Trends Sci. 2025; 22(7): 9996

Genetic Characteristics of Periophthalmus chrysospilos through COI Gene Analysis in Coastal Provinces of the Mekong Delta

Ton Huu Duc Nguyen¹, Gieo Hoang Phan^2,* and Quang Minh Dinh¹

¹Faculty of Biology Education, School of Education, Can Tho University, Can Tho 900000, Vietnam

²Faculty of Agriculture and Rural Development, Kien Giang University, Kien Giang 920000, Vietnam

(^*Corresponding author’s e-mail: [email protected])

Received: 15 February 2025, Revised: 11 March 2025, Accepted: 18 March 2025, Published: 30 May 2025

Abstract

Periophthalmus chrysospilos is an amphibious gobiid species inhabiting intertidal mudflats and mangrove ecosystems across Southeast Asia, with the Mekong Delta as a critical habitat. This study investigates the genetic diversity of P. chrysospilos populations across 4 coastal provinces of the Mekong Delta - Tra Vinh, Soc Trang, Bac Lieu, and Ca Mau - through mitochondrial cytochrome c oxidase subunit I (COI) gene sequence analysis. Genomic DNA was extracted, amplified, and sequenced to assess nucleotide composition, genetic distances, and phylogenetic relationships. The analyzed COI gene sequences were 645 base pairs in length. The results demonstrated a high degree of sequence similarity (99.21 - 99.84 %) between P. chrysospilos populations in the Mekong Delta and those from Singapore. Nucleotide composition analysis revealed a higher proportion of adenine-thymine (AT) content than guanine-cytosine (GC). Furthermore, genetic distances among samples were minimal (0.00 - 0.03), suggesting low levels of genetic differentiation. Phylogenetic analysis clustered all sampled individuals into a single clade, confirming their classification as a single species. These findings indicate limited genetic divergence among populations and highlight the necessity for further investigations into environmental and anthropogenic factors affecting genetic structure. This study contributes to understanding P. chrysospilos genetic diversity and provides insights relevant to conservation and aquaculture management strategies.

Keywords: COI gene sequence, Conservation genetics, Genetic diversity, Mekong Delta, Mudskipper, Periophthalmus chrysospilos, Phylogenetics

Introduction

Periophthalmus chrysospilos, belonging to the family Oxudercidae, inhabits coastal mudflats and mangrove swamps in Southeast Asia [1], with the Mekong Delta (VMD) being one of its key distribution areas [2,3]. This species is ecologically unique and attracts scientific interest because it can thrive in a semi-terrestrial environment, where it can respire through the skin and buccal mucosa [4]. It plays a crucial role in wetland ecosystems by participating in food webs, facilitating nutrient cycling, and maintaining ecological balance [5,6]. Beyond its ecological significance, P. chrysospilos holds potential economic value for sustainable aquaculture. Although not yet widely exploited, some studies suggest that this species could

become a valuable local resource, contributing to the livelihoods of coastal communities [7]. However, natural populations of P. chrysospilos are increasingly threatened by habitat degradation due to unsustainable resource extraction, water pollution, and climate change [8]. In this context, genetic studies, particularly those focusing on genetic diversity, are essential for understanding the species’ adaptability and population sustainability in natural habitats. Insights from genetic research aid in conserving wild populations and support the development of efficient aquaculture strategies, especially as coastal ecosystems face mounting environmental pressures.

The COI gene (Cytochrome c oxidase subunit I) is a part of mitochondrial DNA and is considered one of the most widely used genetic markers in studies of biodiversity and evolution. It encodes a protein that plays a crucial role in the electron transport chain and is directly involved in cellular respiration [9]. With its high conservation across species but enough variation to distinguish between species or even populations, the COI gene has become an ideal choice for DNA barcoding to identify species and investigate genetic diversity [10,11]. The use of the COI gene in population studies of animals, mainly fish, has proven remarkably effective for species identification, as demonstrated in species from the Gobidae family [12], Pangasius genus [13], Channa genus [14], Anguilla genus [15], Papus genus [16], etc. This gene enables scientists to accurately identify species, even from small or incomplete biological samples [17]. In studies of mudskippers, including P. chrysospilos, the COI gene provides significant insight into phylogenetic relationships and genetic traits. Studying the COI gene can offer scientific evidence of species adaptation and evolutionary potential, especially in ecologically distinctive regions like the VMD, where fish populations live under rapidly changing environmental conditions. The application of the COI gene in research on this fish species not only helps determine the genetic diversity of the species in coastal provinces such as Tra Vinh, Soc Trang, Bac Lieu, and Ca Mau but also clarifies the role of environmental factors in shaping and maintaining this diversity.

