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. 2022 Aug 22;15(8):2004–2011. doi: 10.14202/vetworld.2022.2004-2011

The first study of genetic diversity and population structure of Indo-Pacific bottlenose dolphin (Tursiops aduncus) and pantropical spotted dolphin (Stenella attenuata) in the Thai Andaman Sea based on ISSR

Promporn Piboon 1, Anocha Poommouang 1, Kittisak Buddhachat 2,3, Patcharaporn Kaewmong 4, Kongkiat Kittiwattanawong 4, Korakot Nganvongpanit 1,2,✉
PMCID: PMC9615499  PMID: 36313843

Abstract

Background and Aim:

The Indo-Pacific bottlenose dolphin, Tursiops aduncus, and the pantropical spotted dolphin, Stenella attenuata, are protected marine mammals in Thailand; however, knowledge regarding the populations of both species in Thai seas is minimal. We aimed to reveal the genetic diversity and population structure of two species, T. aduncus, and S. attenuata, based on inter-simple sequence repeats (ISSRs).

Materials and Methods:

Samples of stranded T. aduncus (n = 30) and S. attenuata (n = 23) found along Thai Andaman Sea coasts from 1998 to 2018 were used in this study. A total of 17 and 16 ISSR primers that produced clear and polymorphic bands were selected for T. aduncus and S. attenuata, respectively.

Results:

The highest percentages of polymorphic bands for T. aduncus and S. attenuata were 93.750% and 92.857%, respectively. Phylogenetic dendrograms indicated that the population of each species was clustered into three groups. This outcome was consistent with the genetic population structure, as both suggested three genetic clusters (DK = 3). Genetic diversity analysis revealed that the average Shannon’s information index (I) was 1.926 ± 0.066 for T. aduncus and 1.714 ± 0.090 for S. attenuata, which indicate a high level of genetic variation. Further, low fixation index (F) values were observed for T. aduncus and S. attenuata at −0.231 ± 0.024 and −0.312 ± 0.042, respectively, suggesting that inbreeding is unlikely to have occurred for both species over the past decades.

Conclusion:

At least three genetic clusters of both species were found in the Thai Andaman Sea, and the diversity indices of each species indicated that these species are not at a critical level for extinction. However, monitoring their population status should be prioritized to observe any future changes in the level of diversity.

Keywords: cetaceans, diversity, Indo-Pacific bottlenose dolphin, pantropical spotted dolphin, population structure

Introduction

All cetacean species inhabiting Thai seas, including two odontocetes identified as the Indo-Pacific bottlenose dolphin, Tursiops aduncus, and the pantropical spotted dolphin, Stenella attenuata, have been categorized as rare marine animals in Thailand. However, T. aduncus has been recognized as a common species found along the coasts of the Thai Andaman Sea and the Gulf of Thailand [1]. This coastal species is mainly found in shallow waters and exhibits a discontinuous distribution in warm temperate waters and those of the tropical Indo-Pacific Ocean [2, 3]. Meanwhile, S. attenuata is an oceanic species with a pantropical distribution in all oceans between 40°N and 40°S. Moreover, this species has been observed in marine waters along both coasts of Thailand [1, 2, 4]. According to the global conservation status assessment published in the International Union for Conservation of Nature Red List, T. aduncus was listed as a near-threatened species [3]. In contrast, the status of S. attenuata was of the least concern [5]. Furthermore, both species have been listed as protected animals in the Wild Animal Reservation and Protection Act of Thailand [6]; however, information regarding the population of these species in Thai seas is limited. In 2010, 80 T. aduncus and 90 S. attenuata individuals were reported to inhabit the Thai Andaman Sea [7]. All marine mammals worldwide, particularly small cetaceans, are currently impacted by various anthropogenic activities, which include commercial fisheries and other human interactions involving sea waters and generate oceanic pollution in the form of sea garbage and microplastics [8, 9]. The detrimental impacts of commercial fisheries include fisheries bycatch and environmental imbalances caused by prey depletion; these imbalances are associated with competition in the fishery industry [10, 11]. Fisheries bycatch is one of the greatest potential threats to small cetaceans worldwide [12–14]. Tursiops spp. and S. attenuata have been affected by various practices in the fishing industry in several regions of the world. Examples of these species include the offshore population of the common bottlenose dolphin Tursiops truncatus, which occurs in the Pilbara trawl fishery area in the waters of Northwest Australia [15]; T. aduncus, which is often caught using gillnet fisheries in the waters of Japan and Pakistan [16, 17]; and the population of S. attenuata, which is negatively impacted by the tuna purse-seine fishery in the waters of the eastern tropical Pacific Ocean [18, 19]. In the territorial waters of Thailand, fishery industries operate along both coasts of the country [20]. However, the number of dolphins annually affected by fisheries bycatch or commercial fishing gear remains unknown, particularly with respect to T. aduncus and S. attenuata.

