Research Article |
Corresponding author: Kar-Hoe Loh ( khloh@um.edu.my ) Academic editor: Nina Bogutskaya
© 2020 Shyama Sundari Devi Chanthran, Phaik-Eem Lim, Yuan Li, Te-Yu Liao, Sze-Wan Poong, Jianguo Du, Muhammad Ali Syed Hussein, Ahemad Sade, Richard Rumpet, Kar-Hoe Loh.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Chanthran SSD, Lim P-E, Li Y, Liao T-Y, Poong S-W, Du J, Hussein MAS, Sade A, Rumpet R, Loh K-H (2020) Genetic diversity and population structure of Terapon jarbua (Forskål, 1775) (Teleostei, Terapontidae) in Malaysian waters. ZooKeys 911: 139-160. https://doi.org/10.3897/zookeys.911.39222
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A background study is important for the conservation and stock management of a species. Terapon jarbua is a coastal Indo-Pacific species, sourced for human consumption. This study examined 134 samples from the central west and east coasts of Peninsular (West) Malaysia and East Malaysia. A 1446-bp concatenated dataset of mtDNA COI and Cyt b sequences was used in this study and 83 haplotypes were identified, of which 79 are unique haplotypes and four are shared haplotypes. Populations of T. jarbua in Malaysia are genetically heterogenous as shown by the high level of haplotype diversity ranging from 0.9167–0.9952, low nucleotide diversity ranging from 0.0288–0.3434, and high FST values (within population genetic variation). Population genetic structuring is not distinct as shown by the shared haplotypes between geographic populations and mixtures of haplotypes from different populations within the same genetic cluster. The gene flow patterns and population structuring observed among these regions are likely attributed to geographical distance, past historical events, allopatric speciation, dispersal ability and water currents. For instance, the mixture of haplotypes revealed an extraordinary migration ability of T. jarbua (>1200 km) via ancient river connectivity. The negative overall value of the neutrality test and a non-significant mismatch distribution are consistent with demographic expansion(s) in the past. The median-joining network concurred with the maximum likelihood haplotype tree with three major clades resolved. The scarcity of information on this species is an obstacle for future management and conservation purposes. Hence, this study aims to contribute information on the population structure, genetic diversity, and historical demography of T. jarbua in Malaysia.
COI, crescent perch, Cyt b, historical demography, ikan mengkerong, Pleistocene
A population’s genetic structure describes the total genetic diversity in the population, which is shaped by several factors, including the life history, geographical barriers, gene flow, selection and bottlenecks (
Terapon jarbua (Forskål, 1775) is a medium-sized fish commonly known as crescent perch, and it is locally known as “ikan mengkerong” in Malaysia (
Existing reports on T. jarbua are generally limited to their morphometry (e.g., length-weight relationship) and reproductive biology (
The main focus of the current study is on the Malaysian populations: Peninsular (West) Malaysia and East Malaysia (Sabah and Sarawak) which are located in the tropical Indo-west Pacific region (Fig.
Sampling around major landing sites and local markets was conducted in both East and Peninsular Malaysia where 134 samples of various sizes were collected randomly from five wild populations of T. jarbua. Populations were provisionally divided into five groups according to region: 1) Kuala Selangor (KS, N = 31) of west Peninsular which is surrounded by the Straits of Malacca; 2) Kuantan, Pahang (KN, N = 30) of east Peninsular which is adjacent to the South China Sea; 3) Mukah, Sarawak (MH, N = 21) of East Malaysia which is surrounded by the South China Sea; 4) Sandakan (SN, N = 28) and 5) Tawau (TW, N = 24) of East Malaysia which are surrounded by the Sulu Sea and the Celebes Sea, respectively (Fig.
