Mitochondrial <scp>DNA</scp> Diversity and Distribution of <scp>F</scp> raser's ( <scp> <i>Lagenodelphis hosei</i> </scp> ) and Rough‐Toothed Dolphins ( <scp> <i>Steno bredanensis</i> </scp> ): Initial Genetic Data of Rare Cetaceans Stranded Along th

Fourteen species of coastal and oceanic cetaceans from the family Delphinidae have been identified in the Thai Andaman Sea (Department of Marine and Coastal Resources 2023), which forms a significant part of the eastern boundary of the Indian Ocean (Gibson et al. 2007). Opposite that are the Andaman and Nicobar Islands, which connect the Andaman Sea with the Bay of Bengal and Indian Ocean. The sea features a deep central basin with depths exceeding 3000 m, while the coastal areas near Thailand and the Andaman and Nicobar Islands are relatively shallow, averaging around 100 m (Gibson et al. 2007). To the south lies the northern coast of Sumatra and the Strait of Malacca, a channel that connects the Andaman Sea to the South China Sea and the Pacific Ocean (Gibson et al. 2007). The oceanography of the Andaman Sea is characterized by strong seasonal currents. From June to September, the southwest monsoon brings currents from the Bay of Bengal, while from December to May, the northeast monsoon drives currents through the Strait of Malacca (Isa et al. 2020; Wibowo et al. 2022; Gibson et al. 2007). These monsoonal currents also contribute to upwelling of deep water in the Thai Andaman (Yesaki 1981), along with drainage of nutrient-rich water from mangrove forests (Janekarn and Hylleberg 1989), enhancing marine productivity and supporting a high diversity of marine life. Fraser's (Lagenodelphis hosei) and rough-tooth (Steno bredanensis) dolphins are offshore oceanic species found in pantropical regions across all three major oceans (Jefferson 2009; Louella and Dolar 2018). Fraser's dolphins have a distributional range between 30° N and 30° S, primarily in deep waters (Louella and Dolar 2018). Similarly, rough-toothed dolphins prefer deep oceanic waters, but have a slightly broader range, spanning tropical to subtropical areas between 45° N and 35° S (Jefferson 2009). The range of both species also includes the western coast of Thailand along the Thai Andaman (Indian Ocean) and the eastern coast, bordering the Gulf of Thailand (Pacific Ocean) but they are considered rare based on limited numbers of stranding events (Department of Marine and Coastal Resources 2013a, 2013b). Notably, strandings of both species have occurred more frequently along the Thai Andaman coast, where the deeper waters may represent preferred habitat (Department of Marine and Coastal Resources 2013a, 2013b). Both Fraser's and rough-toothed dolphins are classified as Least Concern by the International Union for Conservation of Nature (IUCN) (Kiszka et al. 2019; Kiszka and Braulik 2018) and are listed in Appendix II of the Convention on International Trade in Endangered Species (CITES). In Thailand, both species are protected by the Wildlife Conservation and Protection Act, B.E. 2562 (2019). Stranding events involving these species along the Thai coastline have been recorded since 2006 (Figure 1). Historically, Fraser's dolphin had only been found in the Thai Andaman Sea. However, in 2020, a live stranding was observed in Narathiwat province in the Gulf of Thailand (Department of Marine and Coastal Resources 2020). The rough-toothed dolphin has also now been recorded in both the Gulf of Thailand and the Thai Andaman Sea. Two stranding events of three individuals have been reported: the first in 2008 (a live stranding in Phuket province) (Department of Marine and Coastal Resources 2013b) and the second in 2014 (a dead stranding in Satun province) (Figure 1) (Department of Marine and Coastal Resources 2014). In waters adjacent to Thailand, both species have been identified through strandings and sightings; for example, Fraser's dolphin in the Andaman and Nicobar Islands (Sanctuary Nature Foundation 2023) and Maldives (Ballance and Pitman 1998), Fraser's and rough-toothed dolphins in Myanmar (Thu et al. 2024), Indonesia (Rudolph et al. 1997), and Vietnam (Vu et al. 2020; Van 2009); however, the relationships among these regional populations—and their connection to other populations—remain unclear. Despite a wide oceanic distribution, obtaining population data for both species remains challenging due to difficulties associated with surveying such vast areas. Population genetic studies of Fraser's and rough-toothed dolphins have primarily relied on mitochondrial DNA (mtDNA) and microsatellite markers. For Fraser's