, including the C. porteri holotype (Rothschild 1903), and two from Cerro Fatal (Fig 1). We analyzed nuclear microsatellite and mtDNA genetic data from both sample sets to (1) confirm the genetic distinctiveness of the two tortoise populations, (2) clarify the genetic assignment of the holotype for C. porteri, (3) identify diagnostic genetic characters that define a new species from Cerro Fatal, and (4) determine the holotype for the new species.

Material and Methods Museum specimens Three specimens representing Santa Cruz Island tortoises were obtained from natural history museum collections: a skull from the University of Wisconsin Zoological Collection (UWZS; USW32700), collected in the Cerro Fatal area in 1991; an incomplete carapace section (CF_March2010) from the museum at the Charles Darwin Research Station in Puerto Ayora, Santa Cruz Island, collected in 2010 from Cerro Fatal; and the C. porteri holotype from the London Tring Museum (reg. no. BMNH 1949.1.4.38 or BMNH-1949; formerly Testudo porteri) [22] collected by R.H. Beck in 1902 with site information limited only to island of origin. DNA extractions from museum specimens were performed in two physically isolated laboratories dedicated to the extraction of ancient DNA (aDNA): at Yale University and the University of Crete (Greece). All standard precautions were followed to prevent contamination by extant specimens. Detailed descriptions of the methods used to extract, amplify, and sequence DNA from the bones of the giant Galapagos tortoises are provided in the S1 File. Approximately 700 bp of the mtDNA control region (CR) and 12 microsatellite loci were amplified from all museum specimens using previously published primers and protocols [11, 16, 18, 28]. Genetic analyses To investigate evolutionary relationships of the three museum samples in the context of all available data from extant and extinct giant Galapagos tortoise species, we combined the new mtDNA sequences with 123 unique CR haplotypes from tortoises of all the named extinct and extant species identified by previously published mtDNA studies [1, 2, 11, 13–16, 18, 20] and three outgroup taxa from continental South America (C. chilensis, C. denticulata, and C. carbonaria) [1, 2]. Control region sequences were aligned in MAFFT v.7 [29] using default settings. Bayesian Inference (BI) phylogenetic analysis was conducted in MrBAYES v.3.2.1 [30]. The TrN + G model of nucleotide substitution was used, selected according to the Bayesian Information Criterion (BIC) implemented in jModelTest v. 2.1.1 [31], ignoring the models that include both gamma distribution and invariable sites [32]. Bayesian Inference phylogenetic analysis was run four times (independent random starting trees) with eight chains for each run of 107 Markov chain Monte Carlo (MCMC) generations, sampling from the chain every 100th generation. This generated an output of 105 trees. To confirm that the chains had achieved stationarity, we evaluated “burn-in” by plotting–lnL tree scores and tree lengths against generation number using Tracer v.1.5.0 [33]. The–lnL tree scores stabilized after approximately 2×106 generations and the first 25% of trees were discarded as a conservative measure to avoid the possibility of including stochastically generated, sub-optimal trees. A majority-rule consensus tree was then derived from the posterior distribution of trees, with posterior probabilities calculated as the percentage of samples that recovered any particular node. We also ran the analysis with no data to sample the prior distributions for each parameter to confirm that the priors were not driving the outcomes. To estimate levels of genetic diversity within each of the two tortoise populations, 70 mtDNA sequences of C. porteri from Reserva and 51 from Cerro Fatal tortoises from previous studies were combined with the sequences collected from museum specimens in this study, creating a dataset of 124 mtDNA control region sequences. The number of segregating sites (S) and haplotype (H D ) and nucleotide (π) diversity were computed using DnaSP v. 5.10 [34]. A haplotype network was generated using statistical parsimony [35] implemented in TCS v.1.13 with the 95% confidence criterion enforced [36]. Genotypic data from 12 nuclear microsatellite loci were used to further investigate genetic distinctiveness of the two populations. Our reference database included genotypic data from extant samples collected for previous studies from Santa Cruz Island (Cerro Fatal; n = 21, Reserva; n = 34; [11, 13, 17, 20]) and the three museum samples analyzed in this study. Given that null alleles, stuttering signals or large allelic dropouts could contribute to ‘false positive’ homozygous patterns, the pure Cerro Fatal and La Reserva populations were examined using MICROCHECKER v2.2.3 [37] with no evidences for scoring error due to stuttering and large allelic dropouts or null alleles. To assign the museum samples to a particular taxon, we used the Bayesian clustering method implemented in STRUCTURE v2.3 [38]. Membership coefficients (Q-values) from individuals collected in either Cerro Fatal or Reserva were used to assign individuals to a particular population of origin following a MCMC simulation of 108 steps after an initial ‘burnin’ of 107 steps. The MCMC sampling frequency was set at default. Analyses were run using an admixture model using locality origin as prior information for cluster assignment of extant samples, but not for the museum samples in order to be assigned to one of the two populations. The analysis was repeated 20 times to assess consistency of results. CLUMPP [39] was used to combine and summarize parameter estimates from STRUCTURE, with input files prepared using STRUCTURE HARVESTER [40]. Results were then plotted using DISTRUCT [41]. GENECLASS2 v2.0 [42] was also used to identify migrant individuals, individuals with mixed ancestry, and individuals that do not strongly assign to any population. To compute the probability of each individual’s belonging to a set of reference populations, assignment tests were performed using direct and simulation approaches based on the partial Bayesian method of Rannala and Mountain [43] and by setting the threshold for exclusion of individuals to 0.05. Average allelic richness (corrected for sample size by rarefaction) per locality was calculated in the HIERFSTAT package [44] for R (http://www.R-project.org/). Observed and expected heterozygosity were calculated using Arlequin v3.5.1.3 [45]. Weir and Cockerham’s [46] estimate of F IS (inbreeding coefficient) was calculated using GenePop v4.0.10 [47]. To assess levels of genetic differentiation between the Reserva and Cerro Fatal tortoises, we compared the mtDNA and microsatellite distances between these two populations with those found between other named species of Galapagos tortoises. For these analyses, we excluded introgressed individuals (i.e. used only purebred individuals) as we were interested in estimating the amount of evolutionary divergence between the two taxa. For mtDNA sequence data, we calculated divergences using two metrics: uncorrected p-distance, and maximum likelihood-corrected distances (calculated in PAUP* v4.0b10) [48]. For the mtDNA-based metrics, non-redundant haplotypes were the units of analysis (123 haplotypes from previous studies plus one from this study; see results below), excluding the haplotypes from the individuals that showed signs of introgression (n = 10), yielding 6441 interspecific pairwise comparisons. Similarly, for microsatellite data, we used two metrics that, in combination, can be informative about whether divergences occurred on recent vs. older timescales (i.e., F ST vs. R ST calculated in GENEPOP and R ST CALC v2.2 [49], respectively). For the microsatellite-based metrics, populations were the unit of analysis (i.e., 79 interspecific pairwise comparisons). In addition to Cerro Fatal and Reserva lineages, the taxa for which all possible pairwise comparisons were performed were C. hoodensis, C. chathamensis, C. abingdoni, C. ephippium, C. darwini, C. vandenburghi, C. microphyes, C. guntheri, C. vicina, C. elephantopus, and C. becki. Nomenclatural Acts The electronic edition of this article conforms to the requirements of the amended International Code of Zoological Nomenclature, and hence the new names contained herein are available under that Code from the electronic edition of this article. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix "http://zoobank.org/". The LSID for this publication is: urn:lsid:zoobank.org:pub:065FBB00-835F-421E-860A-D06C15465D1E. The electronic edition of this work was published in a journal with an ISSN, and has been archived and is available from the following digital repositories: PubMed Central, LOCKSS.

