Conserv Genet (2008) 9:709713 DOI 10.

1007/s10592-007-9395-y

SHORT COMMUNICATION

Morphological and genetic variation in the endangered Sulawesi tortoise Indotestudo forstenii: evidence of distinct lineages?
Ian Ives Phillip Quinton Spinks Howard Bradley Shaffer

Received: 21 March 2007 / Accepted: 13 July 2007 / Published online: 7 August 2007 Springer Science+Business Media B.V. 2007

Abstract Limited eldwork and anecdotal evidence indicate that allopatric populations of the endangered Sulawesi tortoise (Indotestudo forstenii) differ in size and presence/absence of a nuchal scute, suggesting that these may constitute separate evolutionary lineages. We examined morphological and genetic variation to determine if the nuchal scute is correlated with size or genetic divergence. Our results indicate a strong correlation between size and nuchal scute such that turtles lacking a nuchal scute were larger than those possessing a nuchal scute. However, we found no correlation between genetic divergence and presence/absence of nuchal scutes, and thus no evidence of genetically differentiated lineages. Keywords Indotestudo Tortoise Nuclear introns Nuchal scute Asian turtle crisis

Introduction The ~316 species comprising the worlds turtle and tortoise fauna (TTWG 2007) is arguably the most endangered major clade of vertebrates on earth. According to the IUCN World Conservation Union (http://www.redlist.org/ 28 December 2006 download) 137 of the 205 species that have been evaluated are in the highest categories of endangerment
I. Ives Massachusetts Audubon Society, P.O. Box235, Cummaquid, MA 02637, USA P. Q. Spinks (&) H. B. Shaffer Section of Evolution and Ecology and Center for Population Biology, University of California, One Shields Ave., Davis, CA 95616, USA e-mail: pqspinks@ucdavis.edu

(Extinct, Extinct in the wild, Critically Endangered, Endangered, Vulnerable), implying that between 67% (137/ 205) and 43% (137/316) of the worlds living species of chelonians are conservation concerns. This is a greater proportion than for any other major vertebrate clade (birds: 12%, mammals: 23%, amphibians: 32.5%, all based on 2004 tallies in Stuart 2004). The most diverse, and most threatened turtle clades occur across Asia, where virtually all species are heavily harvested for the food and traditional medicinal trades (van Dijk et al. 2000). The Sulawesi tortoise, Indotestudo forstenii, is endemic to Sulawesi and the neighboring island of Halmahera. It is considered to be Endangered (EN A-1) by the IUCN based on a drastic reduction in population size in the last decade; it is also listed on Appendix II of CITES http://www.cites.org/. Empirical eld data, and anecdotal evidence, suggests that I. forstenii populations are patchily distributed throughout the western part of both Northern and Central Sulawesi (Hoogmoed and Crumly 1984; Platt et al. 2001; Samedi and Iskandar 2000), where it is legally harvested for the food trade. A long-standing debate with this species has centered on the possible existence of a cryptic species within forstenii, based on discrete variation in the presence or absence of the nuchal (=cervical) scute on the carapace (Fig. 1). In many other groups of turtles, the condition of the nuchal is an important taxonomic character; for example, it is one of the key features differentiating the Austral/Papuan genus Emydura (nuchal scute present) from its close relative Elseya (nuchal scute absent) (Ernst and Barbour 1989). Indotestudo forstenii individuals with and without a nuchal are well known, and limited eld data suggest that tortoises with the two conditions are geographically allopatric and thus may constitute distinct populations or species (Ives 2006). However, preliminary analysis (Iverson et al. 2001) based on a single specimen of