Recent studies on P. chrysospilos primarily focus on its ecological and biological characteristics, including population structure [18], reproduction [19], growth [20], burrow architecture [3], otolith structure [21], etc. These studies have elucidated the ecological significance of this species within mangrove and coastal mudflat ecosystems. Additionally, some research has addressed its tolerance to extreme environmental conditions, such as hypoxic mudflats and high temperatures [4,22]. However, genetic data, particularly regarding genetic diversity, remains limited for this species. In genetic studies, the cytochrome c oxidase subunit I (COI) gene has been widely applied to analyze genetic characteristics in various goby species (Gobiidae), including research on taxonomy, population structure, and geographic differentiation [17]. Nevertheless, studies focusing on P. chrysospilos are still scarce, especially concerning populations in the VMD, where environmental fluctuations may significantly influence the species’ genetic diversity. Therefore, this study aims to analyze the genetic diversity of P. chrysospilos based on COI gene sequences from 4 coastal provinces in the Mekong Delta: Tra Vinh, Soc Trang, Bac Lieu, and Ca Mau. Additionally, the research investigates genetic differentiation among these regions, thereby clarifying the environmental factors that shape the species’ genetic characteristics. This study contributes to a deeper understanding of the genetic makeup of P. chrysospilos, a species uniquely adapted to Southeast Asian mangrove ecosystems. By applying COI gene analysis, the research provides insights into population structure and genetic differentiation and expands the scientific foundation for understanding the role of environmental factors in the evolution and adaptation of this species. Furthermore, the findings enhance the genetic diversity database of the Gobiidae family, a crucial group for conservation and ecosystem management.

Materials and methods

Sample collection and processing

DNA was extracted from 4 fish specimens collected from 4 provinces: Tra Vinh, Soc Trang, Bac Lieu, and Ca Mau (3 fish specimens × 4 sampling sites; Figure 1). All specimens were accurately identified to the species level based on the description provided by Tran et al. [2]. Environmental parameters such as temperature, pH, and salinity were referenced by Dinh and Nguyen [3], as the sampling environments were similar. All sampling procedures were approved by the Scientific Council of the Faculty of Education, Can Tho University (approval code BQ2021-05/KSP) following an assessment of animal welfare. Since DNA extraction was conducted in the laboratory, the specimens were preserved in 90 % ethanol and stored at −20 °C.

Figure 1 Map showing sampling sites [23].

DNA amplification and sequencing

Total DNA was extracted from fish fins according to the manufacturer’s instructions using the TopPURE® Genomic DNA Extraction Kit (ABT, Vietnam). The primer pair FishF1/FishR1, as described by Ward et al. [17], was used to amplify approximately 630 base pairs (bp) of the cytochrome oxidase I (COI) subunit.

Polymerase Chain Reaction (PCR) was conducted in a 50 µL reaction volume containing 40 µL of DEPC water, 5 µL of 10X PCR Buffer, 1.5 µL of each primer (10 pmol/µL), 1 tube of EZ Mix (Phu Sa), and 2 µL of genomic DNA. The thermal cycling conditions were as follows: An initial denaturation at 95 °C for 5 min; 35 cycles of 95 °C for 30 s, annealing at 57 °C for 30 s, and extension at 72 °C for 45 s; followed by a final extension at 72 °C for 5 min and a hold at 25 °C for 2 min. The method assessed PCR product quality using electrophoresis on a 1.5 % TBE agarose gel run at 110 V for 40 min, with the DL2000 marker (Phu Sa company, Vietnam) to estimate product size.

The amplified products were purified with a Purification Kit (Jena Bioscience) as per the manufacturer’s protocol. Using the method outlined by Sanger et al. [24], the purified PCR products were sequenced on an ABI 3500 (ThermoFisher) at PhuSa Biochem LTD in Can Tho City, Vietnam. All sequences were submitted to GenBank for assignment of the accession number after editing. Information on the sequences after registration is shown in Table 1.

