Genetic Diversity Comparison of Pampus minor between Chinese and Malaysian Populations Inferred from mtDNA Cytb

Yuan Li1, Liyan Zhang2, Karhoe Loh3, Ji Feng1, Xinqing Zheng1, Puqing Song1 and Longshan Lin1,*

1Third Institute of Oceanography, State Oceanic Administration, Xiamen 361005, China

2Fujian Institute of Oceanography, Xiamen 361012, China

3Institute of Ocean and Earth Sciences, University of Malaya, Kuala Lumpur 50603, Malaysia

Yuan Li and Liyan Zhang have contributed equally to this paper.

ABSTRACT

Pampus minor is often mistakenly identified as the larva of Pampus argenteus or Pampus cinereus because of its small size. Despite its importance, studies on the population genetics of P. minor are not yet available. In the present study, the mitochondrial Cytb gene was employed to investigate the population genetics of P. minor collected along the coasts of China and Malaysia. The genetic diversity of all P. minor populations was moderate, and two major haplotype lineages were detected that were differentiated approximately 0.3 million years ago. These two haplotype lineages differed significantly in frequency distribution of Chinese and Malaysian populations, showing an imperfect geographical pedigree structure. Results of AMOVA also showed that the genetic differentiation was mainly among populations. According to the distribution of the haplotypes, an ancestral haplotype existed in both the Chinese population and the Malaysian population, further confirming that the Chinese and Malaysian P. minor populations originated from the same refuge in the South China Sea. A historical demographic analysis indicated that P. minor experienced a recent population expansion during the late Pleistocene period. Due to the need of P. minor to adapt to the diverse habitats, unique haplotypes were ultimately formed under the differing pressures of natural selection. This study is expected to provide a basis for future research of the population genetics of P. minor.

Article Information

Received 13 December 2017

Revised 24 February 2018

Accepted 04 April 2018

Available online 27 November 2018

Authors’ Contribution

YL and LL conceived and designed experiments. YL, XZ, PS and JF performed all experiments and wrote the manuscript. LZ and KL analyzed the data.

Key words

Pampus minor, Genetic diversity, Population structure, Population expansion, Cytb.

DOI: http://dx.doi.org/10.17582/journal.pjz/2019.51.1.149.157

* Corresponding author: [email protected]

0030-9923/2019/0001-0149 $ 9.00/0

Copyright 2019 Zoological Society of Pakistan

Introduction

Pampus minor Liu and Li, 1998 belongs to the class Actinopterygii, order Perciformes and family Stromateidae. It is a newly discovered warm-water Pampus species, distributed mainly in the offshore to the south of the Taiwan Strait (Liu and Li, 1998; Liu et al., 2002). Due to the similar external characteristics and small size (adult fish are generally less than 150 mm long), P. minor has often been mistaken for P. argenteus or P. cinereus (Cheng, 1962; Liu and Li, 1998). Zhang (2011) suggested that there is introgressive hybridization between P. argenteus or P. cinereus in the South China Sea, whereas our studies demonstrate that this species is P. minor (Li et al., 2013). There are few studies on P. minor, and those available are limited to morphological (Liu and Li, 1998)and phylogenetic (Guo et al., 2010; Cui et al., 2010, 2011) studies. Basic surveys on the status of fishery resources and the distribution of P. minor have not been reported, let alone studies on its population genetic diversity.

Based on morphology and DNA barcoding studies on a large number of P. minor samples collected, we summarized the identifying morphological characteristics of P. minor as follows (Liu and Li, 1998; Li, 2015): oval body; eye diameter more than 1/2 of the head length; dorsal fins VII-IX 34-39, pectoral fins 22-24, anal fins V-VII 35-39, and caudal fins 18-20; transverse occipital canals and the dorsal branches of the lateral-line canal on top of the head with a truncated rear edge; ventral transverse occipital canals sparse and slightly longer than or equal in length to the dorsal branches; gill rakers, thin (delicate), sparse, 3-4+8-10=11-14; and vertebrae 29-31.

