First Assessment of Mitochondrial DNA Diversity in the Endangered Nile Softshell Turtle, Trionyx triunguis, in the Mediterranean
Abstract
We assessed mitochondrial DNA diversity in Trionyx triunguis from the Mediterranean basin (22 samples) and continental Africa (4 samples). The continental African group comprised 4 different and newly described haplotypes, while the Mediterranean group consisted of only 1 previously known haplotype, with the nucleotide divergence between the 2 groups being 1.5% ± 0.7%.
Trionyx triunguis is the only species of Trionyx, the genus from which the Linnaean family name Trionychidae is formed. Molecular studies show that T. triunguis is most closely related to other “giant” softshells that persist in eastern Asia (Engstrom et al. 2004). Giant softshells are all endangered (Praschag and Gemel 2002; Moll and Moll 2004) because they require large rivers and estuaries, fragile habitats that also happen to be heavily occupied and fished by humans.
Trionyx triunguis is heavily exploited because of its meat, and its habitats have been degraded by pollution and dam construction. Accidental capture and intentional killing also affect the survival of most populations (Gramentz 2005; Türkozan 2007). In fact, T. triunguis has been eradicated from the Nile Delta as well as the rest of Egypt (Schleich et al. 1996; Nada 2002; Baha el Din 2006). The Mediterranean population of T. triunguis has been listed by IUCN as critically endangered (European Reptile and Amphibian Specialist Group 1996) (category CR C2A) and estimated at fewer than 1000 adults (Kasparek 2001; Venizelos and Kasparek 2006). Around the eastern Mediterranean, this species persists in Egypt, Israel, Lebanon, Syria, and especially Turkey, where the largest populations are found. Important nesting populations have been recorded along the Mediterranean coast between Dalyan and Samandağ (Atatür 1979; Gramentz 1993; Kasparek 1994; Türkozan 2009).
Studies on T. triunguis are very limited and restricted mainly to distribution, ecology, ethology, and reproductive ecology studies (Leshem and D'miel 1986; Kasparek and Kinzelbach 1991; Gramentz 1993, 1994; van der Winden et al. 1994; Gidiş and Kaska 2004; Türkozan et al. 2006). However, the need for improved knowledge of the conservation biology of the declining populations prompted us to initiate applied studies that can help guide the management and survival of the species. The aim of this study is to provide a preliminary assessment of the genetic variation in mitochondrial DNA (mtDNA) of T. triunguis in order to guide conservation strategies.
Methods
Tissue samples were collected either from dead hatchlings or live adults between 2005 and 2008. A total of 26 samples were studied from 6 different populations (Fig. 1). Of these sampling localities, 4 were from Turkey, namely Dalyan (DL) (3 specimens), Dalaman (DM) (6 specimens), Anamur (AN) (4 specimens), and Kazanlı (KZ) (3 specimens). Furthermore, we collected tissues from Israel (IS-Alexander river) (6 specimens) and were provided tissue samples from adult captive animals that apparently originated from the sub-Saharan African continent (AF) (4 specimens). The precise localities of the African samples were unknown; however, the partial cytochorome b (cyt b) gene sequences of museum materials (Paris Natural History Museum, France) originating from Gabon and Congo confirmed that our African samples were apparently from sub-Saharan Africa rather than the Nile drainage. We did not use these Gabon and Congo specimens in our study because of problems with degraded DNA; we were not able to obtain whole gene sequences for these samples as we did for the rest of our specimens.
Citation: Chelonian Conservation and Biology 8, 2; 10.2744/CCB-0792.1
We used partial sequences of both cyt b and NAD 4 (also commonly abbreviated ND4) genes of mtDNA that have been used in other phylogenetic studies of trionychids (Weisrock and Janzen 2000; Engstrom et al. 2004). These relatively rapidly evolving markers have been used in order to determine the genetic diversity within and among populations.
Genomic DNA was extracted by standard phenol/chloroform techniques (Sambrook and Russell 2001) using a commercial DNA extraction kit (Invitrogen Inc.). Two mitochondrial genes (ND4 and cyt b) were amplified via the polymerase chain reaction (PCR) using the following primers: ND4—ND4 4672 (F) (5′-TGACTACCAAAAGCTCATGTAGAAGC-3′) (Engstrom et al. 2002), Hist (R) (5′-CCTATTTTTAGAGCCACAGTCTAATG -3′) (Arevalo et al. 1994), and cyt b—DW 2000 (F) (5′-ACAGGCGTAATCCTACTAA-3′) (Weisrock and Janzen 2000), DW 1594 (R) (5′-TCATCTTCGGTTTACAAGAC-3′) (Shaffer et al. 1997). PCR amplifications were performed in 50-µL volumes containing 1X KCl PCR buffer (Fermantas), 1.5 mM MgCl2 (Fermantas), 2.5 mM each dNTP, 0.5 µM each primer (1 µM for cyt b), 1.0 units of Taq polymerase (Fermantas), and 1–2 µL (50 ng DNA) of template DNA. Amplicons were purified using the PCR Purification Kit (Invitrogen). Amplicons were analyzed on an AB3700 or 3730xl automatic sequencer (Macrogen, Applied Biosystems) using the same primers mentioned previously.