Table 1 List of locality information and GenBank’s accession numbers.

Samples	Sites	Length (bp)	Accession number
Periophthalmus chrysospilos-TV	Duyen Hai, Tra Vinh	645	OP764034
Periophthalmus chrysospilos-ST	Tran De, Soc Trang	645	OP764035
Periophthalmus chrysospilos-BL	Dong Hai-Bac Lieu	645	OP764036
Periophthalmus chrysospilos-CM	Dam Doi, Ca Mau	645	OP764037

Data analysis

The sequences were meticulously analyzed using FinchTV 1.4.0 software (http://www.geospiza.com). Interfering bases at both ends were trimmed and edited before further analysis. The ClustalW function in MEGA X, following Kumar, et al. [25], was employed to align the COI sequences. Genetic distances within and between groups were estimated using the K2P model. The optimal nucleotide substitution models were identified using the “Find Best DNA Model” tool in MEGA X, with the model showing the lowest Bayesian Information Criterion (BIC) score selected for phylogenetic analysis [26]. Analyzed phylogenetic relationships among P. chrysospilos individuals using the Maximum Likelihood method based on the Neighbor-Joining model, with 1,000 bootstrap replicates, in MEGA X.

Results and discussion

The sequence and nucleotide percentage of the COI gene

Four DNA samples collected from Tra Vinh, Soc Trang, Bac Lieu, and Ca Mau showed clear, bright bands without any secondary bands when analyzed using 1 % agarose gel electrophoresis. The results of the quality assessment were presented in Figure 2.

Figure 2 Total DNA after electrophoresis. (L: DL2000 DNA marker; TV: Tra Vinh; ST: Soc Trang; BL: Bac Lieu; CM: Ca Mau).

In addition, the total DNA samples were measured for optical density (OD) at 260 and 280 nm wavelengths to assess DNA concentration and the presence of contaminants. The results in Table 2 showed that the OD260/OD280 ratios for the samples in this study ranged from 1.94 to 2.05, indicating that the extracted DNA was high purity and suitable for PCR analysis.

Table 2 OD measurement results in total DNA samples.

Samples	Conc (ng/µl)	A260/A280
Periophthalmus chrysospilos-TV	251.9	2.05
Periophthalmus chrysospilos-ST	461.5	1.97
Periophthalmus chrysospilos-BL	249.0	1.96
Periophthalmus chrysospilos-CM	611.6	1.94

This study subjected the high-quality total DNA samples to PCR amplification to target the COI gene region. The target gene region was successfully amplified using the primers FishF1 and FishR1, producing a product of approximately 645 bp in all 4 samples. Figure 3 showed the PCR products on a 1.5 % agarose gel after electrophoresis, with clear, bright bands and no secondary bands. Therefore, these samples were selected for sequencing to support further analyses.

Figure 3 PCR products after electrophoresis (L: DL2000 DNA marker; (-): negative control; TV: Tra Vinh; ST: Soc Trang; BL: Bac Lieu; CM: Ca Mau).

The sequencing results showed that, in all 4 sequences, the highest percentage of nucleotides was observed for C and T, each accounting for approximately 29 %, followed by A at around 23 %, and the lowest for G at about 19 % (Table 3). When examined by nucleotide pairs, the percentage of AT pairs was higher than that of GC pairs in all sequences. Overall, there were no significant differences in the percentage of each nucleotide type across the samples from the 4 sampling sites.

Table 3 Nucleotide percentage of samples.

No.	Samples	Access code	Size (bp)	% A	% C	% G	% T	% AT	% GC
	Tra Vinh	OP764034	645	22.95	29.15	18.91	28.99	51.94	48.06
	Soc Trang	OP764035	645	23.10	29.15	18.76	28.99	52.09	47.91
	Bac Lieu	OP764036	645	22.95	29.15	18.91	28.99	51.94	48.06
	Ca Mau	OP764037	645	23.10	29.15	18.76	28.99	52.09	47.91

The BLAST results from the NCBI gene database Table 4. It revealed a high degree of similarity between the COI gene sequences of P. chrysospilos and those of P. chrysospilos from Singapore. Specifically, the coverage of the samples was 98 %, with similarity percentages ranging from 99.21 to 99.84 %. These results suggested that the samples collected in the VMD and Singapore may belong to the same species.