123

710 Fig. 1 Maximum likelihood tree based on 1113 bp of cytb. Estimated model parameters conform to the GTR + G + I model of nucleotide sequence evolution. ln L = 2638.27630, rate matrix: AC = 1.5482, A G = 8.5255, AT = 1.1435, C G = 0.0001, CT = 13.3830, and GT = 1. Base frequencies: A = 0.31, C = 0.31, G = 0.12, and T = 0.26. Proportion of invariable sites (I) = 0.4563, and G-shape parameter = 0.8186. Numbers above branches are MP bootstrap support values. Terminals are species followed by sample ID (* denotes sequences downloaded from GenBank), and GenBank accession # for the cytb sequences. (+) and () indicate turtles with and without a nuchal scute, respectively. Upper photograph shows a tortoise with a nuchal scute while bottom photograph shows a tortoise that lacks the nuchal scute. GenBank accession numbers for the nuDNA sequences are EF491694 EF491711. Outgroup branch length not drawn to scale

Conserv Genet (2008) 9:709713

each nuchal type suggested that there is little genetic divergence between them. Given the importance of correctly determining species limits for effective conservation and the imperiled status of the species, we collected morphometric measurements and generated mitochondrial and nuclear DNA sequence data from I. forstenii with and without nuchal scutes to determine whether independent genetic data supports the interpretation that these phenotypically distinct morphotypes represent separate lineages requiring independent conservation efforts. Given the precarious conservation status of I. forstenii, we felt that it was important to bring what evidence we can to bear on this problem.

Methods Determinations from the eld Evidence suggesting phenotypic variability in wild populations of I. forstenii was derived from interviews

conducted by the senior author in the Palu Valley of Central Sulawesi in February 2005. Local turtle hunters and a turtle dealer in the Palu Valley indicated that individuals captured in the north on the Minahasa Peninsula do not have nuchal scutes, while individuals captured in the south in the Palu Valley and other neighboring valleys have a nuchal scute (Fig. 1), and museum specimens collected on the Minahasa Peninsula in 1872, 1896, and 1999 lack nuchal scutes (Table 1) indicating that presence/absence of the nuchal scute might be geographically correlated. We assessed whether turtles with the two nuchal types differed in size by comparing means of straight-line, midcarapace length (MCL), (including the nuchal scute if present) and mass for I. forstenii of both nuchal types. Analyses were performed by sex (males, n = 39; females, n = 32), with the JMP-IN 5.1 statistical software (Sall et al. 2005), using either a t-test or a Wilcoxon signed-ranks test. Of the 39 males, 25 were from a commercial holding facility in Palu and 14 were from a large captive assurance colony located at the Turtle Bank, a non-prot conservation facility in Massachusetts, USA. Of the 32 females, nine

123

Conserv Genet (2008) 9:709713

711

Table 1 Localities for Indotestudo forstenii with and without a nuchal scute, suggesting two distinct populations (northern and southern) with distinguishing phenotypes Museum number Museum specimens R-145099a R-145100a R-145101a 1872.4.6.116 1896.12.9.1b USNM 52973c,d Location In-situ specimense Kulawi, Sulawesi Lempelero, Sulawesi
a b b

Date

Location

Nuchal present?

1999 1999 1999 1872 1896 1915

Cape Santigi, Sulawesi Cape Santigi, Sulawesi Cape Santigi, Sulawesi Boliahoeta, Celebes Buol, North Celebes Boliahoeta, Celebes Coordinates 0126S, 11959E 0139S, 12002E
c

No No No No No did not examine Nuchal scute (Y or N) Yes Yes

Museum specimens were collected on the Minahasa Peninsula, and in situ specimens were observed in the Kulawi Valley by the senior author American Museum of Natural History, NY; DC; The Natural History Museum, London; National Museum of Natural History, Washington,

d e

Specimen on loan and unavailable for examination Coordinates for in situ specimens represent second hand reports

