A Preliminary Inbreeding Study of Malaysian Endangered Southern

River Terrapin (Batagur affinis) from Captive Population of

Botakanan, Perak and Wild Population of Kemaman, Terengganu

N. A. Ismail

, P-N. Chen

, L. Abd Manaf

, A. Ismail

and N. I. Ab Ghani

Jabatan Biologi, Fakulti Sains, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor Darul Ehsan, Malaysia

Turtle Conservation Society of Malaysia, 6513, RAKR Kg. Fikri, 24000 Chukai, Kemaman, Terengganu Darul Iman,

Malaysia

Jabatan Sains Alam Sekitar, Fakulti Pengajian Alam Sekitar, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor

Darul Ehsan, Malaysia

Keywords: Batagur affinis, Conservation genetic, Inbreeding, Malaysia.

Abstract: The effects of inbreeding are important in conservation genetic studies of endangered animals. Inbreeding

can leads to inbreeding depression due to low level of genetic diversity in a population. Consequently affect

population growth and viability. Hence, it is a major concern in many conservation programs of endangered

animals to avoid the effects of inbreeding and at the same time maintain the genetic diversity. To date,

information about inbreeding and genetic diversity can be assessed using molecular markers such as

microsatellites. In this study, a preliminary inbreeding information of Malaysian endangered Southern river

terrapin (Batagur affinis) from two populations: (i) BotaKanan, Perak (captive population), and (ii)

Kemaman, Terengganu (wild population) were studied using nine cross-species microsatellites. It was found

that inbreeding was higher in BotaKanan population than Kemaman population (F

= 0.57 c.f.F

= 0.43).

BotaKanan population also carried lower genetic diversity than Kemaman population (H

= 0.29 c.f.H

0.40). Hence, suggests that including genetics information to improve the current breeding program and

further genetics study to access various life-history traits in BotaKanan population is necessary. In addition,

study to estimate effective population size and to access various life-history traits are also needed for

Kemaman population to avoid inbreeding in the near future.

1 INTRODUCTION

In conservation genetics, preservation of maximum

genetic diversity (minimisation of inbreeding or co-

ancestry or kinship) is an important factor to ensure

the perseverance and evolution of endangered, small

sized population (i.e. small effective population size)

and isolated species (Brook et al, 2002; Blambert et

al, 2016), particularly due to changes in

environmental conditions — mainly under stress

conditions (e.g. Hendrick and Kalinowski, 2000;

Frankham, 2003). Reduced genetic diversity may

negatively impact the adaptive potential for

endangered, small and isolated species, because

alleles are randomly fixed or lost from the species by

drift. Hence, deleterious mutations tend to

accumulate. However, the process is rather slow, and

thus, does not reduce growth and increase extinction

rates in the short term. On the other hand, inbreeding

effect is rather severe and immediate in small sized

and isolated species, especially for species which

experienced bottlenecks (Barrett and Charlesworth,

1991; Saccheri et al, 1996). Small and isolated

species tend to fix considerable fraction of genetic

load by increasing the frequency of individuals with

homozygous for alleles identical by descent

(Witzenberger and Hochkirch, 2011), and increasing

the uncovering of deleterious recessive alleles

(Witzenberger and Hochkirch, 2011; Nielsen and

Slatkin, 2013; Franham et al, 2010). These result in

fitness reduction (i.e. inbreeding depression) which

are uncover through reduce offspring survival

(Coltman et al, 1998; Amos et al, 2001), reduce

fertility, decrease mating success (Alados and Escos,

1991), slow development, increase sterility and

increase susceptibility to environmental/disease/

parasite stress (Coltman et al, 1999; Cassinello et al,

2001; Keller and Waller, 2002; Ambruster and Reed,

500

Ismail, N., Chen, P., Manaf, L., Ismail, A. and Ghani, N.

A Preliminary Inbreeding Study of Malaysian Endangered Southern River Terrapin (Batagur Afﬁnis) from Captive Population of Bota Kanan, Perak and Wild Population of Kemaman,

Terengganu.

DOI: 10.5220/0009905200002480

In Proceedings of the International Conference on Natural Resources and Sustainable Development (ICNRSD 2018), pages 500-506

ISBN: 978-989-758-543-2

2005; Boakes et al, 2007; Charlesworth and Willis,

2009). Furthermore, there is positive association

between inbreeding and extinction, particularly when

the growth rate of the species is low (e.g. house fly

(Byant and Meffert, 1990); Drosophila and the house

mouse (Frankham, 1995); Bicyclus anyana (Saccheri

et al, 1996); Clarkia pulchenella (Newman and

Pilson, 1997); Drosophila (Bijlsama et al, 2000)).