dolphins, one such study utilized samples primarily from the waters of Japan, Taiwan, and the Philippines and found high genetic diversity in the mtDNA control region across all locations, with haplotype diversity values exceeding 0.9 (Chen et al. 2020). The study suggested that differences in oceanographic conditions between Japan and the Philippines may influence patterns of dispersion and isolation, leading to diverse maternal lineages (Chen et al. 2020; Miyazawa et al. 2009). In the Pacific Ocean, significant genetic differentiation was observed between the Philippines and Japan (FST = 0.029, p = 0.026) and between the Philippines and Taiwan (FST = 0.034, p = 0.022) (Chen et al. 2020), which agrees with skull morphological differences among those populations (Perrin et al. 2003). For rough-toothed dolphins, population differentiation was first identified between the Atlantic and Pacific by Da Silva et al. (2015) based on analysis of the mtDNA control region. Within the Atlantic Ocean, three genetically distinct populations were detected: one in the Caribbean Sea, and two in southeastern and southern Brazil (Donato et al. 2019; Da Silva et al. 2015). In the Pacific Ocean, genetic differentiation has also been observed across the North and South Pacific Ocean basins (Albertson et al. 2017). In addition, several insular populations with unique genetic signatures have been identified among the Islands in three archipelagos, Hawaiian Islands, Society Islands, and Samoan Islands, further highlighting population structure within the species (Albertson et al. 2017; Oremus et al. 2012). More recent studies analyzing populations from the Atlantic, Indian, and Pacific Oceans revealed high overall genetic diversity (H = 0.942, π = 0.016) and identified 51 haplotypes, with no shared haplotypes across the Atlantic, Indian, or Pacific Oceans (Albertson et al. 2022). This is suggestive of genetic isolation, likely driven by distinct oceanographic habitats, which has resulted in the delimitation of two subspecies for this species (Albertson et al. 2022). Neither is considered endangered on a global scale; however, regional population information is limited, particularly for the Indian Ocean. To date, there are no published reports of at-sea sightings of Fraser's and rough-toothed dolphins in Thai waters. Therefore, it remains unclear whether the recorded strandings represent isolated pelagic individuals that migrated between regions, or if they indicate the presence of a small, possibly unique, population in Thai waters. To address this uncertainty, we aimed to understand whether individuals found in Thai Andaman Sea are genetically different from those in other parts of their range. By providing new mtDNA control region sequences from stranded individuals in the Thai Andaman Sea, this study represents the first genetic study of both species in Thai waters. Our findings contribute to previous research (Albertson et al. 2022; Albertson et al. 2017; Chen et al. 2020; Da Silva et al. 2015), by filling a gap in genetic data from populations in the Indo-Pacific region, which remains underrepresented in existing studies. A total of 13 skin tissue samples were collected from dead, stranded dolphins along the Thai Andaman coastline between 2006 and 2017 (Fraser's dolphin, n = 6; rough-toothed dolphin, n = 7) by the Phuket Marine Biological Center (PMBC), Phuket, Thailand (Figure 1). These strandings occurred from April to September. All samples were preserved in 95% ethanol and stored at −20°C. Species identification was initially conducted by the veterinarian at the PMBC based on external morphological appearance. Use of the tissue samples in this study was approved by the Animal Use Committee of the Faculty of Veterinary Medicine, Chiang Mai University, Thailand, in 2020 (S14/2563). DNA extraction was performed using DNA extraction kits according to the manufacturer's instructions (DNeasy Blood & Tissue Kit, QIAGEN, Germany) at the Laboratory of the Faculty of Veterinary Medicine, Chiang Mai University. Extracted DNA concentrations were determined spectrophotometrically by measuring absorbance at A260/280. The mtDNA control region was amplified using primers DLF and DLR (Blair et al. 2014). This pair of primers has the ability to amplify the tRNA-Pro gene to the middle of control region, as shown for other cetacean species (Piboon, Poommouang, et al. 2022; Piboon, Kriengsakpichit, et al. 2022; Chantra et al. 2023). PCR reactions were conducted in a total volume of 25 μL consisting of 2.5 μL of 10X PCR buffer and 0.2 μL of Taq DNA polymerase (5 