were from the holding facility in Palu and 23 from the Turtle Bank colony. Genetic samples Turtles used for genetic analysis were members of the Turtle Bank colony. These individuals were wild caught and brought to the United States, either through conscation by US. customs ofcials or through legal purchases between 1997 and 2001. Twelve adult I. forstenii including six with nuchal scutes and six without were sampled by drawing one ml of whole blood from the subcarapacial vein of each turtle (Hernandez-Divers et al. 2002). Blood was stored in lysis buffer and shipped to the University of California at Davis for genetic analysis. Genetic analysis We examined cytochrome b (cytb) mitochondrial DNA (mtDNA) for 12 wild-caught captive I. forstenii, including six individuals with and six without the nuchal scute. We also sequenced three nuclear loci from six of these individuals (three with, three without the nuchal scute). Our nuclear DNA (nuDNA) sequence data included R35 (Fujita et al. 2004), plus TB29 and TB73 (Thomson, unpublished). DNA extraction, and PCR protocols for cytb and R35 follow Spinks et al. (2004). The TB29 and TB73 primers/ PCR protocol are described in Shaffer and Thomson (submitted). Sequences were generated at the UC Davis Division of Biological Sciences DNA sequencing facility http://www.dnaseq.ucdavis.edu/. All fragments were

sequenced in both directions, and aligned within individual turtles. To check for the presence of nuclear mitochondrial pseudogenes (numts), the cytb sequences were converted into amino acid sequences using MacClade 4.06 (Maddison and Maddison 2003). PAUP* v4.0b10 (Swofford 2002) was used to calculate pairwise uncorrected P genetic distances and maximum parsimony (MP) and maximum likelihood (ML) phylogenetic analyses (ten random stepwise heuristic searches using TBR branch swapping). Model parameters for ML analyses were estimated using Modeltest 3.06 (Posada and Crandall 1998) and selected under the Akiake Information Criterion (AIC). We bootstrapped each data set with 100 pseudoreplicates (Felsenstein 1985), but to decrease computation time the ML bootstrap searches were constrained to 1 h per pseudoreplicate.

Results Field and morphological data Five of the six known I. forstenii museum specimens with locality data were examined for presence or absence of a nuchal scute (Table 1). Each specimen was reportedly captured on the Minahasa Peninsula, and all lacked the nuchal scute. One eld survey reported a population of I. forstenii outside the Minahasa Peninsula (Groombridge 1982), but no locality data were available. In addition, the senior author observed 23 I. forstenii with nuchal scutes and 19 individuals without nuchal scutes while visiting a

123

712

Conserv Genet (2008) 9:709713

commercial turtle holding facility in Palu. The owner of the facility, a long time collector of I. forstenii in the region, stated that only individuals south of Palu have a nuchal scute. Locality data could be conrmed for two of the ve individuals encountered in the Palu and Kulawi Valleys (Table 1). Mean comparisons of nuchal types revealed signicant differences for both mass and MCL in both sexes (Table 2). Turtles lacking nuchal scutes had roughly 10% greater MCLs (P = 0.003, males; P = 0.018, females) and were 25% (females, P = 0.024) to 45% (males, P = 0.003) heavier than those with nuchal scutes. Genetic analysis Our mtDNA sequence data set was composed of up to 1113 (bp) of cytb data for 20 tortoises, including 12 sequenced for this analysis plus downloaded tortoise cytb sequences from GenBank for a single Manouria emys (outgroup), and seven additional Indotestudo sequences; two I. forstenii, two I. travancorica, and three I. elongata. Our nuDNA data set consisted of 989 bp of R35, 578 bp of TB29, and 679 bp of TB73 for six tortoises. Within cytb we recovered strong support for the reciprocal monophyly of all Indotestudo species, but very low levels of sequence variation within I. forstenii. Maximum uncorrected P sequence divergence among I. forstenii was 1.3%, with 15 variable and four parsimony-informative positions. Maximum likelihood analysis recovered 19 equally likely trees with log likelihood (lnL) score = 2638.27630. We found no correlation between cytb sequence divergence and presence or absence of the nuchal scute. For example, based on cytb, tortoises I1 and I8, which lack nuchal scutes, were identical to tortoises I5 and I16 which have the nuchal scute (Fig. 1). Analyses of up to 2,246 nucleotides from the three nuclear loci revealed an almost complete lack of variation. Across the three loci, all turtles were identical except for one heterozygote for a single nucleotide polymorphism in TB73 from sample I9.

