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.

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.

Relationship of stranded cetaceans in Thai territorial waters to global populations: Mitochondrial DNA diversity of Cuvier's beaked whale, Indo Pacific finless porpoise, pygmy sperm whale, and dwarf sperm whale.

Cetaceans inhabit oceans throughout the world. Four specific odontocetes, namely Cuvier's beaked whale (Ziphius cavirostris), Indo Pacific finless porpoise (Neophocaena phocaenoides), pygmy sperm whale (Kogia breviceps), and dwarf sperm whale (Kogia sima), have occasionally been found stranded along Thailand's coastal waters (the Andaman Sea and the Gulf of Thailand). Although shared haplotypes of each species for many locations have been found, and some species have revealed genetic structure through haplotype networks, cetaceans in Thai waters have never been investigated in terms of comparing haplotypes to those that have existed before. Herein, we have illustrated the matrilineally phylogeographic relationships among worldwide populations through Bayesian Phylogenetic tree computations using Markov Chain Monte Carlo (MCMC) and Median-Joining Networks (MJNs). Unique haplotypes of the control region mitochondrial DNA of Thai odontocetes were found for all species. Moreover, a high degree of worldwide haplotype diversity (hd) above 0.8 among the four species was detected, while the lowest degree of nucleotide diversity (π) was observed in the Indo Pacific finless porpoise (1.12% ± 0.184%). An expansion of the effective female population size worldwide of three odontocete species was detected using Bayesian Skyline Reconstruction, but this did not include the Indo Pacific finless porpoise. Because Thai seas are located within the Indo Polynesian province, where this biodiversity hotspot exists, we speculate that these odontocetes may also inhabit specific habitats within the Malay Peninsula and Thailand's territorial waters. Therefore, closer attention and monitoring of these cetacean populations will be necessary for future conservation efforts.

Microsatellite Polymorphism and the Population Structure of Dugongs (Dugong dugon) in Thailand.

The dugong (Dugong dugon) is an endangered species of marine mammals, so knowledge of genetic diversity of these populations is important for conservation planning within different habitats. In this study, six microsatellite markers were used to assess the genetic diversity and population structure of 77 dugongs from skin samples of stranded animals collected from 1994-2019 (69 from Andaman Sea and 8 from the Gulf of Thailand). Our results found that dugongs in the Andaman Sea had higher genetic variation than those in the Gulf of Thailand. Populations in Trang, Satun, and some areas of Krabi had highest diversity compared to other regions of Thailand. Bayesian genetic clustering analysis revealed that dugongs in Thailand consist of five genetic groups. Moreover, dugongs in the middle and lower Andaman Sea presented the greatest gene flow compared to other regions. However, based on calculation of inbreeding coefficients (Fis value = 0.239), dugong populations in the Sea of Thailand are experiencing some levels of inbreeding, and so may warrant special protections. These results provide important information for understanding the genetic status of dugongs that can lead to improved management and conservation of this endangered species.

Life Expectancy in Marine Mammals Is Unrelated to Telomere Length but Is Associated With Body Size.

Marine mammals vary greatly in size and lifespan across species. This study determined whether measures of adult body weight, length and relative telomere length were related to lifespan. Skin tissue samples (n = 338) were obtained from 23 marine mammal species, including four Mysticeti, 19 Odontoceti and one dugong species, and the DNA extracted to measure relative telomere length using real-time PCR. Life span, adult body weight, and adult body length of each species were retrieved from existing databases. The phylogenetic signal analysis revealed that body length might be a significant factor for shaping evolutionary processes of cetacean species through time, especially for genus Balaenoptera that have an enormous size. Further, our study found correlations between lifespan and adult body weight (R 2 = 0.6465, p < 0.001) and adult body length (R 2 = 0.6142, p ≤0.001), but no correlations with relative telomere length (R 2 = -0.0476, p = 0.9826). While data support our hypothesis that larger marine mammals live longer, relative telomere length is not a good predictor of species longevity.

Feasibility of melting fingerprint obtained from ISSR-HRM curves for marine mammal species identification.