Table 4 COI gene sequence homology of P. chrysospilos samples with species on GenBank.

No.	Samples	DNA barcoding method
		Species	Access code	Gene size (bp)	Query Cover (%)	Percent identity (%)	Site
1	Tra Vinh - OP764034	P. chrysospilos	MN690438.1	675	98	99.84	Singapore
2	Soc Trang - OP764035	P. chrysospilos	MN690438.1	675	98	99.68	Singapore
3	Bac Lieu - OP764036	P. chrysospilos	MN690438.1	675	98	99.84	Singapore
4	Ca Mau - OP764037	P. chrysospilos	MN690438.1	675	98	99.21	Singapore

Genetic distance

The K2P genetic distance analysis revealed the genetic differentiation among the samples from the 4 sites: Tra Vinh (TV), Soc Trang (ST), Bac Lieu (BL), and Ca Mau (CM). Specifically, the genetic distance between TV and BL was 0.000, indicating no genetic difference between these sites. The distance between TV and ST was 0.002, suggesting a slight difference. Similarly, BL also showed a distance of 0.002 compared to ST and CM, indicating high similarity among these samples. Meanwhile, the distance between CM and ST was 0.003, the largest among the recorded values. However, overall, the genetic distances between the samples were relatively low, suggesting they could belong to the same species.

Table 4 Percent Kimura 2-parameter genetic distances.

Samples	TV	ST	BL	CM
TV
ST	0.002
BL	0.000	0.002
CM	0.002	0.003	0.002

Phylogenetic analysis

The phylogenetic tree was based on the COI gene sequences, revealing that the P. chrysospilos samples from VMD formed a monophyletic group with 2 main clades. The sample from ST(OP764035) separated earliest, while the remaining samples (TV, BL and CM) formed a subclade with high bootstrap support (89). This subclade was further divided into 2 smaller groups: 1 group consisting of TV (OP764034) and BL (OP764036) with a bootstrap value of 61, and the other group being CM (OP764037). Additionally, the P. chrysospilos sample from Singapore (MN690438.1) was placed as an outgroup relative to the VMD group, indicating a genetic difference between the populations in these 2 regions. Furthermore, the P. modestus sample from Malaysia (KX223930) was used as an outgroup, clearly separating from the P. chrysospilos group, confirming species-level differences. These results are consistent with the earlier genetic distance matrix, where TV and BL showed the highest similarity (genetic distance of 0.000) and were placed together in the phylogenetic tree. Meanwhile, the CM sample exhibited greater separation, especially compared to ST, aligning with the highest genetic distance (0.003) recorded between these 2 sites. These results suggest genetic differentiation between P. chrysospilos populations in Vietnam, potentially influenced by geographical or ecological factors affecting gene flow between the regions.

Figure 4 Maximum Likelihood tree based on COI sequence using the Neighbor-Joining model with the bootstrap test (1,000 replicates). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site (TV: Tra Vinh, ST: Soc Trang, BL: Bac Lieu, CM: Ca Mau).

Discussion

The nucleotide composition data of the COI gene in P. chrysospilos also indicated a high degree of conservation across samples, with the percentages of C (29 %) and T (28 - 29 %) being higher than those of A (23 %) and G (18 - 19 %). Furthermore, the nucleotide percentages in this species showed minimal variation between individuals from all 4 sampling sites, suggesting that the findings of this study are consistent with previous research on the COI gene, which exhibits high conservation within species and low variability [27]. Due to this characteristic, the COI gene has been widely used for species identification in various fish species [28-30]. The GC content is an essential indicator for describing nucleotide composition and is related to genome size [31]. This study found that the AT nucleotide pair percentage was higher than that of GC in all samples, aligning with the general characteristics of species within the Oxudercidae family [22]. This result has been reported in several studies conducted in Australia [17], Canada [32], Cuba [33], and various fish species in Taiwan [27].

