Conservation Genet Resour DOI 10.

1007/s12686-013-9915-7

APPLICATION ESSAYS

pagos: molecular tools help Illegal wildlife trade in Gala the taxonomic identication of conscated iguanas and guide their rapid repatriation
Gabriele Gentile Marco Ciambotta Washington Tapia

Received: 6 November 2012 / Accepted: 21 March 2013 Springer Science+Business Media Dordrecht 2013

pagos. All Abstract Four species of iguanas occur in Gala of them are included in the red list of the International Union for the Conservation of Nature and are protected under the Convention on International Trade in Endangered Species of Wild Fauna and Flora. In Ecuador, pagos Islands attempting to remove wildlife from the Gala is a serious environmental crime, punishable under Articles of the Ecuadorian Criminal Code. On July 2012, a tourist pagos while trying to illegally transwas arrested in Gala port four iguanas out of the province. The four iguanas, pagos National Park, conscated by the authorities of Gala needed rapid taxonomic identication and determination of geographic origin for the purposes of repatriation. The phylogenetic analysis of the Cytochrome b sequence data contributed to the unequivocal molecular taxonomic identication of the four conscated iguanas, each of which belonged to the species Conolophus subcristatus. The genetic data unambiguously indicated that the four conscated iguanas had been subtracted from the same population, currently distributed in Baltra and Seymour Norte. The use of molecular tools proved crucial for the rapid assessment of the population of origin. Such a rapid assessment was possible because a large genetic database pagos iguanas. It is recomwas already available for Gala mended that genetic programs be developed for the

pagos complete genetic characterization of wildlife in Gala as well as in other areas of endemism worldwide. Keywords Forensics Conolophus Molecular phylogeny Species identication Haplotype frequency estimation

Introduction The illegal wildlife trade is such a worldwide problem that, for the rst time, it was put on the agenda of the United Nations General Assembly, on September 2012. According to TRAFFIC, the wildlife trade monitoring network (www.trafc.org), the wildlife trade involves hundreds of millions of individual plants and animals from tens of thousands of species. The rapidly-growing illicit international trade in endangered speciesincluding living animals and their productsis now estimated to be worth between $8 and 10 billion per year globally (Haken 2011). For the uniqueness of their biodiversitywhich represents an attractive source of specimens for private collectionsareas of endemism are subject to especial attention from illegal trafckers. pagos (Ecuador), there are certainly high levels In Gala of biodiversity endemism. For example, around 50 native species of reptiles, birds and mammals have been recorded pagos. Of them, more than 50 % are threatened to in Gala various extents (IUCN 2012). pagos iguanas are among the most representative Gala pagos (Fig. 1), four species of the archipelago. In Gala species of iguanas are currently recognized, all of them being endemic to those islands: (1) Amblyrhynchus cristatus Bell (Marine Iguana, distributed across several islands); (2) Conolophus pallidus (Gray) (Barrington Land Iguana, inhabiting only Santa Fe island); (3) C. marthae

G. Gentile (&) M. Ciambotta Department of Biology, University of Rome Tor Vergata, Via della Ricerca Scientica, snc., 00133 Rome, Italy e-mail: gabriele.gentile@uniroma2.it W. Tapia Conservation and Sustainable Development Department, pagos National Park Directorate, Puerto Ayora, Gala pagos, Ecuador Gala

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pagos Pink Land Iguana, occurring Gentile and Snell (Gala only in a limited area of Volcan Wolf, Isabela island). pagos Land Iguana, (4) C. subcristatus Heller (Gala occurring in Fernandina, Isabela, Santa Cruz, Plaza Sur, Seymour Norte and Baltra islands). Habitat destruction, human predation and feral animals caused the extinction of the iguana population in Baltra (Woram 1991). Luckily, in the 1930s, a few adults had been previously transferred from Baltra to Seymour Norte, a nearby island where land iguanas did not occur. They served as the basis for a captive rearing program that allowed the repatriation of land iguanas back to Baltra (Hofkin et al. 2003). On each island, population sizes are small and even the largest estimated population (Santa Fe) currently barely exceeds the size of rquez et al. 2010). The four species 5,000 individuals (Ma of iguanas are included in the red list of the International Union for Conservation of Nature (IUCN) and are protected under the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES). Ecuador ratied the CITES agreement in 1975. According to Ecuadorian law, any attempt to remove wildlife from the pagos Islands is a serious environmental crime, punGala ishable under Articles of the Ecuadorian Criminal Code. On Sunday July 8th 2012, while inspecting the luggage of pagos National Park a tourist, the authorities of the Gala Service (GNPS) detected a suspicious package containing four iguanas, individually wrapped in cotton bags. The four iguanas were alive. Based on traditional morphological criteria, they were determined as all belonging to the species pagos Land Iguana). Consequently, the C. subcristatus (Gala tourist was stopped by the Environmental Police.