Identification of management units is essential in developing conservation strategies for cetaceans, as most of these species are associated with extremely high mobility in marine environments, where geographical barriers are usually undetectable. Currently, genetic information is used to establish relevant management units for cetacean species in several regions of the world. In addition, this information is used in genetic population studies that have used nuclear and mitochondrial DNA markers for short-beaked common dolphins (Delphinus delphis) in Australian waters [21, 22] and the Irrawaddy dolphin (Orcaella brevirostris) inhabiting the Gulf of Thailand [23]. Due to the limited population studies over the past decades, there is scarce information regarding the populations of T. aduncus and S. attenuata inhabiting Thai seas. Consequently, the conservation status of the local population of these species in Thai seas cannot be evaluated. Thus, inadequate studies on local animal populations and inaccurate use of the conservation status could lead to ineffective management practices and may accelerate the extinction of local populations, as observed in the cases of the Antillean Manatee Trichechus manatus manatus, a subspecies occurring in Suriname [24], and the Songkhla Lake dolphin O. brevirostris, inhabiting the Songkhla Lake [12].

To monitor alterations in population diversity that may occur in the future and to collect additional information on the conservation biology of T. aduncus and S. attenuata in Thai seas, it is essential to acquire accurate genetic data. Thus, to the best of our knowledge, this is the first study to provide information on the genetic diversity and population structure of T. aduncus and S. attenuata, which inhabit Thai seas, based on inter-simple sequence repeats (ISSRs) using skin tissue samples from deceased dolphins.

Materials and Methods

Ethical approval

This study was approved by the Animal Use Committee of the Faculty of Veterinary Medicine, Chiang Mai University, Thailand, in 2020 (S14/2563).

Study period and location

This study was conducted from June 2021 to February 2022 at the Faculty of Veterinary Medicine, Chiang Mai University, Thailand.

Samples and DNA extraction

The skin tissue samples of 30 deceased T. aduncus and 23 deceased S. attenuata dolphins, which were stranded along the Thai coasts from 1998–2018, were provided by the tissue bank of the Phuket Marine Biological Center, Phuket, Thailand (Figure-1). All samples were preserved in 95% ethanol at −20°C. DNA was extracted from these samples using DNA extraction kits according to the manufacturer’s instructions (DNeasy Blood and Tissue Kit, QIAGEN, Germany) at the Faculty of Veterinary Medicine, Chiang Mai University. The qualitative and quantitative analyses of the DNA samples were performed using agarose gel electrophoresis and spectrophotometry, respectively [25]. These samples were diluted to a final concentration of 10 ng/mL. Due to the poor quality of some of the DNA samples, 22 T. aduncus samples and 20 S. attenuata samples were used for subsequent analyses.

Figure-1.

Figure-1

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Map showing the location of stranded samples along Thai coasts during 1998–2018 [Source: https://www.rstudio.com/]. Black circles indicate Tursiops aduncus, and yellow triangles indicate Stenella attenuata.