Genomic DNA was extracted using 10% Chelex Resin following the protocol of
PCR was performed using a Mastercycler epgradient S thermalcycler (Eppendorf, Hamburg, Germany) and 25μl reaction mixtures consisting of 12.5μl exTEN 2X PCR master mix (1st BASE, Selangor, Malaysia), 9.5 μl of sterile distilled water, 1μl each of forward and reverse primers, and 1μl of DNA template. PCR cycling conditions were as follow: initial denaturation for 1 min at 96°C, 36 cycles of denaturation at 95°C for 30 s, annealing for 30 s at 44°C (CO1) or 48°C (Cyt b), elongation for 1 min at 72°C, and final elongation for 10 min at 72°C. The amplicons were checked for correct length via electrophoresis on a 1% agarose gel (90V for 25 min). PCR products were sent to Apical Scientific Sdn. Bhd. (Selangor, Malaysia) for purification and DNA sequencing.
Multiple sequence alignment was first performed separately for each gene region using the CLUSTAL X (
Unique haplotypes were quantified and the genetic diversity, nucleotide diversity, and pairwise distance were calculated using DNASP v. 4.0 (
In addition, a neutrality test of the pairwise differences among all populations was performed to infer historical demographic and deviation of sequence variation from evolutionary neutrality. Deviations from neutrality were evaluated using Fu’s Fs (
The 1446 bp concatenated COI (631 bp) and Cyt b (815 bp) sequences were analyzed for 134 individuals obtained in five different locations (Fig.
A total of 83 putative haplotypes were derived from the 134 individuals sequenced with 79 of them being unique haplotypes (95.18%) and four were shared haplotypes (4.82%). The dominant haplotype of Malaysian populations is Hap5 (KS, KN, TW, SN, MH, TAI) while other shared haplotypes are Hap3 (KS, KN, TW, MH, IND), Hap8 (KS and KN) and Hap45 (TW and SN). The population from KS recorded the highest total number of haplotypes (22) of which 19 were unique haplotypes, while Tawau recorded the lowest number of haplotypes (15) with 12 unique haplotypes. The nucleotide diversity (π) of T. jarbua populations in this study ranged from 0.0288 ±0.0158 (mean ±SD) to 0.3434 ±0.1722 while haplotype diversity (h) ranged from 0.9167 ±0.0482 to 0.9952 ±0.0165 (Table
Information and molecular indices of T. jarbua. N, number of samples; NH, number of haplotypes; NUH, number of unique haplotypes; h, haplotype diversity; π, nucleotide diversity; k, average number of pairwise differences.
ID | Populations | N | NH | NUH | h | π | k |
KS | Kuala Selangor, Selangor | 31 | 22 | 19 | 0.9828 ±0.0135 | 0.1817 ±0.0904 | 36.5161 ±16.3285 |
KN | Kuantan, Pahang | 30 | 19 | 16 | 0.9678 ±0.0208 | 0.0288 ±0.0158 | 5.7885 ±2.8485 |
MH | Mukah, Sarawak | 21 | 16 | 14 | 0.9952 ±0.0165 | 0.3434 ±0.1722 | 69.0238 ±31.0005 |
SN | Sandakan, Sabah | 28 | 20 | 18 | 0.9577 ±0.0262 | 0.0487 ±0.0256 | 9.7810 ±4.6212 |
TW | Tawau, Sabah | 24 | 15 | 12 | 0.9167 ±0.0482 | 0.0514 ±0.0271 | 10.3333 ±4.8857 |
Total | 134 | 83 | 79 | 0.9820 ±0.0050 | 0.0248 ±0.0031 | 35.8653 ±12.2638 |
A ML tree was reconstructed based on the 83 haplotypes of this study and four COI + Cyt b sequences from Hainan, Taiwan, India and Philippines which were downloaded from National Center for Biotechnology Information (NCBI) (Appendix
The general topology of the median-joining network (Fig.
Pairwise FST comparisons between populations in Malaysia were significant at the 95% confidence level except for the comparison between TW and SN (Table
The genetic structure of the T. jarbua populations analysed by AMOVA showed little (39.52%) genetic differentiation among regions but high (62.13%) variation within populations (Table
Pairwise FST (below diagonal) and exact P-values (above diagonal) among five populations of T. jarbua based on 1000 permutations of the sequence data set. Numbers in bold represent the highest and lowest value. *Significant at p <0.05 by the permutation test. Overall gene flow (Nm) is 0.82.