P. minor is also distributed in Malaysian waters, where it has been recorded recently. If the same species lives in heterogeneous habitats, different genetic diversity often emerge to allow for adaptation to local environments, ultimately shaping different genetic structures. The habitats of the northern South China Sea and Malaysian waters are significantly different with different surface sea temperatures, salinity and substrate environment (See et al., 2016). Hence, it is of interest to determine whether there is a significant difference in the genetic diversity of the P. minor populations in these waters and whether a significantly divergent genetic structure has formed. Only a few studies on the genetic diversity and genetic structure of pomfret fish can be found, and these focused mainly on the study of P. argenteus (Peng et al., 2009; Zhao et al., 2011a; Archangi et al., 2013) without any relevant study on P. minor. Sequence analysis is a widely used technique that is considered suitable for testing the genetic diversity of some species. In the present study, based on the accurate identification of P. minor, the mitochondrial DNA cytochrome b gene (Cytb), which has a moderate nucleotide mutation rate, was used as a molecular marker (Abbas et al., 2017) to comparatively analyze the genetic diversity, genetic structure, and historical demographics of this species along the coasts of China and Malaysia, thereby increasing the understanding of the status of its genetic diversity and clarifying the species’ geographical distribution pattern. This study is of great practical significance for the protection of P. minor and for the promotion of its sustainable utilization.

 

Materials and methods

Sample collection

Four different geographical populations (96 individuals) of P. minor were collected from China (Beihai, Haikou) and Malaysia (Kuala Selangor, Mukah) in 2016 (Fig. 1; Table I). To ensure the accuracy of the taxonomy, all specimens were identified according to their morphological characteristics (Liu and Li, 1998). Back-muscle tissues were excised and preserved in 95% alcohol for subsequent molecular experiments.

DNA extraction, amplification and sequencing

Genomic DNA was isolated from muscle tissue by proteinase K digestion and extracted with a Qiagen DNeasy kit. The extracted DNA was assessed by 1.5% agarose gel electrophoresis and stored at -20°C for PCR amplification. The mtDNA cytochrome b (Cytb) was amplified with the primers L14734: 5′-AACCACCGTTGTTATTCAACT-3′ (Inoue et al., 2001) and H15149: 5′-CTCAGAATGATATTTGTCCTCA- 3′ (Ohdachi et al., 1997). Each PCR was performed in a 25-μL reaction mixture containing 17.5 μL of ultrapure water, 2.5 μL of 10×PCR buffer, 2 μL of dNTPs, 1 μL of each primer (5 μM), 0.15 μL of Taq polymerase, and 1 μL of DNA template. The PCR was performed under the following conditions: 4 min of initial denaturation at 94°C; 32 cycles of 30 s at 94°C for denaturation, 30 s at 50°C for annealing, and 30 s at 72°C for extension; and a final extension at 72°C for 10 min. After purification of the PCR products, both strands were sequenced. The newly determined Cytb sequences were deposited in GenBank under the accession numbers MF616364–MF616380.

 

Data analysis

All Cytb sequences were aligned and edited manually using DNASTAR software. Molecular diversity indices such as polymorphic sites, haplotype number, transitions and transversions were calculated by ARLEQUIN version 3.5 (Excoffier et al., 2005). Genetic relationships among haplotypes were reconstructed using the neighbor-joining (NJ) method implemented with 1000 replicates in MEGA 5.0 (Tamura et al., 2011). Analysis of molecular variation (AMOVA) was performed using ARLEQUIN to

 

Table I.- Information and molecular indices of P. minor.

ID	Populations	n	Date	NH	NUH	h ± SD	л± SD	k ± SD
TB	Beihai	24	2016.11	5	2	0.6377±0.0606	0.0019±0.0016	0.7717±0.5842
TH	Haikou	24	2016.12	5	2	0.5290±0.1042	0.0014±0.0013	0.5942±0.4921
TKS	Kuala Selangor	24	2016.05	7	3	0.6341±0.0973	0.0048±0.0032	2.0109±1.1731
TSK	Mukah	24	2016.05	7	4	0.5036±0.1226	0.0028±0.0021	1.1558±0.7732
Total		96	-	17	-	0.7818±0.0224	0.0054±0.0033	2.2493±1.2490

NH, numbers of haplotypes; NUH, numbers of unique haplotypes; h, haplotype diversity; л, nucleotide diversity; k, average number of pairwise differences.