Unexpectedly, when facing the charge in court, the defense claimed that there was insufcient evidence to indicate that the four conscated iguanas were actually pagos Land Iguanas (C. subcristatus). This would Gala raise the forensic problem of proper taxonomic identication. Wisely, the park authorities sought independent evidence of the origin of sequestered iguanas for repatriation purposes. Due to the young age of the conscated iguanas, morphological criteria proved ambiguous in the assessment of the original population. As expected, the four iguanas temporarily housed in the facilities of the GNPSwould not accept appropriate food. Prolonged fasting could jeopardize the survival of the young iguanas. Such a scenario would call for an urgent repatriation action. Genetics has contributed to wildlife conservation and forensics for a long time (for a recent review see: Alacs et al. 2010). As noted by those authors, in many wildlife forensic casesincluding the present onethe taxonomic identication of the traded product or specimen to the species level may not be sufcient and the geographic origin needs to be determined. This is certainly possible if a genetic structure within the region of interest exists. In this regard, the use of phylogenetic methods may allow reliable taxonomic identication, while phylogeographic studies may prove crucial in the identication of the original population by assessing the geographic distribution of genealogical lineages. We provide here a clear example of how the use of appropriate molecular toolsin combination with a previous genetic characterization of local wildlifemay prove crucial in performing taxonomic identication and guiding a rapid repatriation action.

pagos Islands. Marine iguanas are present on all of the large islands of the archipelago. Those islands where land iguanas occur or Fig. 1 Gala have occurred historically are in grey. The crosses indicate recent extinctions

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Materials and methods Conscated land iguanas were labeled as A, B, C, and D. Approximately 1 ml of blood was drawn from the caudal vein and preserved in 5 ml of lysis buffer (Rassmann et al. 1997a). DNA was extracted using the QIAamp DNA Mini Kit (Qiagen). The whole sequence of the mitochondrial gene for the Cytochrome b (Cytb) was obtained by PCR (Polymerase Chain Reaction) for the four conscated iguanas. The primers TGLU14121 (50 -CCGAAAAATCCACCTTGTTA TTCAAC-30 ) and TTHRREV (50 -GGGGGTGGTTTAATT CCCAGC-30 ) were used as in Gentile et al. (2009) to PCR amplify a 1200 bp long fragment that included the whole Cytb gene and small fragments of the tRNAs for Glutamic acid and Threonine. The PCR conditions used are available in Gentile et al. (2009). Primers and PCR conditions are highly specic and consistently lead to sharp and clean PCR products. Sequencing products were resolved by capillary electrophoresis using an ABI 3730X automated sequencer at BMR Genomics (University of Padua, Italy). The sequences were edited with SEQUENCHER 4.1.2 (Gene Codes). The alignment obtained using CLUSTAL X (Thompson et al. 1997) was checked by eye. The GenBank accession numbers for the four sequences (1113 bases long) generated for the purposes of this study are KC816547-KC816550. The four sequences were included in a larger database which comprised 150 sequences used in Gentile et al. 2009 (FJ536145FJ536154 for Amblyrhynchus and FJ535863FJ536003 for Conolophus) and 620 Conolophus sequences whose accession numbers will be published in a separate paper. The resulting dataset consisted of sequences of the four conscated iguanas; 262 individuals from Isabela; 101 from Fernandina; 115 from Santa Cruz; 47 from Baltra; 55 from Plaza Sur; 78 from Santa Fe (C. pallidus); 102 from Volcan Wolf (C. marthae); and 10 of marine iguana (A. cristatus). Phylogenetic methods were applied to address the molecular taxonomic identication of the four conscated iguanas. A maximum likelihood (ML) approach was followed: ML trees were sought via a genetic algorithm, as implemented in Treender ver. 10/2008 (Jobb et al. 2004). The model proposer in Treender selected separate models for the rst, second and third positions. For each model, parameters were optimized during the run. The models and estimated parameters were the HKY (Hasegawa et al. 1985) ?C (Ti/Tv = 11.227; a = 0.169), the HKY ? I (Ti/Tv = 14.091; h = 0.899), and the TN ? C (Tamura and Nei 1993) (TAG = 0.753; TCT = 0.207, Tv = 0.001; a = 4.575) for rst, second, and third positions, respectively. Nodal support was tested by bootstrapping (Felsenstein 1985), with 1,000 pseudoreplicates. A. cristatus, the sister taxon of Conolophus (Rassmann 1997;

Rassmann et al. 2004; Wiens and Hollingsworth 2000) was used as an outgroup within the Iguaninae group.