U/μL) (Vivantis, Selangor Darul Ehsan, Malaysia), 0.5 μL of 10 mM dNTPs (Vivantis, Selangor Darul Ehsan, Malaysia), 0.5 μL of 10 μM of each primer and 0.5 μL of DNA templates (50 ng/μL). The final volume was adjusted with deionized water. The PCR conditions were: 95°C for 5 min; 40 cycles of denaturation at 95°C for 30 s, an annealing step at 50°C for 30 s, and extension at 72°C for 1 min; 72°C for 10 min. The PCR products were sequenced by Macrogen (Seoul, South Korea). All 13 dolphin samples (Fraser's dolphin, n = 6 and rough-toothed dolphin, n = 7) were successfully sequenced and deposited in GenBank (Accession numbers MZ401170-175 and MZ401237-243). The mtDNA control region sequences of Fraser's (122 sequences) and rough-toothed dolphins (67 sequences) from other locations available in the GenBank database (Chen et al. 2020; Martien et al. 2024; Albertson et al. 2022; Albertson et al. 2017; Barragán-Barrera et al. 2023; Cunha et al. 2005; Oremus et al. 2012) were combined with our samples to generate a dataset for each species. The final two datasets and the accession numbers of all sequences are presented in Tables S1 and S2. The sequences of each dataset were aligned using Clustal W in MEGA11 (Tamura et al. 2021) and incorporated into their respective alignments, 400 bp for Fraser's dolphin (n = 128, Table S1) and 319 bp for rough-toothed dolphin (n = 74, Table S2). The haplotypes of the Thai samples and all other sequences in each dataset were determined using DnaSP version 6.12.3 (Rozas et al. 2017). To determine the genetic diversity of Thai samples, the number of haplotypes (Nh), the number of polymorphic site (S), haplotype diversity (H), nucleotide diversity (π) were also calculated using DnaSP (Rozas et al. 2017). A Median Joining Network (MJN) for each dataset was constructed using default settings in PopART version 1.7 (Leigh et al. 2015) to represent the haplotype relationships among samples from the Thai Andaman Sea and elsewhere. The conventional pairwise F-statistics (Weir and Cockerham 1984) (FST and ΦST) were estimated to observe the population differentiation between the Thai Andaman Sea and other areas of each species based on 10,000 permutations using Arlequin 3.5.2.2 (Excoffier and Lischer 2010). jModelTest version 2.1.10 (Darriba et al. 2012) was used to select the appropriate nucleotide substitution models for ΦST estimation. The best-fit models were identified under the Akaike Information Criterion (AIC) as GTR+I and TIM1+G for Fraser's and rough-toothed dolphin datasets, respectively. The Tamura-Nei model was then selected as the closest available approximation for both calculations. ΦST values were computed using 10,000 permutations, and significance was assessed at 0.05. In our study, 53 haplotypes with 52 polymorphic sites were identified for Fraser's dolphin dataset, while 54 haplotypes with 38 polymorphic sites were found for the rough-toothed dolphin dataset. For the Thai Andaman samples, both species showed an equal number of haplotypes (Nh = 5), while the number of polymorphic sites was six for Fraser's dolphins and nine for rough-toothed dolphins. Species with larger populations are more likely to have higher genetic diversity because they experience less genetic drift and inbreeding depression compared to smaller populations (Vachon et al. 2018; Pearson 2024). In previous studies, Fraser's and rough-toothed dolphins showed high levels of genetic diversity across most locations (Albertson et al. 2022; Chen et al. 2020). This may be related to high population size in some regions, such as Fraser's dolphin in the Sulu Sea, Philippines (Dolar et al. 2006; Kiszka and Braulik 2018), and rough-toothed dolphin in the eastern Tropical Pacific (Kiszka et al. 2019; Wade and Gerrodette 1993). Our samples also exhibited high haplotype diversity, despite the limited sample sizes for both species. The genetic diversity parameters showed that H was 0.933 ± 0.122 and 0.857 ± 0.137, while π was 0.006 ± 0.002 and 0.010 ± 0.002 for Thai Fraser's and rough-toothed dolphins, respectively. (See genetic diversity parameters of other locations in Table S3.) The Thai Andaman Sea in the Indo-Pacific region is known as a marine biological hotspot (Anrose et al. 2010; Gaither and Rocha 2013; Gibson et al. 2007) and a natural habitat for many marine animal species (Pradip Na Thalang et al. 2023; Kaewmong et al. 2016). Specific oceanographic conditions in particular areas, such as water depth, water temperature, oceanic currents, the abundance of prey species, or even anthropogenic threats, can all contribute to