 

investigate the partition of genetic variation among the populations. Population pairwise FST and exact-P test were also performed using ARLEQUIN. A minimum spanning tree was constructed via the MINSPNEt algorithm as implemented in ARLEQUIN to show the relationship among haplotypes and subsequently drawn by hand.

Historical demography/spatial expansions were inferred by neutrality testing and mismatch distribution analysis, as implemented in ARLEQUIN. Deviations from neutrality were evaluated using Fu’s FS and Tajima’s D. Nucleotide mismatch distributions were applied to evaluate the population growth and spatial range expansion. The values of τ were transformed to estimates of real time since expansion with the equation τ=2×μ×t, where μ is the mutation rate for the whole sequence under study, and t is the time since expansion. In the present study, a sequence divergence rate of 0.2×10−7/site/year was applied to the Cytb sequences of P. minor (Avise, 1994; Sun et al., 2012). Bayesian skyline plots were created with BEAST v.8 (Drummond and Rambaut, 2007).

 

Results

Genetic diversity

The length of the Cytb sequence was 415 bp after manual proofreading and comparison. A total of 14 mutations, 10 parsimony informative sites, and four singleton sites were found in the target fragment. There were 14 transitions, three transversions and no insertions/deletions. The ratio of transitions to transversions was 4.7, indicating that the mutations in the Cytb sequence of P. minor had not reached saturation. The A+T content (62.34%) was higher than the G+C content, indicating a significant AT preference.

In the coding gene, most of the mutations were synonymous substitutions, which occurred mostly at the third locus of the codon. All mutations in the P. minor sequence occurred at the third codon due to the relatively slight pressure of natural selection on the nucleotide mutation of the third codon in the protein-encoded gene. No mutations were detected at the first or second codon due to the strongly limited functionality of these codons. The sequences of all nucleotides encoded 138 amino acids, and no mutated amino acid was detected, showing that all mutations are synonymous substitutions.

The 96 collected P. minor samples represented 17 haplotypes. The Beithai population had five haplotypes, of which two were unique haplotypes; the Haikou population had five haplotypes, of which two were unique haplotypes; the Kuala Selangor population had seven haplotypes, of which three were unique haplotypes; the Mukah population had seven haplotypes, of which four were unique haplotypes (Table I); the Chinese population had seven haplotypes, of which three were common haplotypes and four were unique haplotypes; and the Malaysian population had eleven haplotypes, of which three were common haplotypes and seven were unique haplotypes. Only Hap_2 was common in the three populations, and it was also the only haplotype commonly found in both the Chinese and the Malaysian populations, being identified in 30 individuals. It is possibly an ancestral haplotype. In the P. minor populations of this study, the haplotype diversity (h) was 0.782±0.022, and the nucleotide diversity (π) was 0.005±0.003 (Table I), indicating that for the characteristics of high h and low π, the genetic diversity was moderate.

Genetic structure

The neighbor-joining (NJ) tree was constructed based on the 17 Cytb haplotypes of pomfret. The results showed two major haplotype lineages in the P. minor populations, though the bootstrap values were not high. However, pedigree structure that strictly corresponded to the geographical location was not detected (Fig. 2). Lineage A consisted of seven haplotypes (62 individuals), six of which were all in the Chinese group and Hap_2 from 14 Malaysian individuals and 16 Chinese individuals; Lineage B consisted of 10 haplotypes (34 individuals), which were all in the Malaysian group (Table II).

 

Table II.- Distribution of haplotypes among four P. minor populations in lineages A and B.