Populations | KS | KN | MH | SN | TW |
KS | - | 0.0000* | 0.0000* | 0.0000* | 0.0000* |
KN | 0.2965 | - | 0.0000* | 0.0270* | 0.0090* |
MH | 0.3310 | 0.5353 | - | 0.0000* | 0.0000* |
SN | 0.2681 | 0.0702 | 0.5038 | - | 0.0541 |
TW | 0.2633 | 0.1773 | 0.4844 | 0.0452 | - |
Populations | KS | KN | SN | MH | TW | |
COI | KS | - | ||||
KN | 0.003 | - | ||||
SN | 0.003 | 0.000 | - | |||
MH | 0.015 | 0.019 | 0.019 | - | ||
TW | 0.004 | 0.001 | 0.000 | 0.019 | - | |
Cyt b | KS | - | ||||
KN | 0.008 | - | ||||
SN | 0.008 | 0.001 | - | |||
MH | 0.018 | 0.029 | 0.029 | - | ||
TW | 0.008 | 0.001 | 0.000 | 0.029 | - |
Source of variation | Sum of squares | Percentage of variation | F statistic | P |
Among region (FCT) | 800.958 | 39.52 | 0.3952 | 0.1896 ± 0.0134 |
Among populations within region (FSC) | 11.1610 | -0.14 | -0.0033 | 0.0831 ± 0.0082 |
Within populations (FST) | 1476.92 | 62.13 | 0.3952 | 0.0000 ± 0.0000 |
The overall Tajima’s D value was negative with an insignificant p-value, indicating deviation from evolutionary neutrality. Similarly, the Fu’s Fs test which is based on the distribution of haplotypes, revealed negative but significant p-values for all five populations studied, indicating an excess of rare haplotypes or rare mutations in the population compared to what is expected under a neutral model of evolution. Following the results of Fu’s Fs test, the hypothesis of neutral evolution was rejected.
In the present study, all populations demonstrated bimodal and ragged shaped patterns which points to the population having remained largely constant in size and that the lineage was widespread (
Pairwise number of difference (mismatch distribution) analysis was conducted using the constant population size model to observe the population size changes. The observed frequencies were represented by red dotted line. The frequency expected under the hypothesis of population expansion model was depicted by continuous green line. a Kuala Selangor b Kuantan c Mukah d Sandakan e Tawau f all populations.
Parameter estimates of neutrality tests (Tajima’s D statistic and Fu’s Fs) and mismatch distribution (sum of squares deviation (SSD) and r = raggedness index) for each population. Significance (*p < 0.10) was determined using coalescent simulations.
Neutrality test | Mismatch distribution | ||||
Tajima’s D | Fu’s FS | SSD | r | Curve | |
KS | 1.4817 | -1.252 | 0.0296 | 0.0106 | Bimodal |
KN | -1.2595 | -11.560 | 0.0113 | 0.0333 | Bimodal |
SN | -0.4265 | -6.153 | 0.0301 | 0.0160 | Bimodal |
TW | 1.0793 | -2.075 | 0.0236 | 0.0266 | Bimodal |
MH | 2.1863* | -1.093* | 0.0288 | 0.0142 | Bimodal |
Total | -0.3132 | -24.885* | 0.0247 | 0.0201 | Bimodal |
Species identification was confirmed by morphological observation and DNA sequence data in which the intraspecific COI divergence was within the 2% threshold value (
A population’s genetic structure is affected by genetic drift, local adaptation, and gene flow. In a marine environment, the development of population structure is greatly influenced by factors that affect dispersal, such as ocean currents, historical variance, and geographic distance coupled with differences in dispersal ability and habitat discontinuity (
The haplotype tree (Fig.