shaping dolphin population structure (Vachon et al. 2018). Among the haplotypes found in the Thai Andaman Sea for both species, three unique haplotypes not recorded before were identified for each species: Hap_51 (MZ401173), Hap_52 (MZ401174), and Hap_53 (MZ401175) for Fraser's dolphins (Table S4), and Hap_16 (MZ401243), Hap_17 (MZ401241), and Hap_18 (MZ401237) for rough-toothed dolphins (Table S5). Overall, the MJN of Fraser's dolphins revealed a star-like structure with several central haplotypes, for example, Hap_13, Hap_21, Hap_24, and Hap_29 from which multiple satellite haplotypes radiate, indicating shared ancestral lineages across regions (Figure 2). However, the Thai Andaman haplotypes were not clustered together but distributed within the network. Two unique haplotypes from the Thai Andaman Sea, Hap_51 and Hap_52, were connected with Hap_22, which was found in both Japan and the Thai Andaman Sea (our samples: MZ401170 and MZ401172), differing by only one mutational step (Figure 2). Another unique haplotype (Hap_53) was connected by a single mutational step to Hap_13, which includes individuals from the Pacific Ocean, Caribbean Sea, and Atlantic Ocean. Additionally, Hap_21 (MZ401171), also from the Thai Andaman Sea, was shared with individuals from Japan, Taiwan, the Philippines, and the Maldives (Figure 2). For rough-toothed dolphins, the MJN revealed a major separation between Atlantic and Pacific haplogroups. All haplotypes found in the Thai Andaman Sea were distributed within the Pacific haplogroup but not confined to a single branch. The three unique Thai haplotypes—Hap_16, Hap_17, and Hap_18—were connected to haplotypes from the Indian and Western Pacific regions by one to two mutational steps (Figure 3). Furthermore, Hap_2 (MZ401238–240) and Hap_19 (MZ401242), found in Thai waters, were shared with individuals from both the Indian Ocean and Western Pacific regions (Figure 3). This could suggest that the presence of stranded individuals in the area may be a result of dispersal from a larger population located elsewhere outside the Andaman Sea. Further sampling could help clarify this and potentially reveal additional maternal lineages. Pairwise FST values for our Frasier's dolphins exhibited generally low and nonsignificant levels of genetic differentiation from populations in the Central North Pacific, Maldives, Japan, Taiwan, and the Philippines, likely due to shared haplotypes (Table 1). The ΦST values showed potential signs of forming a distinct subpopulation differing from known populations in the Maldives, Philippines, or Western Pacific (Table 1); however, the evidence is not conclusive. For example, the ΦST value between the Thai Andaman and Central North Pacific populations was relatively high and consistent in magnitude with other comparisons, but the difference only approached statistical significance (ΦST = 0.181, p = 0.06). In addition, the elevated ΦST values observed for the Thai Andaman population relative to other regions are most likely explained by the sample size (n = and the of Within this three unique haplotypes were each differing by a single step from haplotypes in Japan, the Philippines, and Taiwan, in to two haplotypes shared with other these haplotypes are not ΦST is by both haplotype and sample and the presence of several unique haplotypes among only six individuals could ΦST this may suggest that individuals from the Thai Andaman Sea are part of a broader pelagic population with ancestral haplotypes with the broader Indo-Pacific as observed in the MJN shared to the Maldives, Philippines, Taiwan, and The presence of dolphins in Thai Andaman Sea may from that near this areas of the Andaman and Nicobar Islands and parts of the Bay of Bengal, where sightings of a have been (Sanctuary Nature Foundation 2023). Among these the Fraser's dolphin population in waters is considered the and closest to the Thai Andaman Sea and the Gulf of Thailand (Dolar et al. 2006; Kiszka and Braulik 2018), of the Indian Ocean remains studies have shown that the Fraser's dolphins in waters from those in Japan and Taiwan, due to differences in oceanographic conditions (Chen et al. 2020). In the Sulu Sea, the species a for deep offshore waters, with most observed at depths from to m, and in coastal areas where deep waters (Dolar et al. In the Thai Andaman Sea with the limited oceanic from 100 to a of around Phuket (Gibson et al. 2007). These may areas and the presence of dolphins, potentially the of sightings of this species in the region. data on Fraser's dolphins in the Thai Andaman Sea have from stranded