Relationships among Tomistoma schlegelii in Malaysia Based on Cyt b-Control Region Gene Analysis

Tomistoma schlegelii is a slender snout crocodile, secretive in nature which is currently under Appendix I of Convention on International Trade in Endangered Species of Wild Flora and Fauna (CITES). Limited information is currently available on its wild population, especially in Malaysia. Thus this study aims to describe genetic relationship of T. schlegelii populations from Malaysia which was done using partial sequencing of Cytochrome b-control regionmtDNA gene.The study reveals that the genetic diversity among T. schlegelii is high, ranging from 0.16% to 3.34%, suggesting healthy populations. Analysis showed that there is gene flow among populations (Da = 1.71% to 2.21%) within Western Sarawak, Peninsular Malaysia, and other geographical regions coherent with Sundaland theory, suggesting that there is ancient river system connecting the two regions of Peninsular Malaysia and West Borneo when the Sunda Shelf was exposed. Unique haplotypes had been observed in Northern Sarawak (SAM01 and SAM02) as well as in Sumatera; thus each T. schlegelii deserved its own management strategies to ensure the survival of the species.


Introduction
Tomistoma, Tomistoma schlegelii, is a vulnerable slender snout crocodile, which is currently under Appendix I of Convention on International Trade in Endangered Species of Wild Flora and Fauna CITES (IUCN 2014) with an estimated population of less than 2500 individuals left in the wild [1].The population is endemic to Malaysia and Indonesia [1,2].Reports on T. schlegelii sightings in Kalimantan and Sumatera from the 1990s to 2000s were published by [2][3][4] while in Malaysia sightings have been rare since the late 1980s [5][6][7][8].
The main threat of T. schlegelii is habitat loss due to deforestation, illegal logging, palm oil plantation, urbanization, fishing activities, and forest fires at peat swamps [2][3][4].Currently, wild specimens could be found in captivity in zoos and farms and by individuals who acquired T. schlegelii illegally.Zoos and farms in Malaysia have approximately 77 specimens [9] while Singapore, Thailand, and Indonesia have collectively 88 specimens.Zoos and farms in Europe and the United States of America hold approximately 57 specimens.In Malaysia and Indonesia, study of T. schlegelii concentrated on surveys of T. schlegelii natural habitats and related issues involving conservation.
Genetic information on T. schlegelii is available; for example, [10][11][12] had sequencing Cyt b from T. schlegelii as part of the classification of Family Crocodylia.Besides that, [13] reported based on Cyt b gene that T. schlegelii haplotype in Peninsular Malaysia is unique.Thus, the aim of this study is to infer genetic relationship among T. schlegelii from captive and wild specimens using partial DNA sequencing of the mtDNA control region (Cyt b-CR) gene.The findings are useful for further support of conservation of T. schlegelii in Malaysia and other geographical regions.
Approximately, 50-100 ng of the template DNA was amplified in a 25 l reaction mixture containing 50 mM 10x buffer, 2 mM MgCl2, 0.4 mM of dNTPs (Promega), 0.2 mM of each primer, and 0.5 U of Taq DNA polymerase (Promega).The cycle parameters involved predenaturation step at 95 ∘ C for 5 min, followed by 35 cycles of denaturation at 95 ∘ C for 30 s, annealing at 47 ∘ C for 30 s, and extension at 72 ∘ C for 60 s with the final extension step at 72 ∘ C for 5 min.All PCR products were subjected to gel electrophoresis using 2% agarose gel stained with two drops of ethidium bromide with TAE buffer for 100 to 120 minutes at 80 V with 100 bp DNA ladder used to estimate the size of the bands.The gel was visualized under the UV light and photographed using UV camera.The PCR products were directly sent to First Base Sdn Bhd to be sequenced.
Multiple alignments of the Cyt b-CR gene sequences were constructed using the CLUSTAL X program v2.0 [16]; the stop codon sequence was removed and subsequently aligned by eye.The phylogenetic tree was constructed using the MEGA 4.0 software [17], besides PAUP version 4.0b [18] and Mr. Bayes version 3.1.2[19].This software was also used to calculate genetic divergence values based on Kimura 2 parameter approach.The measurement of population genetic parameters such as genetic diversity (the probability that two randomly chosen mtDNA sequences differed in the sample) and nucleotide diversity (per nucleotide site, i.e., the probability that two randomly chosen homologous nucleotides differ in the sample) [20] was estimated from the mtDNA dataset using DNASP 4.0 [21].To be noted, the samples from GenBank abbreviations followed the voucher numbers stated in [13].
International Journal of Zoology 3

