Mitochondrial DNA Control Region Research Articles

Riverscape features determining the genetic structure of the Yellow‐Spotted Amazon River Turtle (Podocnemis unifilis) in Brazilian Amazonia

AbstractLandscape characteristics can influence gene flow depending on the species ability to disperse. This imposes different levels of resistance to movement, determining the genetic structure and diversity of populations. Waterfalls and rapids in the Amazon basin have been suggested as contributing factors to the diversification and genetic structure of freshwater species, including turtles. Thus, along some main rivers of the Brazilian Amazon: Juruá, Madeira, Amazonas, Trombetas, Tapajós, Xingu, Tocantins, and Araguaia, we evaluated the spatial distribution of genetic diversity and characterized the population structure of Podocnemis unifilis, a widely distributed and endangered Amazonian turtle, using the mitochondrial DNA control region. We tested for isolation by geographic distance and by resistance models through an integrative approach using genetic, geographic, and ecological data, comparing these matrices with genetic distance. We found heterogeneous levels of genetic diversity and no spatial patterns. Results supported the isolation by resistance hypothesis, where the movement of individuals was influenced by freshwater, vegetational, climatological, and geomorphological variables, explaining the genetic distance of P. unifilis. Rapids and waterfalls impose greater resistance although they are barriers with different levels of permeability to the P. unifilis gene flow. Thus, the idiosyncrasies of each sub‐basin must be considered in the interpretation of the patterns found in assessing phylogeographic history.Abstract in Portuguese is available with online material.

Mixed stock analysis identifies natal origins of green turtles at foraging grounds in southeastern Australia

IntroductionIdentifying critical habitats for marine turtles and connectivity between genetic stocks and the foraging grounds they use is a conservation priority worldwide. Green turtles (Chelonia mydas) are impacted by a wide range of anthropogenic threats that can vary by geographic region and the ontogenetic stage of the individual. In Australia, the strengthening of the East Australian Current due to climate change is increasing ocean temperatures, particularly in southern New South Wales (NSW), which is having large-scale impacts on the distribution and abundance of marine resources. Green turtles are frequently observed in temperate southern NSW waters, but our knowledge of their habitat use, migration patterns and the impact of threatening processes is limited.MethodsTo assess the origins of green turtles from foraging grounds in southeastern Australia, samples were obtained from green turtles of all size classes (post-hatchlings to adults) that had stranded along an ~870 km expanse of the NSW coast and Lord Howe Island between 1997 and 2021. Mitochondrial DNA control region sequences for 283 individuals were compared to 25 potential source genetic stocks in the Indo-Pacific using mixed-stock analysis.ResultsA total of 26 haplotypes were identified in NSW, of which 14 had been previously observed at a rookery, eight had been identified previously, but not at a rookery (i.e. orphan haplotypes), and four were previously undescribed. Mixed-stock analysis revealed that NSW waters support multiple genetic stocks but are dominated by those of the southern Great Barrier Reef and New Caledonia genetic stocks. A small proportion of green turtles originated from more distant stocks in the Indo-Pacific region.DiscussionUnderstanding the connectivity between green turtle rookeries and foraging grounds provides an opportunity to assess the impact of anthropogenic threats to turtle stocks, and in turn, prioritize management actions for the conservation of green turtles across regional, national and international jurisdictions.

Multiple secondary contacts and historic hybridization between glacial lineages of charr (Salvelinus, Salmonidae) in northeastern Asia

AbstractArctic regions of northeastern Asia represent areas of secondary contact of the glacial phylogenetic lineages of charrs belonging to the genus Salvelinus (Salmoniformes: Salmonidae). However, the post‐glacial dispersion of charr across Arctic regions is poorly understood, as knowledge of populations from Chukotka and its neighbouring areas remains limited. Specifically, there is no clear understanding of which charr species inhabit significant regions of Chukotka from the sea coasts to the Kolyma drainage. In this study, we explored the affiliation of lacustrine charrs from the Chukotka area with (1) the Arctic lineage of Taranetz' charr (Salvelinus taranetzi); (2) the Bering lineage of Northern Dolly Varden (Salvelinus malma malma) and (3) the Siberia and Atlantic lineages of Arctic charr (Salvelinus alpinus). We analysed sequence variation of the mitochondrial DNA control region (mtDNA CR; 960 base pairs) and genotyped seven microsatellite loci of nuclear DNA from charr collected at 13 sampling sites. We found different consequences of secondary contact: (1) complete fixation of introgressed mtDNA (mitochondrial capture) and (2) preservation of several mtDNA lineages with the absence of contemporary gene flow between resident populations. Combining the distribution patterns, phylogenetic network topology and knowledge of the glaciation history of the region, we propose two zones of secondary contact of the glacial lineages in Chukotka–Kolima‐Chukotka River system and Paleo‐Amguema River–from where charrs with introgressed genomes spread throughout this range. However, in some cases, the process of foreign mtDNA capture likely occurs in a more localised manner.