Hence, justify the importance to access inbreeding in

endangered, small sized and isolated species.

To access inbreeding, information from studbook

record and/or molecular genetics data can be used

(Pemberton, 2004). The studbook record usually

comprises of basic information such as birth and

death dates, weight, identification number,

translocation record and selected life-history traits

with incomplete pedigree or questionable paternity

assignment data (usually breeding individuals and

their offspring) of the breeding population/species.

Hence, inbreeding can be significantly under or

overestimate based on studbook data alone (Ito et al,

2017; Willis, 2001). The pedigree data from

studbook record can only be used to calculate

inbreeding coefficients (F) based on the assumption

that founders are unrelated and non-inbred, and

individuals of unknown origin have a high level of

relatedness. Meanwhile, molecular genetics analysis

can provides much more realistic inbreeding

assessment. It measures the inbreeding (F

) based on

the deviation of the observed heterozygote relative to

the expected heterozygote under random mating

(Hardy-Weinberg equilibrium). Using this method,

highly polymorphic genetic markers such as Single

Nucleotide Polymorphisms (SNPs), microsatellites

(SSRs; Simple Sequence Repeats; e.g. (Solkkoe and

Toonen, 2006)), Amplified Fragment Length

Polymorphism (AFLP; e.g. (Gardiner et al, 2017)),

and Random Amplified Polymorphic DNA (RAPD)

are use. Among those markers, microsatellite is the

most commonly used (Solkkoe and Toonen, 2006).

This is due to it is a DNA marker which can provides

high information content for small sized or recently

bottlenecked species (Hedrick, 1999).

In Malaysia, Malaysian Southern river terrapin

(Batagur affinis; Figure 1) which is one of

endangered species (UCN, 2018) have been

conserved and protected by government (Department

of Wildlife and National Park (PERHILITAN)) and

non-government (Turtle Conservation Society of

Malaysia (TCS)) agencies of Malaysia. Three

conservation centres at three states (i.e. Perak,

Terengganu and Kedah) were built. Those

conservation centres are: (i) the BotaKanan

Conservation Centre, Perak, (ii) the Terrapin

Conservation Centre, Kemaman, Terengganu, and

(iii) the Wildlife Conservation Centre, Bukit Pinang,

Kedah. Those conservation centres are focusing on

captive breeding and/or raising hatchlings (Moll et

al, 2015). Most hatchlings are releaseas head-start

hatchlings to wild populations with aim to increase

the numbers of individuals’B. affinis in the wild

populations. However, the numbers of individuals in

the wild populations are kept on declining (Moll et

al, 2015). There are currently known non-biological

factors including illegal meat and eggs trading as an

exotic delicacy, sand mining activities at its nesting

river banks and aquaculture activities along its

residential rivers that cause the decline number of

individuals in the wild populations (Moll et al,

2015). However, to our awareness, no genetic factors

including inbreeding had been reported for B. affinis.

Therefore, a preliminary study to understand

inbreeding which is important genetic information

using cross-amplified microsatellite markers was

conducted to access inbreeding and genetic deversity

in two populations of B. affinis: (i) BotaKanan, Perak

(captive population), and (ii) Kemaman, Terengganu

(wild population).

Figure 1: A female Malaysian Southern river terrapin

(Batagur affinis).

2 MATERIALS AND METHODS

Tissue sample collection

Two populations of B. affinis were used in this

study: the captive population of BotaKanan, Perak

and the wild population of Kemaman, Terengganu

(Figure 2). A total of 30 pieces of tissues from the

swimming web of the rear foot of randomly chosen

adults were sampled from the BotaKanan

Conservation Centre, Perak. Whilst a total of 30

pieces of tissues from the sculte of randomly chosen

hatchlings were sampled from the Kemaman River

Terrapin Conservation Centre, Terengganu. All

tissues were collected with the help of staffs at each

conservation centre under special permit (T-00465-

16-16) and NRE 600-2/2/21 Jld.4 (12). Those tissues

were kept in separate 1.5 ml microcentrifuge tube

containing 70% ethanol at room temperature.

A Preliminary Inbreeding Study of Malaysian Endangered Southern River Terrapin (Batagur Afﬁnis) from Captive Population of Bota

Kanan, Perak and Wild Population of Kemaman, Terengganu

501

Figure 2: Two sampling populations of B. affinis: (a) the captive population of BotaKanan, Perak, and (b) the wild

population of Kemaman, Terengganu. (Source: Google Satellite, 2018).