Results and Discussion
A total 700 bp of mtDNA control region (Cyt b-CR) gene of T. schlegelii had been successfully amplified using L14930 primers for all specimens.The sequence matched T. schlegelii with accession number HM593977 [13].
The genetic distance values among individuals were 0.16% to 3.34% (Table 2).The highest genetic distance values had been recorded between sample Langkawi and Serian localities probably because of geographical distance, while samples from Miri and Langkawi have the lowest genetic distance which is 0.16% perhaps due to the same gene pool.The range value was large due to Cyt b-CR region having a high rate of evolution in mtDNA [13].
The analysis also revealed that high genetic distance values were recorded between T. schlegelii populations in which the highest variation of 3.34% was recorded in Langkawi specimen (Table 2).Langkawi population also shows the high genetic distances when compared to Samarahan population as 2.05% to 2.37% genetic distances were recorded among T. schlegelii.The result was different with the findings of [13] which revealed a low level of inter-and intrapopulation genetic distances of 0.08% to 0.18%, respectively.According to [23], the high genetic distance could be explained by several factors including small population size, pass bottleneck event, and physical barriers among population.The genetic variation and structure also increased due to geographical distance [24].Hence, the population genetic analysis is conducted to further understand the genetic structure of T. schlegelii from Malaysia and other regions.
Among the 68 individuals sequenced (12 from this study and 56 from GenBank) 7 haplotypes were identified in total with 3 haplotypes being shared among them as shown in Table 3, namely, H1, H2, and H5.The sharing of haplotype indicates there is gene flow among them.Moreover, nucleotide divergence analysis revealed that high nucleotide diversity () ranges from 0.3% to 0.9% within T. schlegelii population (Table 4).Among populations, high nucleotide Phylogenetic analyses of T. schlegelii from Sarawak produced same tree topologies for Neighbour-Joining (NJ) (not shown), Maximum Likelihood (ML) (not shown), maximum parsimony (MP) (Figure 2), and Bayesian inference tree (not shown).They revealed a monophyly T. schlegelii with respect to outgroups G. gangeticus, C. johnsoni, and C. palustris with the bootstrap value of 98% (MP), 99% (NJ), 100% (ML), and 1.0 Bayesian Posterior Probability.The monophyly status of T. schlegelii with respect to outgroup was congruent with [13].
Five T. schlegelii from Langkawi (LT01, LT02, LT03, LT04, and LT05) are closely related to three samples from Peninsular Malaysia (PM06 from Zoo Negara and two other samples, namely, PM011 and PM013, from Melaka Zoo whose locality is in Selangor) in Subclade II with bootstrap value of 99% (MP), 99% (NJ), 100% (ML), and 0.9 BPP.The owner of CAL reported that all T. schlegelii were bought from Singapore in 1980 with unknown locality.This however cannot determine whether the origin of T. schlegelii in Langkawi is Selangor or other places although all T. schlegelii as mentioned are placed in Subclade II.Subclade III comprises specimens from Kalimantan, Peninsular, and Java while Subclade IV comprises all other specimens which form largest subclade.However, with low bootstrap value (50% (MP); 62% (NJ); 54% (ML), and 0.5 BPP) in between Subclade 4 and Subclade 1, the tree is not resolved.More research needs to be done so the information is helpful for the farm in terms of historical information and could be raised during education briefing to the tourists.

Conclusion
In conclusion, genetic diversity among T. schlegelii is high, ranging from 0.16% to 3.34%, suggesting healthy populations.Analysis showed that there is gene flow among populations (Da = 1.71% to 2.21%) within Western Sarawak, Peninsular Malaysia, and other geographical regions coherent with Sundaland theory, suggesting that there is ancient river system connecting the two regions of Peninsular Malaysia and West Borneo when the Sunda Shelf was exposed.Unique haplotypes had been observed in Northern Sarawak as well as in Sumatera; thus each T. schlegelii deserved its own management strategies to ensure the survival of the species.

Figure 1 :
Figure 1: Locations of T. schlegelii populations involved in this study.

Table
: Details of Cyt b-CR gene sequences obtained from GenBank involved in this study.

Table 2 :
Summary of genetic distance in percentage (%) for mtDNA Cyt b-CR gene sequences of T. schlegelii based on the locality in Malaysia.

Table 3 :
Analyses of mtDNA Cyt b-CR gene sequences among T. schlegelii.

Table 4 :
Measures of haplotypes and nucleotide diversity within populations of T. schlegelii analyzed by location.

Table 5 :
Measures of nucleotide diversity () and net nucleotide divergence (Da) among populations of T. schlegelii analyzed by location.