ISSR amplification

A total of 34 ISSR primers from the University of British Columbia (Microsatellite UBC primer set 9, University of British Columbia, Vancouver, Canada) were initially screened to yield DNA fingerprints. Overall, 17 and 16 primers that produced reproducible bands were selected for the ISSR amplification of T. aduncus and S. attenuata, respectively; these amplified products were used for subsequent analyses (Table-1 for primer lists). Polymerase chain reaction (PCR) was conducted in 25 ml of reaction volumes containing 1× ViBuffer S (16 mM (NH2)4SO4, 50 mM Tris-HCl, 1.75 mM MgCl2, and 0.01% Triton™ X-100, New Jersey, USA), 0.2 mM dNTP (Vivantis, Selangor Darul Ehsan, Malaysia), 0.2 mM ISSR primer, 1 U Taq DNA polymerase (Vivantis), and 10 ng/ml of the DNA sample. The PCR amplifications were performed in PTC-200 using a DNA Engine Thermal Cycler (Bio-Rad Laboratories, Inc., CA, USA) under the following conditions: Pre-denaturation at 95°C for 5 min; 40 cycles comprising a denaturation step at 95°C for 30 s, annealing step at 55°C for 45 s, and extension step at 72°C for 1 min; and a final extension step at 72°C for 10 min. The PCR products were stained using RedSafe™ nucleic acid staining solution (iNtRON Biotechnology, Gyeonggi-do, South Korea). They were separated using 2% agarose gel (PanReac AppliChem ITW companies, Darmstadt, Germany) electrophoresis through a PowerPac 200 (Bio-Rad) containing 1× Tris-acetate-ethylenediaminetetraacetate buffer at 120 V for 30 min. Finally, the PCR products were visualized under ultraviolet light using a GelMax 125 Imager (UVP, Cambridge, England).

Table-1.

The list of ISSR primers and the nucleotide sequences of ISSR primers used in this study, the number of total bands, polymorphic bands, and the percentage of polymorphic bands revealed by ISSR.

Species Markers Primer sequence No. of total bands No. of polymorphic bands PPB
Indo-Pacific bottlenose dolphin (Tursiops aduncus) UBC807 (AG)8T 8 3 37.500
UBC808 (AG)8C 15 14 93.333
UBC809 (AG)8G 11 9 81.818
UBC811 (GA)8C 12 11 91.667
UBC817 (CA)8A 13 12 92.308
UBC818 (CA)8G 11 9 81.818
UBC823 (TC)8C 9 8 88.889
UBC825 (AC)8T 14 11 78.571
UBC826 (AC)8C 14 13 92.857
UBC827 (AC)8G 14 13 92.857
UBC835 (AG)8YC 10 9 90.000
UBC847 (CA)8RC 11 10 90.909
UBC848 (CA)8RG 12 11 91.667
UBC861 (ACC)6 16 15 93.750
UBC874 (CCCT)4 15 13 86.667
UBC880 (GGAGA)3 15 14 93.333
UBC881 (GGGTG)3 13 10 76.923
mean 85.580
Pantropical spotted dolphin (Stenella attenuata) UBC807 (AG)8T 11 10 90.909
UBC808 (AG)8C 9 8 88.889
UBC809 (AG)8G 10 9 90.000
UBC811 (GA)8C 9 8 88.889
UBC817 (CA)8A 9 8 88.889
UBC818 (CA)8G 12 11 91.667
UBC825 (AC)8T 14 13 92.857
UBC826 (AC)8C 11 10 90.909
UBC827 (AC)8G 11 10 90.909
UBC835 (AG)8YC 14 13 92.857
UBC844 (CT)8RC 4 1 25.000
UBC847 (CA)8RC 4 3 75.000
UBC848 (CA)8RG 9 7 77.778
UBC874 (CCCT)4 10 8 80.000
UBC880 (GGAGA)3 10 7 70.000
UBC881 (GGGTG)3 13 9 69.231
Mean 81.486
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PPB=Percentage of polymorphic bands, ISSR=Inter-simple sequence repeat