Data collection

All DNA was isolated using ReliaPrep™ gDNA

Tissue Miniprep System kit by following the

manufacture protocol. All DNA extractions were

eluted in 100 μl of Nuclease-Free water and kept at -

C. Then, Polymerase Chain Reactions (PCRs)

were carried out using nine cross-species

microsatellites (four microsatellites of Burmese

Roofed Turtle [B. trivittata]: (Love et al, 2013), and

five microsatellites of Yellow-headed Sideneck

[Podocnemis unifilis]: (Fantin et al, 2007; Table 1)

to obtain genotyping data for inbreeding study. The

following was PCR protocol of B. trivittata

microsatellites: Pre-denature at 95

C for 4 min.

Followed by 30 cycles of denaturation at 94

C for 1

min, annealing ranging from 50 to 65

C for 1 min,

extension at 72

C for 1 min and final extension at

C for 20 min. Whilst the PCR protocol of P.

unifilis microsatellites were performed as

followed:initial denaturation at 94

C for 1 min, 45

cycles of denaturation at 92

C for 50 sec, annealing

temperature ranging from 50 to 70

C for 1 min,

extension at 72

C for 1 min, and final extension for

20 min. The PCRs volume for all nine cross-

amplified microsatellites (hereafter loci) contained 3

µl of DNA template, 0.7 µl of MgCl

, 1.0 µl of

buffer, 1.2 µl of Taq polymerase, 0.8 µl of dNTPs

mix, 0.6 µl of each forward and reverse

microsatellite primers and 2.1 µl of ddH

O. The

PCR products were then visualized on 3% agarose

gel electrophoresis and run for 90 min at 78 V with

20 bp size marker as allele size reference to obtain

genotyping data by manually scored.

All genotyping data were checked using

MICROCHECKER (van Oosterhout et al, 2004) for

null alleles or allelic dropout. Then, CREATE

software (Coombs et al, 2008) was used to convert

the genotyping data into a readable format for

inbreeding and genetic diversity analysis. Finally,

inbreeding (F

value: (Weir and Cockerham, 1984))

and genetic diversity (H

) in the two populations

were calculated using GENEPOP on the web

(http://genepop.curtin.edu.au/).

3 RESULTS

Generally, the Malaysian B. affinis from wild

population of Kemaman population comprised of

less inbred individuals as compared to captive

population of BotaKanan population (F

= 0.43

c.f.F

= 0.57; Table 1). This was showed from all of

nine cross-amplified loci, Kemaman population had

four loci with positive F

value (Table 1). Whilst

BotaKanan population had seven loci with positive

value (Table 1). The less inbred Kemaman

population also showed higher level of genetic

(b) Kemaman, Terengganu

(a) BotaKanan, Perak

ICNRSD 2018 - International Conference on Natural Resources and Sustainable Development

502

diversity than BotaKanan population (H

= 0.40

c.f.H

= 0.29). The Kemaman population also

showed three loci with observed heterozygote value

of more than 0.50 (H

> 0.50: Puni_1E1, Puni_1H9

and Puni_2C11; Table 2). Whilst the BotaKanan

population only showed two loci (H

> 0.50:

Puni_1H9 and Puni_2A9; Table 2). However, both

populations showed two out of the nine tested loci

were in heterozygote deficit (H

= 0.00; Table 2).

With both showed similar heterozygote deficit at one

locus (Puni_1A5), but, at locus Puni_2A9 for

Kemaman population and Puni_1E1 for BotaKanan

population. In addition, BotaKanan population had

lower value of observed heterozygosity than

expected heterozygosity (mean H

= 0.29 <H

0.46; Table 2). Whereas Kemaman population had

slightly higher value of observed heterozygosity

than expected heterozygosity (mean H

= 0.40 >H

= 0.39; Table 2).

Table 1: The F

values of all nine cross-amplified loci for

Kemaman and BotaKanan populations of Southern river

terrapin.

Locus

value

Kemaman BotaKanan

Batr16 0.89 0.51

Batr25 0.49 0.99

Batr30 0.98 0.28

Batr39 0.32 0.91

Puni_1A5 - -

Puni_1E1 -0.52 1.00

Puni_1H9 -0.07 0.57

Puni_2A9 - -0.41

Puni_2C11 -0.87 0.28

Table 2: The observed heterozygosity values (H

) and

expected heterozygosity values (H

) of all nine cross-

amplified loci for Kemaman and BotaKanan populations

of Southern river terrapin.

