# Relationships amongTomistoma schlegelii in Malaysia Based on Cytb-Control Region Gene Analysis

Authors: Muhammad Amirul Arib Md Adzhar; Ruhana Hassan
Journal: International Journal of Zoology
 (2017)

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## Abstract

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 ofT. schlegeliipopulations from Malaysia which was done using partial sequencing of Cytochromeb-control region mtDNA gene. The study reveals that the genetic diversity amongT. schlegeliiis 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 eachT. schlegeliideserved its own management strategies to ensure the survival of the species.


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## Body

## 1. 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 onT. schlegeliisightings in Kalimantan and Sumatera from the 1990s to 2000s were published by [2–4] while in Malaysia sightings have been rare since the late 1980s [5–8].The main threat ofT. schlegeliiis habitat loss due to deforestation, illegal logging, palm oil plantation, urbanization, fishing activities, and forest fires at peat swamps [2–4]. Currently, wild specimens could be found in captivity in zoos and farms and by individuals who acquiredT. schlegeliiillegally. 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 ofT. schlegeliiconcentrated on surveys ofT. schlegeliinatural habitats and related issues involving conservation.Genetic information onT. schlegeliiis available; for example, [10–12] had sequencing CytbfromT. schlegeliias part of the classification of Family Crocodylia. Besides that, [13] reported based on Cytbgene thatT. schlegeliihaplotype in Peninsular Malaysia is unique. Thus, the aim of this study is to infer genetic relationship amongT. schlegeliifrom captive and wild specimens using partial DNA sequencing of the mtDNA control region (Cytb-CR) gene. The findings are useful for further support of conservation ofT. schlegeliiin Malaysia and other geographical regions.


## 2. Materials and Methods

A total 12T. schlegeliisamples are obtained from 4 localities (Figure 1), from Miri Crocodile Farm (n=5), from Crocodile Adventureland Langkawi (CAL) (n=5), Samarahan (n=1), and Serian (n=1). Other data were obtained from GenBank (Table 1).Table 1
Details of Cytb-CR gene sequences obtained from GenBank involved in this study.
Samples
GenBank accession number
Locality
Collection site
SAR003–SAR005
HM593977–HM593980
Sarawak
MWC
SAR021–SAR023
HM593996–HM593998
Sarawak
Miri
PM006–PM009
HM593981–HM593984,
Peninsular Malaysia
ZN
PM029-PM030
HM593999-HM594000
PM010–PM013
HM593985–HM593988
Peninsular Malaysia
MZ
PM014–PM016
HM593989–HM593991
Peninsular Malaysia
TZ
PM017–PM020
HM593992–HM593995
Peninsular Malaysia
MZT
SZ01–SZ010
HM594005–HM594014
Unknown
SZ
HK1–HK4
HM594001–HM594004
Unknown
HKW Park
SUM36–SUM38
HM594015–HM594017
Sumatera
PTE
KAL39–KAL43
HM594018–HM594022
Kalimantan
CVS
J045–J054
HM594023–HM594032
Java
CWRC
MWC: Matang Wildlife Centre; ZN: Zoo Negara; MZ: Melaka Zoo; MZT: Mini Zoo Temerloh; TZ: Taiping Zoo; SZ: Singapore Zoo; HKW: Hong Kong Wetland; PTES: PT Eka Nindya, Sumatera; CVS: CV Surya Raya Farm, EK; CWRC: Cikananga WRC, Java.Figure 1
Locations ofT. schlegeliipopulations involved in this study.Total genomic DNA was extracted using the modified cetyltrimethylammonium bromide (CTAB) method [14] with the presence of the proteinase K. The amplification of mtDNA control region (Cytb-CR) gene fragment was conducted using oligonucleotide primers L14930 (5′-AGC GGG CAA AAT AGA AAA CTGA-3′, forward) and H15630 (5′-ATA GAG ATG CCG GGA TTA CGAA -3′, reverse) [13]. PCR followed [15] using programmable gradient-enabled thermocycler (Bio-Rad MyCycler™ Thermal Cycler).Approximately, 50–100 ng of the template DNA was amplified in a 25μl 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 Cytb-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].