Discussion The results from both mtDNA and nuDNA sequence data revealed that individual I. forstenii with and without the nuchal scute had nearly identical cytb haplotypes, with the average cytb uncorrected P sequence divergence among I. forstenii being 0.17%. This is consistent with previous, more limited mtDNA data (Iverson et al. 2001). In addition, the nuDNA sequences were virtually identical across all six of the specimens sequenced. This extremely low level of sequence divergence, coupled with the observation that turtles with and without nuchal scutes did not form exclusive groups strongly suggests that presence or absence of the nuchal scute is not correlated with genetic divergence. Thus, even though many endemic Sulawesi taxa show range fragmentation and intra-island diversication (Evans et al. 2003), we found no such evidence among the I. forstenii individuals examined. Although the four genes examined show a lack of differentiation among nuchal types, our limited morphometric results indicate that two similar, but morphologically distinct forms of I. forstenii exist (Table 2). Whether these differences represent recently evolved populations or environmentally induced variation remains an open question. The Minahasa Peninsula and the Palu Valley have similar temperatures but marked differences in rainfall. The hills adjacent to the Palu Valley, where the senior author documented that the species occurs, receive <2,000 mM of rain annually, while many of the localities on the Minahasa Peninsula where the species has been recorded receive >2,500 mM (Whitten 1987). Limited resources as a result of a dryer climate could result in reduced growth rates and smaller body size of tortoises in and around the Palu Valley. This effect has been hypothesized for another tortoise species, Testudo weissingeri, which was originally recognized as a dwarf population of T. marginata-like animals restricted to the Manai Peninsula of Greece, but was elevated to species based on subtle morphological variation (Bour 2001, cited in Fritz et al.

Table 2 Comparison of carapace length and mass measurements between I. forstenii with and without nuchal scutes Measurement Carapace length Carapace length Mass Mass Sex M F M F Nuchal scute absenta 214.6, 27.0 (164.6260.8) n = 19 208.3, 12.8 (189.7227.3) n = 17 1362.6, 449.3 (6822,250) n = 19 1253.05, 197.2 (9801,650) n = 17 Nuchal scute presenta 190.1, 21.6 (145.2235.2) n = 20 186.5, 33.4 (115.4223.2) n = 15 942.2, 229.9 (6001,374) n = 20 1003.9, 378.3 (3001,615) n = 15 tb 3.14 2.489 2.593 (Z test) 2.376 P 0.003 0.018 0.003 0.024

The total sample of 71 turtles was comprised of individuals from a commercial holding facility in Palu, Sulawesi and an assurance colony in the US
a b

Mean, standard deviation (minimummaximum), sample size t-Test or Wilcoxon rank-sum test