Locus

Kemaman

BotaKanan

Batr16 0.38 0.60 0.40 0.65

Batr25 0.31 0.39 0.03 0.35

Batr30 0.03 0.47 0.45 0.53

Batr39 0.30 0.57 0.05 0.52

Puni_1A5 0.00 0.00 0.00 0.00

Puni_1E1 0.70 0.46 0.00 0.45

Puni_1H9 0.97 0.54 0.57 0.65

Puni_2A9 0.00 0.00 0.60 0.43

Puni_2C11 0.93 0.51 0.50 0.58

Mean 0.40 0.39 0.29 0.46

3.1 Discussion

B. affinis can be found at three states of Malaysia:

Kedah, Perak and Terengganu. In this preliminary

inbreeding study, using nine cross-amplified

microsatellites, two populations of B. affinis were

selected (captive population of BotaKanan, Perak

and wild population of Kemaman, Terengganu). It

was confirmed that Kemaman population had lesser

loci with positive F

value as compared to

BotaKanan population (Kemaman population: four

loci c.f. BotaKanan population: seven loci). This

suggests that the wild population of Kemaman,

Terengganu was less inbred as compared to the

captive population of BotaKanan, Perak (F

= 0.43

c.f. F

= 0.57). In addition, the result of genetic

diversity (H

) supported the finding of inbreeding

value) in this study. It was showed that the less

inbred wild population of Kemaman had higher

genetic diversity than captive population of

BotaKanan (H

= 0.40 c.f.H

= 0.29).

Higher inbreeding and number of loci with

positive F

values in captive population of B. affinis

from BotaKanan as compared to wild population of

Kemaman suggests that mating between siblings is

likely high among individuals in BotaKanan. This is

probably due to the current captive population of

BotaKanan that consists of about 200 adult B.

affinis. But, detail pedigree record either from

studbook data and/or molecular analysis of those

200 adults is not available. Hence the probability of

identical by descent among those 200 adults cannot

be clearly determined. Though, the result of F

values from this study clearly suggests that there is

probability of at least 30 from those 200 adults are

inbreed. All of those adults were captured from the

nearby Perak River and keep in two main ponds

(known as Pond A and B) at the BotaKanan River

Terrapin Conservation Centre, BotaKanan, Perak. In

addition, among those 200 adults, not all of them are

fertile (N

: genetic effective population size;

personal conversation with the staffs of

PERHILITAN Perak). Hence, it is necessary to

further study and improves the current conservation

program of B. affinis at BotaKanan. Apparently,

there is no assurance about the appropriate number

of individuals to be kept for ensuring the persistence

of a population (Berger, 1990). Yet, the conservation

genetics proposes the ‘50/500’ rule. Suggesting the

genetic effective population size should be

maintained above 50 at all times to avoid inbreeding,

and above 500 to retain evolutionary potential

(Witzenberger and Hochkirch, 2011;Harmon and

Braude, 2010). Because these values would tolerate

A Preliminary Inbreeding Study of Malaysian Endangered Southern River Terrapin (Batagur Afﬁnis) from Captive Population of Bota

Kanan, Perak and Wild Population of Kemaman, Terengganu

503

the effect of random drift to fix deleterious alleles in

short term (i.e. inbreeding depression), whilst

maintaining genetic diversity to minimise the effect

of selection over long term (Harmon and Braude,

2010). However, the implementation of ‘50/500’

rule depends on the number of founders and

population history such as a population's network

(Harmon and Braude, 2010). On the other hand, the

inbreeding situation of B. affinis from Kemaman

population is perhaps less urgent than is the case of

B. affinis from BotaKanan, which probably consist

of more genetic effective population size than the

captive population of BotaKanan. Though the

current exact number of genetic effective population

size, and detail pedigree record either from studbook

data and/or molecular data is not available for

Kemaman population because of no such study had

been conducted.

In addition, the lower genetic diversity (referring

to heterozygosity) in captive population of

BotaKanan as compared to the wild population of

Kemaman had supported the high inbred result. Low

genetic diversity has also been reported in several

threatened species elsewhere in the world (e.g.

(González-Pérez et al,2004; Spitzweg et al, 2018).