The analysis of the COI gene sequences of P. chrysospilos collected from VMD, including Tra Vinh, Soc Trang, Bac Lieu, and Ca Mau, revealed a low level of genetic diversity. This genetic homogeneity may be attributed to the ecological characteristics and distribution of the species. P. chrysospilos inhabit mangrove forests and coastal mudflat ecosystems, where the environmental conditions are similar, and there is little geographic isolation between the sampling sites [3]. Consequently, the species’ ability to move between regions could reduce genetic differentiation and maintain population homogeneity. Compared to previous studies, the genetic diversity of P. chrysospilos in the VMD is lower than in other goby species in the region. For example, a study on P. novemradiatus in Malaysia found higher intra-species genetic distances (0.002 - 0.012) [34], indicating more substantial differentiation between populations. Similarly, studies on Pangasius krempfi, P. mekongensis, and P. elongatus in the Mekong River recorded higher genetic diversity (K2P = 0.009 - 0.012), likely due to the Mekong River’s flow’s influence, creating ecological isolation between populations [13]. Compared to other P. chrysospilos populations from different regions, the BLAST results of this study indicated a high similarity (99.21 - 99.84 %) between the COI sequences of samples from the Mekong Delta and those from Singapore. This suggests that the populations in these 2 regions may share a common origin or have had genetic exchanges in their evolutionary history. Several factors could influence the low genetic diversity of P. chrysospilos in the Mekong Delta, including strong gene flow between regions, the absence of significant geographic barriers, and the species’ widespread distribution. Without significant geographical barriers, such as rivers or mountain ranges, individuals can freely move and interbreed between coastal areas, leading to high genetic homogeneity [12]. Additionally, environmental stability may limit selective pressures, resulting in minimal genetic differentiation between populations [5]. Although low genetic diversity helps maintain population stability, it also challenges conserving the species. Genetic homogeneity may reduce the adaptability of P. chrysospilos to environmental changes [35], such as salinity, temperature, or water pollution [8]. Therefore, to conserve and manage P. chrysospilos populations, strategies for protecting natural habitats, especially mangrove forests and coastal mudflats, are necessary. Expanding research to include more areas, including neighboring coastal regions, will provide a more accurate assessment of the species’ genetic differentiation across a broader range.

The habitat plays a crucial role in shaping aquatic species’ adaptation and genetic differentiation [36,37], especially for those living in coastal and mangrove ecosystems. In this study, the genetic analysis of P. chrysospilos revealed high genetic similarity among the samples from VMD in the phylogenetic tree, which may reflect the strong gene flow between areas with similar ecological conditions. However, the slight differences observed between the populations in Soc Trang (ST) and the other sites and the genetic differentiation from the Singapore population suggest that environmental factors may have influenced the species’ adaptation over time. The coastal waters of the VMD display significant fluctuations in salinity and temperature due to tidal regimes, seasonal saltwater intrusion, and river flow from the Mekong [3]. P. chrysospilos is a species known for its ability to adapt to brackish and saline waters [38]. However, strong salinity fluctuations could exert selective pressure, leading to minor genetic differences between populations living under different environmental conditions. Previous studies have shown that ecological pressures can lead to geographic genetic differentiation in some fish species. For instance, Shen et al. [39] found that populations of Mugil cephalus living in areas with different salinity levels exhibited significant genetic differences, even though the geographic distance between populations was insignificant. Similarly, studies on P. modestus have shown genetic differences between populations living in environments with more significant salinity fluctuation than those in more stable environments [22]. Additionally, water pollution from aquaculture and industrial activities can reduce population size, increase genetic drift, and affect adaptation [35]. Furthermore, habitat fragmentation due to mangrove deforestation and coastal infrastructure development can limit movement and mating between populations, leading to a decline in genetic diversity over time [36]. Although clear genetic differentiation has not yet been observed, if these environmental impacts continue, they may negatively affect the species’ ability to adapt and long-term survival. Therefore, habitat conservation, genetic diversity monitoring, and expanded research will help maintain the future stability and sustainable development of P. chrysospilos. This suggested that environmental variability, especially in salinity, may be a key factor driving the genetic differentiation of P. chrysospilos populations in the Mekong Delta and potentially between populations in other regions. Adapting this species to such fluctuating conditions highlights its resilience but also underscores the importance of considering environmental factors in the conservation and management of aquatic species.