Haplotype		Total	TB	TH	TKS	TSK
Lineage A
	Hap_1	26	10	16
	Hap_2	30	11	5	14
	Hap_3	1	1
	Hap_4	1	1
	Hap_5	2	1	1
	Hap_6	1		1
	Hap_7	1		1
Lineage B
	Hap_8	22			5	17
	Hap_9	1			1
	Hap_10	2			1	1
	Hap_11	1			1
	Hap_12	1			1
	Hap_13	2			1	1
	Hap_14	2				2
	Hap_15	1				1
	Hap_16	1				1
	Hap_17	1				1

 

Based on the 17 haplotypes of P. minor, the haplotype network diagram was constructed by minimum spanning tree (MST) methods (Fig. 3). The results showed a significant pedigree structure in the topology of the network, which is consistent with the results of the NJ tree. The frequency distribution of the two haplotype lineages in the P. minor populations was calculated (Table II). The results showed that Hap_1, Hap_2, and Hap_8 had a relatively high distribution frequency and were the main haplotypes. Except for Hap_2, the frequency distributions of the two haplotype lineages in the geographical groups were significantly divergent; in other words, an imperfect geographical structure was present.

The net genetic distance between the two haplotype lineages was calculated to be 0.0059, and Lineages A and B differentiated approximately 0.3 million years ago based on a mitochondrial Cytb differentiation rate of 2% per million years.

The results of the population pairwise FST show that except for the moderate differentiation between the North Sea population and the Haikou population, the FST values of the remaining populations were higher than 0.25, exhibiting great differentiation (Wright, 1965), and the statistical test between all groups showed significant differences (Table III). The results of the exact-P test showed that except for the North Sea population and the Haikou population, which yielded an insignificant P value in the statistical test, the P values between all other group pairs in the statistical test were significant (Table III). This result suggested significant genetic differentiation between all group pairs except for the North Sea population and the Haikou population, which showed only weak genetic exchange.

 

Table III.- Results of the pairwise FST (below diagonal) and exact-P tests (above diagonal) among four populations of P. minor.

Populations	TKS	TSK	TB	TH
TKS		0.0000*	0.0000*	0.0000*
TSK	0.3952*		0.0000*	0.0000*
TB	0.4211*	0.7877*		0.1334
TH	0.3019*	0.7535*	0.0809*

*, Significant at P<0.05 by the permutation test.

 

Table IV.- AMOVA of P. minor populations based on mitochondrial Cytb sequences.

Source of variation	Sum of squares	Percentage	F statistic	P
One gene pool
Among populations	54.719	56.52	FST= 0.5652	0.000
Within populations	52.125	43.48
Two gene pools (TB and TH) (TSK and TKS)
Among groups	40.448	45.23	FCT=0.45233	0.346
Among populations within groups	14.271	17.84	FSC=0.32573	0.000
Within populations	92	36.93	FST=0.63072	0.000

 

The genetic structure (Table IV) of the P. minor populations was detected using analysis of molecular variance (AMOVA). First, all P. minor groups were analyzed as one gene pool. The results showed that the genetic differentiation index (FST) between the P. minor populations was large, with a genetic difference of 56.52%, and the statistical test result was significant. In contrast, the genetic difference within a population was only 43.48%. Second, to further confirm the genetic structure of the P. minor population, the four populations were grouped into two gene pools according to the geographical distribution of P. minor: the Chinese group (TB and TH) and the Malaysian group (TSK and TKS). The results showed that the genetic variation between the two lineages was 45.23%, but the statistical test was not significant. The genetic variation within a population between groups was only 17.84%, and the statistical test was significant. The genetic variation within a group was 36.93%, and the statistical test was significant.

Historical demographics

Although two lineages were detected in the P. minor populations, no obvious geographical structure existed in the distribution of the different populations, which may be the result of non-equilibrium in genetic variation after isolation and differentiation. Because of the significant differentiation existed, the two lineages were analyzed separately for their historical demographics. Both the distribution of mismatched nucleotides in all sequences of P. minor and the two lineages showed a single peak, respectively.

The results of the neutrality test showed (Table V) that the values of Fu’s FS were negative, and the statistical tests were significant (P<0.05), indicating that P. minor experienced a population expansion event. Furthermore, the values of the D test were negative, but the statistical tests were not significant. However, the sum of squared deviations (SSD) and Harpending’s raggedness index (HRI) statistical tests were not significant (P>0.05), indicating no significant deviation from the expected distribution under the population expansion model. Therefore, the expected distribution can be used for the historical demographic analysis of population (Fig. 4).