FST values are often used to infer gene flow, in which a lower FST value indicates low genetic divergence and higher gene flow. FST values below 0.05, as observed between SN and TW populations, indicate negligible genetic divergence, probably due to active exchange of genetic material between populations through breeding. Furthermore, the pairwise divergence between these populations is not statistically significant. According to
Populations from the same region, i.e., TW and SN of Sabah, were the least genetically variable (Tables
Another interesting finding of this study is the occurrence of shared haplotype between the populations from Peninsular and East Malaysia, India, Hainan, Philippines and Taiwan. Common haplotypes between localities and mixed haplotypes of different lineages in some populations in the current study can be explained by the biogeographical history of Southeast Asia (historically known as the Sundaland). Southeast Asia is believed to have experienced simultaneous glaciation and consequent deglaciation along with its associated decrease and increase of seawater levels during the Pleistocene period, which greatly influenced continental and oceanic configuration (
The MH population is the most genetically distinct with the highest between-group mean distances, haplotype and nucleotide diversity among the five populations. Geographical isolation of allopatric populations restricts gene flow between two populations, which in turn allows the evolution of a genome adapted to local condition (
Among the four populations, MH is genetically closest to KS. Geological evidence suggests that the river systems of Sarawak were historically interconnected with most major river systems of Peninsular Malaysia via the Sunda River during Pleistocene glaciation (about 10000 years ago), thus allowing gene flow among these drainages (
Historical demographic expansions were determined by analysing the frequency distributions of pairwise differences between sequences (
The mismatch distribution is generally displayed as a multimodal pattern for populations showing demographic equilibrium. In contrast, a unimodal pattern depicts populations which have experienced recent demographic expansion (
To summarize, we found 1) high haplotype diversity but low nucleotide diversity among T. jarbua populations in Malaysia; 2) significant results suggesting population expansion of T. jarbua in this region; 3) despite the three genetic clusters observed in the haplotype tree and median-joining network, no obvious population structuring was detected among geographically distinct populations. Common haplotypes among populations and haplotypes from several populations in each genetic cluster indicate high genetic connectivity among the populations. This study assesses the genetic diversity and population structure of T. jarbua in Malaysia for appropriate conservation and management strategies. Conservation of crescent grunter at its natural variation level is required as it forms a diverse group of taxa with 83 haplotypes distributed across Malaysia. The haplotype composition surveyed in the present study may provide a baseline for future comparisons to monitor the temporal variability of haplotype frequency and population structure. This study also has indirectly revealed the dispersal power of T. jarbua through its high mobility and rapid adaptability to a newly colonized area. Further studies can be conducted using larger sample size and temporal replicates, samples collected from other areas of geographical distributions, and sequence data from other mtDNA genes or information based on nuclear DNA. This research contributed useful data for future large scale biogeographical and taxonomic studies of this species.
The fish species that was employed in this study is not categorized as endangered species under the IUCN list and all the samples were collected from fish markets and landing sites.
This study was supported by the University of Malaya, Research University Grant (RU009E-2018), Top 100 Universities in The World Fund (TU001-2018), IF030B-2017; Ministry of Science and Technology (108-2119-M-110-005) and the China-ASEAN Maritime Cooperation Fund project “Monitoring and conservation of the coastal ecosystem in the South China Sea”. We would also like to thank Surajwaran Mangaleswaran, an English professional for checking on the language used in this paper.