The reasons contribute to this are population size

and variability (Nielsen and Slatkin, 2013;González-

Pérez et al,2004; Spitzweg et al, 2018). Low genetic

diversity in the captive population of BotaKanan

probably due to this population had been started

with either low number of founders (bottleneck) or

highly associated founders (i.e. maximisation of the

effective population size). Both number and

association of founders are major factors

determining the gene pool. Low number of founders

can significantly increase the accumulation of

deleterious alleles and consequently lead to

inbreeding depression (Witzenberger and Hochkirch,

2011; Nielsen and Slatkin, 2013; Franham et al,

2010). Whist highly associated founders increase the

chance for inheriting homozygous alleles at each

locus because of the same alleles are passed to

offspring from common ancestors (Witzenberger

and Hochkirch, 2011). Witzenberger and Hochkirch,

2011 proposed that a minimum of 15 founders of

census population size (N

) seemed to be sufficient

to maintain good genetic diversity to minimise

inbreeding. Whereas Franham et al, 2010 proposed

that 20 – 30 founders of genetic effective population

size were needed to maintain good genetic diversity

to minimise inbreeding. In conservation genetics,

preservation of maximum genetic diversity

(minimisation of inbreeding or co-ancestry or

kinship) is an important factor to ensure the

perseverance and evolution of endangered, small

sized (i.e. small effective population size) and

isolated species (Brook et al, 2002; Blambert et al,

2016), particularly due to changes in environmental

conditions — mainly under stress conditions (e.g.

(Hendrick and Kalinowski, 2000; Frankham, 2003)).

In this preliminary study of B. affinisfrom

BotaKanan and Kemaman populations, only

molecular data was used to access the inbreeding

and genetic diversity information. Though the

acquired information was sufficient to suggest the

present of higher inbreeding and lower genetic

diversity in BotaKanan population as compared to

Kemaman population, more detail study regarding

this matter would be necessary to improve the

current conservation programs of Malaysian B.

affinis. Combination of molecular data and studbook

data to measure relationships between individuals

will provide better conservation genetics information

(e.g. inbreeding, genetic diversity, life-history traits,

demographic and stochastic trends, and etc.) in small

sized population, isolated and recently bottlenecked

species (Pemberton, 2004). The Malaysian B. affinis

is a small sized population species due to its low

turnover (i.e. 25 years old to reach age of

maturation, or earlier at 22 years old at carapace

size of 510 mm, annually reproduce and 23 to 30 of

average number of eggs per clutch (Chan and Chen,

2011), isolated because it is a fresh water species

with restricted pattern of distribution, and probably

experienced recent bottleneck due to environmental

change in their local habitat — including the effects

of sand mining, dam construction and aquaculture

activities (Moll et al, 2015). By including molecular

data, reliable information about allele distribution

(i.e. assessing the frequency of heterozygous,

homozygous dominant and homozygous recessive

alleles) which evident for genetic diversity that

significantly relates to inbreeding, as well as life-

history traits, demographic and stochastic trends can

be assessed. Also, molecular data can provide more

accurate pedigree information than studbook data.

Whereas by including studbook data, inbreeding

coefficients (F) based on the assumption that

founders are unrelated and non-inbred, and

individuals of unknown origin have a high level of

relatedness information can be evaluated. Hence,

including conservation genetics information in the

current conservation programs of Malaysian B.

affinis (i.e. conventional conservation practice) will

help to improve the population size of this species in

the wild populations, as well as in the captive

populations.

ICNRSD 2018 - International Conference on Natural Resources and Sustainable Development

504

4 CONCLUSION

In conclusion, mating between relatives (inbreeding)

is high in captive population of BotaKanan, Perak as

compared to the wild population of Kemaman,

Terengganu. Hence, further conservation genetic

studies, combining both molecular data and

studbook data should be conducted at the

BotaKanan, Perak population to access better

genetic information, particularly information of

inbreeding and life-history in the current population.

Whereas further molecular, parentage and life-

history studies in the Kemaman, Terengganu

population should be estimated to ensure inbreeding

is not a problem in the near future.

ACKNOWLEDGEMENT

We would like to thank the staffs of Department of

Wildlife and National Park (PERHILITAN) of

Perak, the Turtle Conservation Society of Malaysia

(TCS), the villagers of Kg. TokKapor, Kg. Pasir

Gajah and Kg. Dadong in Kemaman, and

anonymous reviewers for their supportive comments

on the earlier versions of this paper. This research

was supported by the Geran Putra-Insentif Putra

Berkumpulan (GP-IPB/2014/9441102), and

conducted under the ‘Permit KhasJabatan

PERHILITAN Semenanjung Malaysia’ (NRE 600-

2/2/21 Jld.4 (12)).

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