The τ value for the distribution of the unmatched nucleotide provides a rough estimated time of population expansion. The τ of Lineage A was larger than that of Lineage B, at 0.844 (95% CI: 0.279-4.184) and 0.125 (95% CI: 0.0488-2.361), respectively (Table V). According to the differentiation rate of 2% per million years and the τ value, the time of population expansion in P. minor for Lineages A and B was estimated to be 2.01×105 and 1.51×105 years ago, respectively, which were in the late Pleistocene period. The ratio of the θ0 after and before the expansion was infinite, indicating that the size of the effective maternal population of P. minor after the expansion experienced a sharp increase.

The Bayesian skyline plots revealed a detailed demographic history of population size changes, from which we could see that both lineages A and B had undergone population expansion in the late Pleistocene. The effective population size of lineage A increased sharply after the last glacial maximum (LGM) approximately 5.5×104 years before the present, and the effective population size of lineage B increased slowly from 1.7×105 years ago (Fig. 5).

 

Table V.- Summary of the molecular diversity, neutral test and goodness-of-fit test for P. minor.

	n	NH	k ± SD	Tajima’s D		Fu’s Fs		Goodness-of-fit test
				D	P	Fs	P	τ	θ0	θ1	SSD	HRI
Lineage A	62	7	0.6996± 0.5360	-0.796	0.262	-3.013	0.046	1.67	0.000	99999	0.024ns	0.178ns
Lineage B	34	10	1.4510± 0.9041	-1.254	0.090	-4.377	0.004	1.25	0.000	99999	0.439ns	0.165ns
All	96	17	2.2493± 1.2490	-0.480	0.369	-5.783	0.033	--	0.000	4.082	0.022ns	0.057ns

NH, numbers of haplotypes; h, haplotype diversity; л, nucleotide diversity; k, average number of pairwise differences; ns, P>0.05.

 

Discussion

Genetic diversity is the basis of species diversity and ecological diversity. Species diversity and genetic diversity form the basis of ecosystem diversity. The research of genetic diversity is increasing attention from scholars in China and other countries. The genetic diversity of species directly affects the adaptability of the species to the environment. The higher the diversity is, the greater the potential for the evolution of the species and the stronger the ability of the species to adapt to environmental changes. In contrast, the possibility of degradation or extinction of the species also exists (Rosel et al., 1995).

Compared to the genetic diversity of the Cytb gene sequence in Trachidermus fasciatus (h=0.97±0.011) (Gao et al., 2013) and P. argenteus (h=0.775±0.041) (Zhao et al., 2011b), the genetic diversity of Chinese and Malaysian P. minor populations was moderate. From the perspective of historical evolution, for marine fish, the large number of populations explains the maintenance of higher h in natural populations. According to the research findings, the overall genetic diversity of the P. minor population (h=0.7818±0.0224; л=0.0054±0.0033) belongs to the second of the four types of marine fish, which was proposed by Grant and Bowen (1998), i.e., a high h and a low л. A fish population of this type may have experienced a historical expansion event, i.e., a rapid expansion from a small effective group into a large group in a short period of time. In the process of group expansion, with the increase in the number of groups, the h was improved, but not enough time has passed to accumulate the variants produced in the nucleotide sequence, resulting in a genetic diversity pattern with a high h and a low л (Grant and Bowen, 1998).

Stepien (1999) believes that fish along the continental shelf maintain long-term stable numbers and that a large effective population is responsible for the high h. Although pomfret resources have declined due to overfishing (Jin et al., 2005), the large amount of resources and the large number of effective populations have maintained moderate genetic diversity in P. minor. In addition, P. minor is widely distributed and highly adaptable to prey, whose spawning grounds exhibit different characteristics according to the different waters of the distribution; the environmental conditions are also not homogeneous (Wu et al., 2012). These life history features and the environmental heterogeneity of P. minor can promote rapid population growth, resulting in slight selective pressure on the population, which may lead to the accumulation of more genetic mutations and a rich genetic diversity.