Results The analysis of the Cytb sequences from iguanas A, B, C, and D returned two haplotypes, here labeled 35 and 36. Haplotype 35 was shown by iguanas A, B, and D, while haplotype 36 was found in iguana C. Such haplotypes are different from each other by three substitutions and are not new, as they were already present in the database used in Gentile et al. (2009). In fact, haplotype 35 corresponds to GenBank records FJ535863, FJ535864, FJ535865, FJ535867, FJ535868, FJ535890, and FJ535891. Haplotypes 36 corresponds to records FJ535866, FJ535910, FJ535911, and FJ535912. As in Gentile et al. (2009), the present phylogenetic analysis (Fig. 2) strongly supported the monophyly of Conolophus (bootstrap value = 96). As expected, based on Gentile et al. (2009), all conscated iguanas clustered within the monophyletic clade of C. subcristatus from the central islands. Such a clade was strongly supported by bootstrap (bootstrap value = 96). In particular, haplotype 35 was found only among the individuals from Baltra while haplotype 36 was found only among those from Baltra and Seymour Norte.

Discussion Mitochondrial DNA can be effective for the identication of species. However, and as stated by Alacs et al. (2010): When the appropriate gene region is chosen based on its rate of mutation, the levels of genetic diversity within and among species need to be sufciently characterized before the gene can be applied to species identication of wildlife for forensic applications. In this regard, previous studies (Rassmann 1997; Rassmann et al. 1997a, b; 2004; Hanley and Caccone 2005; Gentile et al. 2009) have conrmed that the data on the sequence of the mitochondrial gene for the Cytb are especially suitable for (1) unequivocally distinguishing the sister taxa Amblyrhynchus and Conolophus from the rest of genera included in the family Iguanidae; (2) unequivocally distinguishing Amblyrhynchus and Conolophus from each other; and (3) resolving genetic relationships within the genus Conolophus. Additionally, those studies also demonstrated that some potential limitations in the use of mitochondrial DNA sequence data, related to the maternal mode of inheritance, paternal leakage (resulting in the coexistence of two or more different mtDNA genomes in the organism), the possible occurrence of nuclear pseudogenes of mtDNA, do not apply in the case of Amblyrhynchus and Conolophus or else are negligible.

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Conservation Genet Resour Fig. 2 Phylogenetic analysis: ML tree. The numbers indicate the nodal support by bootstrap

It might be argued that the use of the sole mtDNA could be insufcient in addressing possible hybridization between marine and land iguanas, as maternal inheritance might hide the genetic contribution made by a heterospecic iguana parent. However, this is not the case as hybridization only occurs in Plaza Sur. Here, given the small size of the island, marine and land iguanas undergo forced proximity. Rare hybrid mating (always between male marine iguanas with female land iguanas) can occur as the breeding seasons of the two species partly overlap on this island. Rassmann et al. (1997b) observed only F1 (rst generation) hybrids and concluded that F1 hybrids are sterile. Finally, the morphology of F1 hybrids is very distinctive and distinct from both of the parental (A. cristatus and C. subcristatus) species. Results in Gentile et al. 2009 were the baseline of the phylogenetic expectation of the present work. That paper addressed the molecular phylogeny of the four species of iguanas. Indeed, C. marthae was described and named in a subsequent paper by Gentile and Snell (2009), but it had already been acknowledged as a good species in Tzika et al. (2008) and Gentile et al. (2009). Several works (Rassmann 1997; Rassmann et al. 2004; Gentile et al. 2009) recognized Conolophus as a monophyletic clade. Rassmann et al. (2004) produced genetic data supporting the existence of two monophyletic clades within C. subcristatus. Gentile et al. (2009) provided further evidence in this respect. These two clades group the populations inhabiting the western (Isabela and Fernandina) and central islands (Seymour Norte, Baltra, Plaza Sur, Santa Cruz and its associated islet Venecia), respectively. The existence of these two separate and divergent clades has been discussed as a ground for taxonomic rearrangement, within the genus Conolophus, in Snell et al. (1984), Rassmann et al. (2004) and in the meeting of the IUCN Iguana Specialist Group held in Antigua Guatemala, in November 2011. It is clear