Statistical analysis

The ISSR amplification results of the DNA samples from each species were analyzed separately. The bands or loci that were clearly observed and unambiguous among the ISSR amplified fragments were scored for the presence (1) or absence (0) of bands. A binary matrix was generated to determine the level of polymorphism in each primer, which was represented by the percentage of polymorphic bands calculated using the formula established by Ng and Tan [26] as follows:

graphic file with name Vetworld-15-2004-g002.jpg

Phylogenetic dendrograms were then created using the DARWIN software version 6.0 (https://darwin.cirad.fr/) [27, 28] and Interactive Tree of Life web-based tools (https://itol.embl.de) such as clustering analysis. The groups obtained from this process were then used for genetic diversity and differentiation in population analysis.

Genetic diversity and differentiation in populations

Genetic diversity parameters, including Shannon’s information index (I), observed heterozygosity (Ho), expected heterozygosity (He), and the fixation index (F), were determined using the GENALEX program, version 6.5 (https://biology-assets.anu.edu.au/GenAlEx/Welcome.html) [29]. Further, the genetic differentiation between the populations of each species was investigated using Nei’s genetic distance through GENALEX.

Population structure

All samples of each species were analyzed using distance and model-based clustering methods to reveal population affinity and structure. The program STRUCTURE version 2.3.430 (Pritchard Lab, Stanford University, California, USA) [30, 31] was used to cluster individuals into populations based on ISSR genotypes, with assumed admixture and correlated allele frequencies. The LOCPRIOR model was used to infer cryptic population structure [32]. For both species, three runs for 1–10 clusters (K value) each were conducted to assess the presence of population structure with a Markov chain Monte Carlo burn-in length of 100,000 iterations and a run length of 1,000,000 iterations. Chain convergence was assessed by comparing the results obtained from three different chains. The ΔK statistics determined from the K values were then plotted using STRUCTURE HARVESTER (https://taylor0.biology.ucla.edu/structureHarvester/) [33] to identify the optimal number of clusters in the data. Outputs from the STRUCTURE HARVESTER were graphically modified using DISTRUCT (Rosenberg lab, Stanford University, California, USA) [34].

Results

ISSR polymorphism and phylogenetic dendrograms

Overall, 213 DNA fragments of T. aduncus were obtained using 17 ISSR primers (185 were polymorphic), whereas 160 DNA fragments of S. attenuata were obtained using 16 ISSR primers (135 were polymorphic) (Table-1). The highest and the lowest percentages of polymorphic bands for T. aduncus were 93.570% (UBC861) and 37.500% (UBC807), respectively (Table-1). In addition, the primers UBC825 and UBC835 generated the highest percentage of polymorphic bands for S. attenuata at 92.867%, whereas the primer UBC844 generated the lowest percentage of polymorphic bands at 25.000% (Table-1). Phylogenetic dendrograms of both species of dolphins are shown in Figures-2a and b. Each species was clustered into three main groups.

Figure-2.

Figure-2

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Phylogenetic dendrograms: (a) Tursiops aduncus and (b) Stenella attenuata.

Genetic diversity and differentiation

All values (I, Ho, and He) of T. aduncus indicated higher degrees of genetic variation in the a3 group than in the other groups (Table-2). Similarly, S. attenuata b2 group showed the highest genetic diversity for all indicators (I, Ho, and He). In the overall analysis, the Ho, He, and F of T. aduncus were 1.000, 0.817, and −0.231, respectively (Table-2). Meanwhile, S. attenuata showed Ho, He, and F values of 1.000, 0.772, and −0.312, respectively (Table-2). For T. aduncus, the results of pairwise Nei’s genetic distance analysis revealed that the lowest distance was present between the a2 and a3 groups (0.072). In contrast, the greatest distance was observed between the a1 and a3 groups (0.103) (Table-3). For S. attenuata, the results of pairwise Nei’s genetic distance analysis indicated that the closest relationship was between the b2 and b3 groups (0.110), and the most distant relationship was observed between the b1 and b3 groups (0.305) (Table-3).