recorded between April and September. This to the and monsoon (Gibson et al. et al. 2020; Wibowo et al. which brings strong and in currents from the Bay of Bengal that may the and Additionally, in prey or in water due to coastal and offshore upwelling may offshore dolphins to the of stranding along the coast et al. et al. 2020; et al. 2010). In offshore areas, particularly the central Andaman deeper waters (Gibson et al. the that these dolphins deeper offshore and only the coast the monsoon the of monsoon and currents may from deeper offshore waters the Thai possibly the seasonal of For rough-toothed dolphins, our FST with the findings of Albertson et al. which suggest that populations in the Indian and Pacific Oceans are not genetically particularly between the Indian Ocean and western Pacific Ocean, a of haplotype structure among these populations (Table 2). However, significant differentiation was observed between the Thai Andaman population and those from the Central Pacific, Pacific, Atlantic, and Western Atlantic (Table 2). The ΦST values were consistent with the FST with the of the Central Pacific (Table that these individuals are possibly from pelagic populations within the Indian Ocean or western This is by the that samples from the western and central Indian Ocean 1 in Albertson et al. not significant genetic differentiation from the Thai Andaman samples Indian Furthermore, Albertson et al. significant ΦST differentiation between Indian Ocean populations and both Central and Pacific However, in our study, populations from the western and central Indian Ocean, Thai Andaman in the eastern Indian Ocean, were genetically distinct only from the of our samples may gene or shared ancestral haplotypes between the Indian Ocean and Western and Central Pacific, while the Pacific remains more genetically some populations of rough-toothed dolphins in the Pacific population with genetic differentiation even between relatively (Albertson et al. 2017; Oremus et al. our findings suggest that populations in the Indian Ocean may broader and dispersal genetic across oceanic regions, even across regions of the Indian Ocean. However, this remains as sampling for rough-toothed dolphins within the Indian Ocean is A more of population and structure broader particularly from underrepresented areas such as the and southern Indian Ocean. such data the of structure across the Indian Ocean be with the in sample size for both species, obtaining additional samples in the and genetic data could a of their population structure and Further of these populations in Thai waters are to determine whether or areas as for these species. Additionally, the samples of Fraser's and rough-toothed dolphins from the Gulf of Thailand are for and studies. These data may reveal further population potentially populations in the Western In our study on two species of pelagic tropical marine that are rare in the Thai Andaman Sea. Our revealed the presence of unique haplotypes for both species, to date, have only been identified in the Thai Andaman region. Notably, Fraser's dolphin showed a potential of a genetically distinct limited dispersal from known populations in the Maldives, Philippines, or western Pacific but more samples are to this In rough-toothed dolphins in the region are likely to be part of a connected pelagic population spanning the Indian and western Pacific These findings the for genetic and sampling across the Indo-Pacific to understand population and for offshore cetaceans in this region. Piboon, and and Kaewmong and and and and and and All have and to the published version of the the value of this research to the of Veterinary Medicine, the Faculty of Veterinary Medicine, Chiang Mai University, Chiang Thailand and the from Phuket Marine Biological Center (PMBC), Phuket, for their which to the of this This was by the This study was approved by the Animal Use Committee of the Faculty of Veterinary Medicine, Chiang Mai University, Thailand, in 2020 (S14/2563). The no of The had no in the of the in the or of in the of the or in the to the The data that the findings of this study are available in the Information of this Table The accession number of Fraser's dolphin sequences used in this Table The accession number of rough-toothed dolphin sequences used in this Table The genetic diversity parameters of Fraser's and rough-toothed dolphins datasets based on mitochondrial DNA control region. Table The haplotypes of Fraser's dolphins. Table The haplotypes of rough-toothed dolphins. The is not for the or of supporting information by the be to the for the