123

Conserv Genet (2008) 9:709713

713 Hernandez-Divers SM, Hernandez-Divers SJ, Wyneken J (2002) Angiographic, anatomic and clinical technique descriptions of a subcarapacial venipuncture site for chelonians. J Herpetol Med Surg 12:3237 Hoogmoed MS, Crumly CR (1984) Land tortoise types in the Rijksmuseum van Natuurlijke Historie with comments on nomenclature and systematics (Reptilia: Testudines: Testudinidae). Zool Med Leiden 58:341259 Iverson JB, Spinks PQ, Shaffer HB, McCord WP, Das I (2001) Phylogenetic relationships among the Asian tortoises of the genus Indotestudo (Reptilia: Testudines: Testudinidae). Hamadryad 26:272275 Ives I (2006) Conservation of Sulawesis two endemic chelonians, Leucocephalon yuwonoi and Indotestudo forstenii; a preliminary investigation into in-situ and ex-situ conservation concerns. Thesis, Antioch University New England Maddison DR, Maddison WP (2003) MacClade 4: Analysis of phylogeny and character evolution, version 4.06. Sinauer, Sunderland, Massachusetts Platt SG, Lee RJ, Klemens MW (2001) Notes on the distribution, life history, and exploitation of turtles in Sulawesi, Indonesia, with emphasis on Indotestudo forstenii and Leucocephalon yuwonoi. Chelonian Conserv Biol 4:154159 Posada D, Crandall KA (1998) Modeltest: testing the model of DNA substitution. Bioinformatics 14:817818 Sall J, Creighton L, Lehman A (2005) Jump start statistics. 3rd edn. SAS Institute Inc. Thomson Brooks Cole Samedi ML, Iskandar DT (2000) Freshwater Turtle and Tortoise Conservation Utilization in Indonesia. In: van Dijk, Stuart and Rhodin (eds), Asian Turtle Trade: Proceedings of a Workshop on Conservation and Trade in Freshwater Turtles and Tortoises in Asia, Chelonian Research Monographs, Number 2, pp 106111 Spinks PQ, Shaffer HB, Iverson JB, McCord WP (2004) Phylogenetic hypotheses for the turtle family Geoemydidae. Mol Phylogenet Evol 32:164182 Stuart SN (2004) Status and trends of amphibian declines and extinctions worldwide. Science 306(5702):17831786 Turtle Taxonomy Working Group (in press) An annotated list of modern turtle terminal taxa (with comments on areas of instability and recent change). Chelonian research monographs 4 Swofford DL (2002) PAUP*. Phylogenetic analysis using parsimony (*and other methods). Version 4. Sinauer Associates, Sunderland, Massachusetts van Dijk PP, Stuart BL, Rhodin AGJ (Eds) (2000) Asian turtle trade proceedings of a workshop on conservation and trade of freshwater turtles and tortoises in Asia Phnom Penh, Cambodia, 14 December 1999. Chelonian Research Monographs 2. Chelonian Research Foundation, Lunenberg, MA Whitten AJ, Mustafa M, Henderson GS (1987) The Ecology of Sulawesi. Gadja University Press, Yogyakarta, Indonesia

2005). However, based on mitochondrial and nuclear DNA data, Fritz et al. (2005) rejected weissingeri as a distinct evolutionary lineage, and suggested that the reduced size of T. weissingeri was a result of suboptimal environmental conditions (Bringse et al. 2001, cited in Fritz et al. 2005). Although we cannot say with certainty, we similarly interpret interpopulation variation associated with nuchal variation in I. forstenii as either representing environmentally induced variation, or possibly very shallowly diverged populations resulting from recent population divergence. In summary, the phenotypic variation of I. forstenii, while statistically signicant, does not appear to represent diverged genetic lineages. It may represent shallowly diverged genetic differentiation, environmentally induced phenotypic variation, or both. Due to the continued harvest of this species from the wild and the urgent need for its protection, we feel that immediate conservation attention should be placed on the species as a whole, rather than for each nuchal morphotype. A concerted effort by Indonesian and international organizations to protect and effectively manage the remaining hotspots for biological diversity within Sulawesis West Central and Northwest areas of endemism, would be an important starting point.

References
Ernst CH, Barbour RW (1989) Turtles of the world. Smithsonian Press, Washington Evans BJ, Supriatna J, Andayani N, Setiadi MI, Cannatella DC, Melnick DJ (2003) Monkeys and toads dene areas of endemism on Sulawesi. Evolution 57:14361443 Felsenstein J (1985) Condence limits on phylogenies: an approach using the bootstrap. Evolution 39:783791 Fritz U, Siroky P, Kami H, Wink M (2005) Environmentally caused dwarsm or a valid speciesIs Testudo weissingeri Bour, 1996 a distinct evolutionary lineage? New evidence from mitochondrial and nuclear genomic markers. Mol Phylogenet Evol 37:389401 Fujita MF, Engstrom TN, Starkey DE, Shaffer HB (2004) Turtle phylogeny: insights from a novel nuclear intron. Mol Phylogenet Evol 31:10311040 Groombridge B (1982) The IUCN Amphibia-Reptilia Red Data Book. Part 1. Testudines. Crocodylia, Rynchocephalia. IUCN Publ., Gland, Switzerland, 426 pp

123