Conclusion Genetic and morphological data confirm the existence of two tortoise species on Santa Cruz Island. We describe the tortoises from Cerro Fatal as a new species, C. donfaustoi. The recognition of C. donfaustoi as a new species has important conservation implications for both taxa. The revised taxonomy reduces the range of C. porteri, with a population of several thousand individuals, to occupying only the western and southwestern parts of Santa Cruz Island. It also confines C. donfaustoi to the eastern part of Santa Cruz Island, with a much smaller population size estimated currently at ca. 250 individuals. From a conservation standpoint, recognition of this new species will help promote efforts to protect and restore it, given that its low abundance, small geographic range, and reduced genetic diversity make it vulnerable. In particular, further investigation is needed to better determine C. donfaustoi‘s population size and structure, range, movement patterns, location of nesting zones, and habitat requirements, as well as ongoing threats and effective ways to mitigate them. In an age of increasing human occupation of much of the higher elevations on Santa Cruz Island, maintaining the two species’ biological isolation is critical. Of particular importance is ensuring that no human-mediated transport of tortoises occurs between the two sides of Santa Cruz Island given that the two species’ ranges are now linked via a single agricultural zone.

Supporting Information S1 File. Detailed descriptions of the methods used to extract, amplify, and sequence DNA from the bones of the giant Galapagos tortoises. https://doi.org/10.1371/journal.pone.0138779.s001 (DOCX)

Acknowledgments We wish to thank Tom Fritts and Cruz Marquez who pointed out the Cerro Fatal tortoises and urged us to look at them from a genetic point of view, as he suspected that they were distinct from the Reserva tortoises. We also would like to thank all the long time collaborators who have contributed to the discovery of this new species because they were instrumental in collecting and analyzing previously published genetic (L. Beheregaray, C. Hyseni, C. Ciofi) and morphological (J. Claude) data. We also would like to extend a special thanks to Steve Blake for input on the conservation threats faced by the Santa Cruz tortoises and for collecting the carapace from a Cerro Fatal tortoise now in the Charles Darwin Research Station (CDRS) museum. We wish to thank CDRS, the University of Wisconsin Zoological Collection, and the Tring Museum for allowing us to sample the museum specimens in their care. We are very grateful to Peter Paul Van Dijk for advice regarding the nomenclature issue in relation to the C. porteri holotype. We want to acknowledge the continued support of the Galapagos National Park Directorate, which has facilitated this work in many ways and advances daily the protection and restoration of these lineages. A special thanks is due to the many park rangers whose efforts in the field were instrumental to gathering these data. We wish to express our gratitude to the editor and the reviewer for providing comments that significantly improved an earlier version of the manuscript. This work was supported by funding from the Yale Institute for Biospheric Studies, the Turtle Conservation Society, Galapagos Conservancy, and the Paul and Bay and Eppley foundation to AC.

Author Contributions Conceived and designed the experiments: AC. Performed the experiments: NP DLE. Analyzed the data: NP DLE RCG MAR. Contributed reagents/materials/analysis tools: NP DLE AC. Wrote the paper: NP DLE RCG YC EB MAR GJWC SG WT JPG LJC AC.