An important factor influencing genetic diversity is the level of connectivity between populations. As P. chrysospilos is a species living in coastal and mangrove ecosystems, its ability to move between areas is not restricted by significant geographical barriers. This facilitates continuous gene flow between populations, reducing genetic differentiation across regions [12]. However, when compared with populations from Singapore, the higher genetic differentiation could reflect geographic isolation and environmental differences between the 2 regions. Previous studies have also shown that isolation between populations living in different ecosystems can lead to significant genetic differences. For example, the study by Ward et al. [17] on goby species in Australia showed that populations living in distinct ecosystems exhibited notable genetic differentiation. In summary, this study’s results suggest that strong gene flow helps maintain genetic stability among P. chrysospilos populations in the Mekong Delta; selective pressures from local environmental factors may gradually create minor genetic differences between populations living in different ecological conditions. These findings highlight the interplay between gene flow and environmental pressures in shaping the genetic structure of populations, emphasizing the importance of understanding both genetic connectivity and local ecological factors in conservation and management strategies for aquatic species.

With the low genetic diversity observed in Periophthalmus chrysospilos, the conservation of this species requires intervention measures to maintain and enhance genetic diversity while considering the role of environmental factors in shaping genetic variation. Firstly, habitat protection strategies such as mangrove forest restoration and minimizing the impact of coastal development activities should be implemented to maintain ecological corridors that connect populations and promote gene flow. Regular genetic monitoring using techniques like microsatellites or SNPs also helps assess genetic diversity loss and identify timely intervention measures [40,41]. Furthermore, environmental factors play a significant role in shaping the species’ genetic variation. Conditions such as salinity fluctuations, temperature, and water pollution can exert selective pressures on populations, leading to changes in allele frequencies across generations. Further research into the adaptability of P. chrysospilos to these environmental changes will provide valuable information for conservation efforts. At the same time, developing sustainable aquaculture programs with appropriate breeding selection processes can help maintain and enhance genetic diversity without depleting the natural gene pool. Finally, collaboration between scientists, regulatory bodies, and local communities is key to ensuring the long-term sustainability of P. chrysospilos [42,43].

The findings from the genetic diversity study of P. chrysospilos provide valuable insights for developing conservation strategies and sustainable management of this species. While the genetic homogeneity across populations in the VMD contributes to the stability of the population, it also reduces the species’ ability to adapt to environmental changes. Climate change, salinity intrusion, and environmental pollution can significantly affect populations, especially if they lack genetic diversity to cope with new conditions [8]. Some studies have indicated that fish species with low genetic diversity are more vulnerable to environmental impacts. For instance, Zhu et al. [14], in their study on Channa species (Channa argus, Channa maculata, Channa asiatica, and Channa striata), found that populations with higher genetic diversity showed better resilience when environmental conditions changed. This suggests that maintaining genetic diversity in P. chrysospilos populations could enhance the species’ resilience to environmental changes in their habitats. Expanding research to include more areas will provide a more accurate assessment of the genetic differentiation of this species across a broader range. This would contribute to the development of more effective conservation strategies based on a comprehensive understanding of the species’ genetic structure in Southeast Asia. Conservation efforts can help ensure the long-term survival and adaptability of P. chrysospilos by focusing on maintaining genetic diversity of this species in the face of ongoing environmental challenges.

Conclusions

This study provides valuable insights into the genetic diversity of P. chrysospilos populations in the coastal provinces of the Mekong Delta using COI gene analysis. The results indicate a low level of genetic differentiation among populations from Tra Vinh, Soc Trang, Bac Lieu, and Ca Mau, suggesting high genetic homogeneity. Despite the overall genetic similarity, the Soc Trang population exhibited slight variations compared to other sampling sites. The phylogenetic analysis also revealed a close genetic relationship between P. chrysospilos populations in Vietnam and Singapore, highlighting a potential shared evolutionary history. The findings emphasize the importance of conserving P. chrysospilos populations in the Mekong Delta, particularly in the face of environmental changes and habitat degradation. Further research incorporating a larger sample size and additional genetic markers would provide a more comprehensive understanding of the species’ genetic structure and adaptive potential. This knowledge will contribute to developing effective conservation and management strategies for P. chrysospilos and other mangrove-associated species.

Acknowledgments

This work is funded by The Vietnam National Foundation for Science and Technology Development (NAFOSTED) under grant number 106.05-2019.306.

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