Nucleotide composition (%) | ||||||||||||||||||
ID | COI | Cyt b | Combine | |||||||||||||||
A | T | C | G | A + T | C + G | A | T | C | G | A + T | C + G | A | T | C | G | A+T | C+G | |
KS | 22.4 | 28.8 | 30.9 | 17.9 | 51.2 | 48.8 | 23.5 | 28.7 | 32.9 | 14.9 | 52.2 | 47.8 | 22.9 | 28.9 | 32.0 | 16.2 | 51.8 | 48.2 |
KN | 22.6 | 28.9 | 30.7 | 17.8 | 51.5 | 48.5 | 23.5 | 28.7 | 32.8 | 14.9 | 52.2 | 47.7 | 23.0 | 29.0 | 31.9 | 16.1 | 52.0 | 48.0 |
MH | 22.5 | 28.4 | 31.4 | 17.8 | 50.9 | 49.2 | 23.7 | 28.4 | 32.9 | 15.0 | 52.1 | 47.9 | 23.0 | 28.6 | 32.2 | 16.2 | 51.6 | 48.4 |
SN | 22.6 | 28.9 | 30.7 | 17.8 | 51.5 | 48.5 | 23.5 | 28.7 | 32.8 | 14.9 | 52.2 | 47.7 | 23.0 | 29.0 | 31.9 | 16.1 | 52.0 | 48.0 |
TW | 22.6 | 28.9 | 30.8 | 17.8 | 51.5 | 48.6 | 23.5 | 28.7 | 32.9 | 14.9 | 52.2 | 47.8 | 23.0 | 28.9 | 32.0 | 16.1 | 51.9 | 48.1 |
Total | 22.2 | 29.2 | 30.9 | 17.7 | 51.4 | 48.6 | 23.6 | 28.7 | 32.9 | 14.9 | 52.3 | 47.8 | 23.0 | 28.9 | 32.0 | 16.1 | 51.9 | 48.1 |
HAI | 22.5 | 28.9 | 30.7 | 17.8 | 51.4 | 48.5 | 23.4 | 28.8 | 32.9 | 14.8 | 52.2 | 47.7 | 23.1 | 28.9 | 32.0 | 16.1 | 52.0 | 48.1 |
IND | 22.3 | 29.5 | 30.5 | 17.7 | 51.8 | 48.2 | 23.6 | 28.8 | 32.8 | 14.8 | 52.4 | 47.6 | 22.9 | 29.7 | 31.6 | 15.8 | 52.6 | 47.4 |
PHI | 22.9 | 28.9 | 30.7 | 17.5 | 51.8 | 48.2 | 23.4 | 28.6 | 32.9 | 15.1 | 52.0 | 48.0 | 23.2 | 28.7 | 32.0 | 16.1 | 51.9 | 48.1 |
TAI | 22.5 | 28.9 | 30.7 | 17.8 | 51.4 | 48.5 | 23.6 | 28.7 | 32.9 | 14.8 | 52.3 | 47.7 | 23.1 | 28.8 | 32.0 | 16.1 | 51.9 | 48.1 |
Polymorphic site analysis based on COI, Cyt b and combined gene. C, conserved site; V, variable site; Pi, parsimony informative sites; S, singleton sites.
ID | COI (631 bp) | Cyt b (815 bp) | Combine (1446 bp) | |||||||||
C | V | Pi | S | C | V | Pi | S | C | V | Pi | S | |
KS | 602 | 29 | 20 | 9 | 742 | 73 | 53 | 20 | 1344 | 102 | 73 | 29 |
KN | 618 | 13 | 6 | 7 | 793 | 22 | 14 | 8 | 1411 | 35 | 20 | 15 |
MH | 575 | 56 | 54 | 2 | 716 | 99 | 95 | 4 | 1291 | 155 | 149 | 6 |
SN | 617 | 14 | 7 | 7 | 787 | 28 | 20 | 8 | 1404 | 42 | 27 | 15 |
TW | 619 | 12 | 9 | 3 | 798 | 17 | 14 | 3 | 1417 | 29 | 23 | 6 |
Total | 557 | 74 | 60 | 14 | 687 | 128 | 111 | 17 | 1244 | 202 | 171 | 31 |
Location | Accession number | Publication | |
COI | Cyt b | ||
Hainan | NC027281 | NC027281 |
|
Taiwan | KP204162 | KP152133 |
|
Kochi, India | KC774674 | KC774717 | Lenka et al. 2014 (Unpublished) |
Philippines | KF999840 | KF999856 |
|
Malaysia | MN529663–MN529796 | MN529797–MN529930 | This study |
Frequency distribution of haplotypes according to localities. Highlighted columns indicate shared haplotypes.