The distribution of genetic diversity will be affected not only by evolutionary forces (e.g., migration) but also by historical events, habitat discontinuities and an unstable number of populations can cause differentiation among species groups (Stepien, 1999). Both the NJ tree and haplotype network showed significant genetic differentiation in P. minor populations. The results of AMOVA also showed that the genetic differentiation was mainly attributable to the genetic variation among populations. The two lineages differentiated approximately 0.3 million years ago, and this differentiation may have been affected by the Pleistocene ice age. Many studies have shown that the Pleistocene ice age had a huge impact on the genetic structure of marine fish populations (Liu et al., 2007; Shen et al., 2011; Zhao et al., 2011a, b; Wu et al., 2012; Gao et al., 2013).

At the end of the Quaternary Period, the global climate experienced a series of glacial and interglacial changes. Climate fluctuations occurred over a period of approximately 100 kyr in the past 800 kyr (Lambeck et al., 2002). The end of the fourth glacial period was approximately 420 kyr ago (Petit et al., 1999), which is consistent with the differentiation time of the two haplotype lineages of P. minor. With the arrival of the fourth ice age, the sea level dropped approximately 120-140 m, and the P. minor population may have been isolated in a refuge in the South China Sea; after the ice age, as the sea level rose, the P. minor populations in the refuge would have expanded toward the coasts of China and Malaysia. The NJ tree and haplotype network showed that Hap_2 was the only common haplotype in the Chinese and Malaysian populations, found in the highest number of individuals and identified as the ancestral haplotype, which further verified that the Chinese and Malaysian P. minor populations cohabitated the same refuge in the South China Sea. After the population expansion event, the P. minor populations resided in the different sea areas and adapted to the diverse habitats resulting in many new mutations, the accumulation of rich haplotype diversity, and the formation of a unique haplotype. However, time was not sufficient for mutations to occur in the nucleotide. According to the distribution of haplotypes, except for the ancestral haplotype, all remaining haplotypes observed in the Chinese and Malaysian P. minor populations were significantly differentiated, and different geographical regions shaped their own unique haplotypes, respectively. The results of the FST and exact P tests also showed significant genetic differentiation between the Chinese and Malaysian P. minor populations, which further verified the accumulation of genetic variation that allowed adaptation to the living environments. As Malaysia is at low latitude, with high water temperatures, more diversified habitats, and ocean currents bringing rich sources of plankton by monsoon, the Malaysian P. minor population was under slight selective pressure and accumulated more genetic variation, thus showing higher genetic diversity.

Based on the above results, we conclude that the expansion period of P. minor from the refuge to the coasts of China and Malaysia was short. With the passing of time, the Chinese and Malaysian P. minor populations will accumulate enough genetic variation to achieve complete differentiation. Similar genetic structure have been detected from P. chinensis in the similar distribution areas. Due to the lack of the basic biological information on P. minor and insufficient understanding of its floating time and spawning characteristics, the real cause of this genetic structure in P. minor cannot be accurately speculated. In future studies, more P. minor specimen will be collected from Vietnam, the Malay Peninsula, and the Strait of Malacca to investigate its exactly genetic diversity and genetic structure with more mtDNA markers and/or genomic approaches such as SNPs survey by using RAD-seq method.

 

Acknowledgements

The present study could not have been performed without assistance from Dr. Binbin Shan, Dr. Xiang Zhang and Dr. Wentao Niu during the collection of the P. minor specimens. We are very grateful to Sze-Wan Poong, Sze-Looi Song and Yoon-Yen Yow for helping to revise the manuscript. We sincerely thank the reviewer for the constructive criticisms and valuable comments, which were of great help in revising the manuscript. The research was funded by the Scientific Research Foundation of TIO, SOA (2016010), the National Programme on Global Change and Air-Sea Interaction (GASI-02-SCS-YSWspr/aut), the Provincial Research Institutes of Basic Research and Public Service Special Operations (2017R1006-5), the China-ASEAN Maritime Cooperation Fund Project (HX150702, HX161101) and the Air-Ocean-Land Interactions by the University of Malaya (UM RU009-2015).

 

Statement of conflict of interest

The authors declare no conflicts of interest including in the implementation of the research experiments and the writing of this manuscript.

 

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