Currently, species identification of stranded marine mammals mostly relies on morphological features, which has inherent challenges. The use of genetic information for marine mammal species identification remains limited, therefore, new approaches that can contribute to a better monitoring of stranded species are needed. In that context, the ISSR-HRM method we have proposed offers a new approach for marine mammal species identification. Consequently, new approaches need to be developed to identify individuals at the species level. Eight primers of the ISSR markers were chosen for HRM analysis resulting in ranges of accuracy of 56.78-75.50% and 52.14-75.93% in terms of precision, while a degree of sensitivity of more than 80% was recorded when each single primer was used. The ISSR-HRM primer combinations revealed a success rate of 100% in terms of discrimination for all marine mammals included in this study. Furthermore, ISSR-HRM analysis was successfully employed in determining marine mammal discrimination among varying marine mammal species. Thus, ISSR-HRM analysis could serve as an effective alternative tool in the species identification process. This option would offer researchers a heightened level of convenience in terms of its performance and success rate. It would also offer field practice to veterinarians, biologists and other field-related people a greater degree of ease with which they could interpret results when effectively classifying stranded marine mammals. However, further studies with more samples and with a broader geographical scope will be required involving distinct populations to account for the high degree of intraspecific variability in cetaceans and to demonstrate the range of applications of this approach.

Genetic diversity in a unique population of dugong (Dugong dugon) along the sea coasts of Thailand.

Dugong (Dugong dugon) populations have been shrinking globally, due in large part to habitat fragmentation, degradation and ocean pollution, and today are listed as Vulnerable by the IUCN. Thus, determining genetic diversity in the remaining populations is essential for conservation planning and protection. In this study, measures of inter-simple sequence repeat (ISSR) markers and mtDNA D-loop typing were used to evaluate the genetic diversity of 118 dugongs from skin samples of deceased dugongs collected in Thai waters over a 29-year period. Thirteen ISSR primers revealed that dugongs from the Andaman Sea and Gulf of Thailand exhibited more genetic variation in the first 12 years of the study (1990-2002) compared to the last decade (2009-2019). Dugongs from the Andaman Sea, Trang, Satun and some areas of Krabi province exhibited greater diversity compared to other coastal regions of Thailand. Eleven haplotypes were identified, and when compared to other parts of the world (235 sequences obtained from NCBI), five clades were apparent from a total 353 sequences. Moreover, dugongs from the Andaman Sea were genetically distinct, with a separate haplotype belonging to two clades found only in Thai waters that separated from other groups around 1.2 million years ago. Genetic diversity of dugongs in present times was less than that of past decades, likely due to increased population fragmentation. Because dugongs are difficult to keep and breed in captivity, improved in situ conservation actions are needed to sustain genetically healthy wild populations, and in particular, the specific genetic group found only in the Andaman Sea.

Age relationships with telomere length, body weight and body length in wild dugong (Dugong dugon).

The ability to estimate age and determine the growth status of free-ranging dugongs (Dugong dugon) is vital to providing insight into the basic biology of this endangered species. Currently, age estimation in dugong carcasses relies on counting dentin growth layer groups (GLGs) in tusks, but a disadvantage is they need to be intact. We explored whether measures of telomere length could be used as an alternative approach to age estimation in dugongs given that in other species, telomere length and age are inversely related. In this study, relative telomere length (rTL) was measured by qPCR in skin samples from 24 dugongs of varying ages determined by counts of GLGs. In addition, relationships between age by GLG counts and body weight and length and were examined. Our findings indicate that age estimated by GLGs was negatively correlated with telomere length using the logistic formula with a rate of telomere attrition of approximately 0.036 rTL/year between the ages of 5-20 years. By comparison, both body weight and length were positively correlated with GLG-based age, with growth rates of ~8.8 kg/year for weight and ~3.58 cm/year for length, respectively. After that, growth rates slowed substantially and then plateaued. The results suggest that physical maturity in dugongs occurs at 20 years of age and that measures of rTL might serve as a tool for age estimation in dugongs, living and deceased.