Haplotype | Total | KS | KN | TW | SN | MH | PHI | TAI | HAI | IND |
---|---|---|---|---|---|---|---|---|---|---|
Hap_1 | 3 | 3 | ||||||||
Hap_2 | 1 | 1 | ||||||||
Hap_3 | 17 | 4 | 4 | 7 | 1 | 1 | ||||
Hap_4 | 1 | 1 | ||||||||
Hap_5 | 21 | 5 | 6 | 1 | 5 | 3 | 1 | |||
Hap_6 | 1 | 1 | ||||||||
Hap_7 | 1 | 1 | ||||||||
Hap_8 | 2 | 1 | 1 | |||||||
Hap_9 | 1 | 1 | ||||||||
Hap_10 | 1 | 1 | ||||||||
Hap_11 | 1 | 1 | ||||||||
Hap_12 | 1 | 1 | ||||||||
Hap_13 | 1 | 1 | ||||||||
Hap_14 | 1 | 1 | ||||||||
Hap_15 | 1 | 1 | ||||||||
Hap_16 | 1 | 1 | ||||||||
Hap_17 | 1 | 1 | ||||||||
Hap_18 | 1 | 1 | ||||||||
Hap_19 | 1 | 1 | ||||||||
Hap_20 | 1 | 1 | ||||||||
Hap_21 | 1 | 1 | ||||||||
Hap_22 | 1 | 1 | ||||||||
Hap_23 | 1 | 1 | ||||||||
Hap_24 | 4 | 4 | ||||||||
Hap_25 | 1 | 1 | ||||||||
Hap_26 | 1 | 1 | ||||||||
Hap_27 | 1 | 1 | ||||||||
Hap_28 | 1 | 1 | ||||||||
Hap_29 | 1 | 1 | ||||||||
Hap_30 | 1 | 1 | ||||||||
Hap_31 | 1 | 1 | ||||||||
Hap_32 | 1 | 1 | ||||||||
Hap_33 | 1 | 1 | ||||||||
Hap_34 | 1 | 1 | ||||||||
Hap_35 | 1 | 1 | ||||||||
Hap_36 | 1 | 1 | ||||||||
Hap_37 | 1 | 1 | ||||||||
Hap_38 | 1 | 1 | ||||||||
Hap_39 | 1 | 1 | ||||||||
Hap_40 | 1 | 1 | ||||||||
Hap_41 | 5 | 5 | ||||||||
Hap_42 | 1 | 1 | ||||||||
Hap_43 | 1 | 1 | ||||||||
Hap_44 | 1 | 1 | ||||||||
Hap_45 | 2 | 1 | 1 | |||||||
Hap_46 | 1 | 1 | ||||||||
Hap_47 | 1 | 1 | ||||||||
Hap_48 | 1 | 1 | ||||||||
Hap_49 | 1 | 1 | ||||||||
Hap_50 | 1 | 1 | ||||||||
Hap_51 | 1 | 1 | ||||||||
Hap_52 | 1 | 1 | ||||||||
Hap_53 | 1 | 1 | ||||||||
Hap_54 | 1 | 1 | ||||||||
Hap_55 | 1 | 1 | ||||||||
Hap_56 | 1 | 1 | ||||||||
Hap_57 | 1 | 1 | ||||||||
Hap_58 | 2 | 2 | ||||||||
Hap_59 | 1 | 1 | ||||||||
Hap_60 | 1 | 1 | ||||||||
Hap_61 | 1 | 1 | ||||||||
Hap_62 | 1 | 1 | ||||||||
Hap_63 | 1 | 1 | ||||||||
Hap_64 | 2 | 2 | ||||||||
Hap_65 | 1 | 1 | ||||||||
Hap_66 | 1 | 1 | ||||||||
Hap_67 | 1 | 1 | ||||||||
Hap_68 | 1 | 1 | ||||||||
Hap_69 | 2 | 2 | ||||||||
Hap_70 | 1 | 1 | ||||||||
Hap_71 | 1 | 1 | ||||||||
Hap_72 | 1 | 1 | ||||||||
Hap_73 | 2 | 2 | ||||||||
Hap_74 | 1 | 1 | ||||||||
Hap_75 | 1 | 1 | ||||||||
Hap_76 | 3 | 3 | ||||||||
Hap_77 | 1 | 1 | ||||||||
Hap_78 | 1 | 1 | ||||||||
Hap_79 | 1 | 1 | ||||||||
Hap_80 | 1 | 1 | ||||||||
Hap_81 | 1 | 1 | ||||||||
Hap_82 | 1 | 1 | ||||||||
Hap_83 | 1 | 1 | ||||||||
Hap_84 | 1 | 1 | ||||||||
Hap_85 | 1 | 1 |