Table-2.

Diversity index values (mean±SE) for ISSR markers evaluated on Tursiops aduncus and Stenella attenuata.

Species Group I ± SE H0 ± SE He ± SE F ± SE
Indo-Pacific bottlenose dolphin (Tursiops aduncus) a1 0.974 ± 0.062 1.000 ± 0.000 0.598 ± 0.021 −0.705 ± 0.059
a2 1.206 ± 0.079 1.000 ± 0.000 0.661 ± 0.024 −0.544 ± 0.058
a3 1.912 ± 0.083 1.000 ± 0.000 0.823 ± 0.018 −0.227 ± 0.032
All 1.926 ± 0.066 1.000 ± 0.000 0.817 ± 0.014 −0.231 ± 0.024
Pantropical spotted dolphin (Stenella attenuata) b1 0.477 ± 0.083 0.688 ± 0.120 0.344 ± 0.060 −1.000 ± 0.000
b2 1.464 ± 0.069 1.000 ± 0.000 0.725 ± 0.018 −0.394 ± 0.037
b3 1.321 ± 0.136 0.938 ± 0.063 0.661 ± 0.052 −0.459 ± 0.068
All 1.714 ± 0.090 1.000 ± 0.000 0.772 ± 0.022 −0.312 ± 0.042
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I=Shannon’s information index, Ho=Observed heterozygosity, He=Expected heterozygosity, F=Fixation index, ISSR=Inter-simple sequence repeat, SE=Standard error

Table-3.

Pairwise population matrix of Nei’s genetic distance of Tursiops aduncus and Stenella attenuata.

Group a1 Group a2 Group a3
Indo-Pacific bottlenose dolphin (Tursiops aduncus) 0 — — Group a1
0.094 0 — Group a2
0.103 0.072 0 Group a3
Group b1 Group b2 Group b3
Pantropical spotted dolphin (Stenella attenuata) 0 — — Group b1
0.286 0 — Group b2
0.305 0.11 0 Group b3
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Population structure

The results from three independent runs indicated that the population structure of both species comprised three genetic clusters (ΔK = 3). The a1 and a2 groups of T. aduncus had a dominant genetic cluster (Figure-3a). The b2 and b3 groups of S. attenuata represented two dominant genetic clusters (Figure-3b).

Figure-3.

Figure-3

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Admixture bar plot estimation of the dataset. The population structure consisted of three genetic clusters for both species indicated by different colors: (a) Orange, pink, and blue for Tursiops aduncus; (b) green, yellow, and brown for Stenella attenuata. Each individual specimen of each species is represented by a thin vertical line, which is separated into ΔK colored segments to represent the individual’s estimated membership fractions in each of the ΔK segments.

Discussion

The results of this study provide genetic information on the population structure and genetic diversity of T. aduncus and S. attenuata inhabiting Thai seas. To the best of our knowledge, this is the first study to analyze the available skin tissue samples of two stranded species of dolphins along the coasts of Thailand. Three genetic clusters of both species were detected using population structure analysis. Although the diversity indices of each species revealed high genetic variation, further genetic studies using on-field or biopsy samples are needed to obtain the exact location of each population.

In this study, we found that the UBC primer set 9 produced DNA fingerprints that could be used to investigate the genetic diversity of T. aduncus and S. attenuata. The average percentages of the polymorphic bands for T. aduncus and S. attenuata were high at 85.58% and 81.48%, respectively. These values were higher than those of several land mammals, such as cattle, goats, sheep, and Asian elephants, which were included in the previous studies [27, 35]. These UBC primers could be used to effectively obtain DNA fingerprints used in previous genetic diversity studies for numerous plants, such as bryophytes [36] and rhubarb [37], as well as for animals, such as the Asian elephant [27], dugong [38], grass carp [39], porpoises [40], and Limnonectes [41].