Sequence analyses were aligned using BioEdit 7.0.9 (Hall 1999). Multiple-sequence alignments were done with CLUSTALW (Thompson et al. 1994) using the default parameters. The computer-generated alignment was further adjusted manually. Genealogical relationships among haplotypes were constructed using TCS (Clement et al. 2000), with statistical parsimony algorithm described by Templeton et al. (1992) to estimate the number of differences among haplotypes as a result of a single substitution with a 90% statistical confidence as the parsimony connection limit and also the most probable ancestral haplotype. For this analysis, combined mtDNA cyt b and ND4 genes were used to construct an unrooted parsimony network.
We estimated net nucleotide divergence (Da) and standard deviations between phylogroups (Nei 1987) with the Jukes–Cantor correction (Jukes and Cantor 1969) in the Dnasp program (Rozas et al. 2003).
Results
The cyt b and ND4 fragments amplified in 26 T. triunguis samples had nucleotide lengths of 805 and 732 base pairs, respectively. Five mitochondrial DNA haplotypes were found, 4 of which are reported here for the first time (TT-A2-GU003980 for ND4, GU003977 for cyt b; TT-A3-GU003981 for ND4, GU003978 for cyt b; TT-A4-GU003981 for ND4, GU003979 for cyt b; TT-A5-GU003982 for ND4, GU003979 for cyt b), while haplotype TT-A1 was identical with one previously reported by Engstrom et al. (2004). The identified new haplotypes have been deposited in GenBank. Nucleotide and haplotype diversity was 0.004 and 0.29, respectively. Twenty-seven polymorphic sites were detected, consisting of 25 transitions and 2 transversions (Table 1). According to the network analysis (Fig. 2) of 5 haplotypes (Posada and Crandall 2001), 2 highly divergent groups of haplotypes were supported. Of these, the African group (AF) comprised 4 different and newly described haplotypes, while the Mediterranean group (DL, DM, AN, KZ, IS) consisted of only 1 haplotype, TT-A1. Apparently, haplotype TT-A1 is fixed to the Mediterranean region. Haplotype TT-A3 was identified as ancestral based on root probability density criterion (Templeton 1998). The nucleotide divergence between populations from the Mediterranean basin and continental Africa was 1.5% ± 0.7%.
Citation: Chelonian Conservation and Biology 8, 2; 10.2744/CCB-0792.1
Discussion
Our study represents the first assessment of genetic variation within T. triunguis. The existence of a single haplotype in the Mediterranean likely reflects migration among river systems through the sea, with gene flow among the populations studied. In fact, there are many records of T. triunguis being found well out to sea from the coasts of Africa and Turkey, in the Aegean and Mediterranean (Pritchard 1979; Taşkavak et al. 1999; Oruç 2001; Taşkavak and Akçınar 2008). The apparent interconnectedness of Turkish populations of T. triunguis may benefit regional conservation efforts since the translocation of individuals in headstarting or other release efforts may not suffer from inadvertent genetic pollution. Nevertheless, future studies using rapidly evolving markers from the nuclear genome (microsatellites) will be necessary to clarify this pattern. Maternally inherited markers showed strong population structure, suggesting isolation between Africa and the Mediterranean. This differentiation, as well as that among African populations, may reflect nest site fidelity, as is seen in various species of sea turtles (Bowen and Karl 1996).
In sea turtles, females show strong philopatry (as evidenced by mtDNA), but this is not reflected in bisexually inherited nuclear markers. Additional studies using nuclear markers as well as known-provenance specimens from the sub-Saharan African continent are sorely needed. The recognition of distinct mitochondrial haplotypes from African specimens suggests that, with known locality material, it may be possible to genetically identify the provenance of trade specimens.
In conclusion, all Mediterranean individuals of T. triunguis belong to same population, and these specimens are clearly different from those of “continental Africa” of unknown origin. Considering the widespread distribution of T. triunguis in large parts of Africa and the biogeographical circumstances there, the species may have more isolated populations, some possibly more isolated than the Mediterranean sample presented in this study, such as the Lake Turkana population.
Map of Mediterranean basin showing sampling sites, mtDNA haplotypes, and observed number of haplotypes.
Genealogical relationships among 5 haplotypes of T. triunguis estimated by TCS (Clement et al. 2000). Small, white circles represent hypothetical haplotypes not found in the sample.