## 3. Results and Discussion

A total 700 bp of mtDNA control region (Cytb-CR) gene ofT. schlegeliihad been successfully amplified using L14930 primers for all specimens. The sequence matchedT. schlegeliiwith accession number HM593977 [13].The overall frequency distributions of nucleotides at the 1st, 2nd, and 3rd codon position were as follows: T = 29.5%, C = 28.6%, A = 29.9%, and G = 12.0%; T = 26.0, C = 33.6%, A = 29.1%, and G = 11.2%; T = 30.0%, C = 26.4%, A = 32.6%, and G = 11.3, respectively. A compositional nucleotide bias analysis revealed no significant bias (p=1.29) across theT. schlegeliisequence data. Among the 700 bp of the partial mtDNA control region (Cyt b-CR) gene, only 32 (4.5%) sites were variables with 6 singleton sites, leaving 26 (3.7%) potentially parsimoniously informative characters, indicating that this gene is a reliable marker to infer genetic variations at the population level and 668 (95.5%) sites were conserved. The variable site had also shown that the transition (16 sites) occurred more than transversion (10 sites). Similarly, reports claimed that transition occurs more than transversion in animal mitochondrial genomes [22].The genetic distance values among individuals were 0.16% to 3.34% (Table2). 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 Cytb-CR region having a high rate of evolution in mtDNA [13].Table 2
Summary of genetic distance in percentage (%) for mtDNA Cyt b-CR gene sequences ofT. schlegeliibased on the locality in Malaysia.
Samarahan
Serian
Miri
Langkawi
Samarahan
—
Serian
0.31
—
Miri
2.21–2.53
0.16–1.25
—
Langkawi
2.05–2.37
2.53–3.34
0.31–0.62
—The analysis also revealed that high genetic distance values were recorded betweenT. schlegeliipopulations 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 amongT. 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 ofT. schlegeliifrom 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 Table3, 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 (π) ranges from 0.3% to 0.9% withinT. schlegeliipopulation (Table 4). Among populations, high nucleotide diversity (π) was observed which is from 0.3% to 1.9% and 1.7% to 2.0% net nucleotide divergences (Da) (Table 5). The net nucleotide divergence (Da) (Table 5) values were higher when comparing Samarahan with Miri (1.78%) than Samarahan with Langkawi (1.71%) because Samarahan and Langkawi are separated by more than 1200 km distance. It could be due to more genetic drift in Miri population compared to Langkawi from Samarahan specimen. The highest net nucleotide divergence (Da) (Table 5) can be observed among Miri and Langkawi (2.21%) suggesting high genetic differentiation among the population being separated with about 1400 km from each other.Table 3
Analyses of mtDNA Cyt b-CR gene sequences amongT. schlegelii.
Species
Haplotype
Locality
Samples
T. schlegelii
Hap_1
Sarawak, Peninsular Malaysia, unknown, Sumatera
SZG9 SZG2 J052 J053 J049 J054 J048 HK2 HK1 SZG6 SZG3 SAR003 SAR002 SAR022 PM016 PM019 PM020 SUM037 SUM038 SUM036 PM010 SZG4 SAR023 PM015 PM009 SAR021 SAR005 SAR004 SZG1 HK3 SZG10 HK4 SZG5 J045 SZG7 PM008 SZG8 PM007 J047 PM012 PM014 PM017 PM018
T. schlegelii
Hap_2
Langkawi (in this study) and Peninsular Malaysia
LTS02 LTS05 LTS01 PM006 PM011 PM013 LTS03 LTS04
T. schlegelii
Hap_3
Sarawak (in this study)
MTS01 MTS05 MTS04 MTS02 MTS03
T. schlegelii
Hap_4
Sarawak (in this study)
SAM01
Hap_5
Kalimantan and unknown
J046 J050 KAL041 J051 KAL040 KAL039 KAL043 KAL044
T. schlegelii
Hap_6
Peninsular Malaysia
PM29 PM30
Hap_7
Sarawak (in this study)
SAM02Table 4
Measures of haplotypes and nucleotide diversity within populations ofT. schlegelii analyzed by location.
Locality
H
Percent (%) pairwise divergence
Gene diversity
Nucleotide diversity
Samarahan
1
0.0
0.01 ± 0.13
0.003 ± 0.03
Serian
1
0.0
0.03 ± 0.23
0.002 ± 0.03
Miri
1
0.4–0.9
1.00 ± 0.18
0.009 ± 0.01
Langkawi
1
0.4
1.00 ± 0.50
0.004 ± 0.01Table 5
Measures of nucleotide diversity (π) and net nucleotide divergence (Da) among populations of T. schlegelii analyzed by location.
Locality
Distance (km)
Nucleotide diversity (π)
Net nucleotide divergence (Da)
Samarahan/Miri
800
0.0156
0.0178
Samarahan/Langkawi
1200
0.0031
0.0171
Samarahan/Serian
40
0.0141
0.0103
Miri/Langkawi
1400
0.0191
0.0221
Miri/Serian
760
0.0022
0.0212
Langkawi/Serian
1200
0.0032
0.0220Phylogenetic analyses ofT. schlegeliifrom 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 monophylyT. schlegeliiwith respect to outgroupsG. gangeticus, C. johnsoni,andC. palustris with the bootstrap value of 98% (MP), 99% (NJ), 100% (ML), and 1.0 Bayesian Posterior Probability. The monophyly status ofT. schlegelii with respect to outgroup was congruent with [13].Figure 2
Maximum parsimony of the 50% majority rule consensus tree of mtDNA Cyt b-CR gene sequence ofT. schlegelii. Bootstrap values and Bayesian posterior probabilities (BPP) are accordingly indicated below the branch nodes.There is only single clade in the phylogenetic tree (Figure2) with 4 subclades:T. schlegelii from wild population in this study, namely, SAM01 and SAM02, in the same subclade with all 5 semiwildT. schlegelii from Miri, namely, MTS01, MTS02, MTS03, MTS04, and MTS05 (Subclade 1), with 98% (MP), 99% (NJ), 100% (ML), and 1.0 BPP.FiveT. 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 allT. schlegelii were bought from Singapore in 1980 with unknown locality. This however cannot determine whether the origin ofT. schlegelii in Langkawi is Selangor or other places although allT. 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.


## 4. Conclusion

In conclusion, genetic diversity amongT. 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 eachT. schlegelii deserved its own management strategies to ensure the survival of the species.



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*Source: 5431041-2017-11-15.xml*