that morphological and genetic data strongly support the elevation of the two clades to species rank. Unsurprisingly, the Cytb sequence data clearly identied the four conscated iguanas as belonging to the genus Conolophus. This is obviously in agreement with the morphological traits of the four iguanas. Species identication was also possible. In fact, the two haplotypes 35 and 36were already known and exclusive to the C. subcristatus gene pool (Gentile et al. 2009). As expected, all those iguanas carrying haplotypes 35 and 36, including iguanas A, B, C, and D, clustered within the C. subcristatus central islands clade. The phylogenetic analysis conrmed the deep divergence between the western and central islands clades, which did not share mtDNA haplotypes. Although the haplotypes shown by the four iguanas were found only in Baltra and Seymour Norte, it might be argued that such haplotypes could exist in the C. subcristatus populations from other islands (especially in those within the central islands clade), but they were missed because of their low frequency in those populations. It is instructive to estimate the probability that the four conscated iguanas might all be collected together in an island other than Baltra (or Seymour Norte), or separately, with each iguana on a different island. This can be done beginning with the estimation of the frequency of the unseen haplotypes. There are several different methods for the estimation of unseen haplotypes (see Egeland and Salas 2008, for a recent review). Here we focused upon the estimation of the highest possible frequency given a certain sample size. This approachin this caseis more conservative than attempting to estimate the possible true frequency. Following such a conservative approach, the frequencies of 35 and 36 can be estimated by using the smallest sample size available (in order to maximize the possible estimated frequency of each haplotype). This rationale can then be easily applied to other populations.

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The smallest sample size used for the genetic characterization of the Conolophus populations of each island was 55 (Plaza Sur, see above). Thus, if both haplotypes 35 and 36 were present in the Plaza Sur population but either were missed by random sampling, the highest frequency (pi) at which they could occur in the population is: pi  xi u=n u; u[0

where the haplotype is observed xi times in a sample of size n and u = 1 corresponds to the addition of the unseen haplotype (u) to the sample. As noted by Egeland and Salas (2008), such an approach tends to overestimate the true frequency for rare haplotypes, since it corresponds to the case where the unseen haplotype appears as the next sample. However, and as said above, this is conservative for our purposes. In fact, given the sample size, p35 B 3/59 and p36 B 1/59, that is p35 B 0.05 and p36 B 0.02. In terms of genetic samplingwhich is how the haplotypes of the four iguanas were drawn from the gene pool of the source population(s)the four iguanas were collected randomly, as no a priori individual genetic information was available to the collector. Thus, the highest probability (p) of randomly collecting four iguanas, three carrying haplotype 35 and one carrying haplotype 36, is approximately:
6 p  p3 35 p36  2:5 10

conscated iguanas. Such a rapid assessment was possible as a large genetic database was already available for pagos iguanas. It is recommended that genetic proGala grams be developed for the complete genetic characteriza pagos wildlife. Similar programs should be tion of Gala preferred in other areas of endemism worldwide. Finally, the Cytb sequence data seems particularly appropriate for the bar-coding purposes of iguanas. The suitability of Cytb for the purposes of the barcode programs of other reptiles should be thoroughly tested.
Acknowledgments We thank the editor and two anonymous reviewers for their valuable criticism. We gratefully thank the Mohamed bin Zayed Species Conservation Fund for nancial support pagos to GG. We acknowledge and thank the authorities of the Gala National Park Service for their continuous effort in preventing the pagos. illegal trade of wildlife in Gala

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This value indicates the highest probability that all four conscated iguanas were collected in Plaza Sur, admitting the existence of haplotypes 35 and 36 in that island. The p value could be smaller in case it is hypothesized that the four iguanas were collected together in Santa Cruz island (central clade). A similar rationale applies if the four iguanas were collected from separate populations (both cases imply lower haplotype 35 and 36 frequencies). The probability (p) that the four iguanas were collected in an island of the western clade (Fernandina and/or Isabela) would be even smaller, if any. On the whole, the order of magnitude of the p values here reported can be considered to be negligible and it can be safely stated that the four conscated C. subcristatus iguanas were collected in Baltra or Seymour Norte. In conclusion, the phylogenetic analysis of the Cytb sequence data contributed to the unequivocal molecular taxonomic identication of the four conscated iguanas, which belonged to the species C. subcristatus. The genetic data unambiguously indicated that the four conscated iguanas had been subtracted from the same population, currently distributed in Baltra and Seymour Norte. In fact, the land iguanas from Baltra and Seymour Norte share the same very recent origin and the same gene pool. The use of molecular tools proved crucial for the rapid assessment of the original population of the four

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