According to the results of our study, the population structures of both species indicate that at least three genetic clusters of dolphin populations live along the Andaman coast. The Groups a1 and a3 of T. aduncus exhibited greater genetic relatedness to each other than to Group a2. This may be due to the fact that Groups a1 and a3 are likely to have a common ancestor in their recent evolutionary history. Similarly, according to S. attenuata, Groups b1 and b3 were more genetically related to each other than Group b2. Only one sample of S. attenuata obtained from the Gulf of Thailand (END194) was included in the population structure analysis, which revealed that the sample had not been separated from the other samples obtained from the Thai Andaman Sea and was determined to belong to Group b2. These results suggest a connection between S. attenuata populations inhabiting the Thai Andaman Sea and those inhabiting the Gulf of Thailand, as this oceanic species is also found around the Malay Peninsula, including the Malacca straits and in the waters of Singapore [5, 42]. Importantly, genetic information on these neighboring populations has not yet been elucidated.

The numbers of genetic clusters for dolphin populations differ in various regions because differences in marine environmental conditions can influence the diversification of each population. These conditions include water depth, ocean currents, sea surface temperatures, and the recycling of biological materials [43–45]. The three genetic clusters of the population of T. aduncus inhabiting the Thai Andaman Sea that were observed in our study differed from those of the previous studies conducted by Natoli et al. [46], which reported only one genetic cluster for a population inhabiting the KwaZulu-Natal coast of South Africa. In addition, Kiszka et al. [47] found that the population of T. aduncus in regions around Mayotte and adjacent shallow reef banks had at least two genetic clusters. Further, S. attenuata population inhabiting the Thai seas was categorized into three genetic clusters. This outcome is similar to that of the three genetic clusters of S. attenuata reported in a study conducted in Hawaiian waters by Courbis et al. [48].

A negative F value for both dolphin species in our study indicated high heterozygosity levels; thus, inbreeding is unlikely to be a problem for these dolphin populations. Both populations are unlikely to become extinct in the Thai Andaman Sea within a short period. Although the genetic diversity indices obtained in this study indicated a high level of variability, further genetic studies should be performed by including more samples. These studies should also include on-field or biopsy samples because using carcass-based studies to analyze population genetic structure can underestimate the genetic diversity in populations [49]. Importantly, genetic studies on the dolphin populations that inhabit the Gulf of Thailand would be precious. At present, the conservation status of the local populations of these two species inhabiting Thai seas is challenging to assess because of the scarce information on the abundance, core habitat, specific threats, and the number of dead animals of these species over the past decades.

Conclusion

This study showed that ISSR can be used to successfully determine population structures and analyze the genetic diversity of T. aduncus and S. attenuata populations in the Thai Andaman Sea. At least three genetic clusters of both species were found, and the diversity indices of each species indicated that they are not at a critical level for extinction. However, due to the inadequate information on these populations and without proper monitoring plans, the abundance and genetic variability of these marine mammals may be significantly reduced in the near future. Thus, future studies are needed to address the existing knowledge gaps and to establish conservation plans for these populations.

Authors’ Contributions

PP: Conducted the study, analyzed the data, and wrote the manuscript. AP: Conducted the study and analyzed the data. KB: Supervised the laboratory work and wrote the manuscript. PK and KK: Provided the samples and relevant data. KN: Designed the study, analyzed the data, and wrote and finalized the manuscript. All authors have read and approved the final manuscript.

Acknowledgments

We dedicate the value of this research to the Phuket Marine Biological Center, for providing us with samples, data, and additional support in analyzing our results. The authors are grateful for research funding from the Excellence Center in Veterinary Bioscience (Grant no. 001-2022), Chiang Mai University, Chiang Mai 50200, Thailand, and the CMU Presidential Scholarship (Grant no. 001-2020), Chiang Mai University, Thailand.

Footnotes

The authors are grateful for research funding from the Excellence Center in Veterinary Bioscience (Grant no. 001-2022)

Competing Interests

The authors declare that they have no competing interests.

Publisher’s Note

Veterinary World remains neutral with regard to jurisdictional claims in published map and institutional affiliation.

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