Does the Artificial Enhancement and Release Activity Affect the Genetic Diversity of Marbled Rockfish Sebastiscus marmoratus in Zhoushan Waters?

Artificial enhancement and release activity is an important method in the restoration of fishery resources. In order to understand the possible genetic effect of hatchery-released populations on wild populations during the artificial enhancement and release activities of Sebastiscus marmoratus in Zhoushan waters, we utilized mitochondrial DNA control region sequences to examine the genetic diversity in four S. marmoratus populations, including one farmed population, one released population and two wild populations. A total of 68 haplotypes from 123 individuals were detected, including 3 shared haplotypes. Haplotype diversity ranged from 0.944 to 0.980, with a mean of 0.966. The nucleotide diversity ranged from 0.020 to 0.025, with a mean of 0.022. Analysis of Molecular Variance (AMOVA) indicated that the primary genetic variation occurs within populations and the index of genetic differentiation between populations (FST) among the four populations showed no differentiation. The results indicate that the current artificial enhancement and release has not impacted the S. marmoratus population in Zhoushan waters. Continued long-term monitoring is essential to protect the high-quality germplasm resources of S. marmoratus.

Capillary-Electrophoresis-Based Species Barcoding of Big Cats: CR-mtDNA-Length Polymorphism.

This study aimed to provide an overview of the methodological approach used for the species determination of big cats. The molecular system described herein employs mitochondrial DNA control region (CR-mtDNA)-length polymorphism in combination with highly sensitive and precise capillary electrophoresis. We demonstrated that the described CR-mtDNA barcoding system can be utilized for species determination where the presence of biological material from big cats is expected or used as a confirmatory test alongside Sanger or massive parallel sequencing (MPS). We have also addressed the fact that species barcoding, when based on the analysis of mtDNA targets, can be biased by nuclear inserts of the mitochondrial genome (NUMTs). The CR-mtDNA barcoding system is suitable even for problematic and challenging samples, such as hair. CR-mtDNA-length polymorphisms can also distinguish hybrids from pure breeds.

Determining evolutionary origin and phylogenetic relationships of mallard-like ducks of Oceania, greater Indonesia, and the Philippines with ddRAD-seq data

AimWe aim to determine the evolutionary origins and population genetics of mallard-like ducks of Oceania, greater Indonesia, and the Philippines. LocationOceania, greater Indonesia, and the Philippines. TaxonMallard (Anas platyrhynchos), Pacific black duck (A. superciliosa spp.), and Philippine duck (A. luzonica) MethodsThousands of nuclear ddRAD-seq loci and the mitochondrial DNA control region were assayed across individuals representative of each species’ range. We assessed population structure and phylogenetic relationships, as well as estimated demographic histories to reconstruct the biogeographical history of each species. ResultsPhilippine and Pacific black ducks represent unique genetic lineages that diverged from the mallard 1–2 million years ago. We find no support for the Philippine duck representing a hybrid species as once posited; however, their low levels of genetic diversity requires further attention. We find a lack of substructure among Philippine ducks. However, we found pronounced differentiation between subspecies of Pacific black ducks, especially between A. s. superciliosa from New Zealand and A. s. rogersi from Australia, Tasmania, Papua New Guinea, and Timor-Leste. Anas superciliosa pelewensis gave mixed results; individuals from the Solomon Islands were differentiated from the other subspecies, but those from the island of Aunu’u, American Samoa, were genetically more similar to A. s. rogersi than A. s. pelewensis samples from the Solomon Islands. Finally, we find limited evidence of interspecific gene flow at evolutionary scales, and mallard introgression among contemporary samples. Main conclusionsMallard-like ducks radiated across Oceania, greater Indonesia, and the Philippines within the last 2 million years. Only the Pacific black duck showed unique sub-structuring that largely followed known sub-species ranges, except for A. s. pelewensis. We posit that the high interrelatedness among Solomon Island samples suggests that their genetic distinctiveness may simply be the result of high levels of genetic drift. In contrast, we conclude that mainland Australian Pacific black ducks were the most likely source for the recent colonization of American Samoa. As a result, our findings suggest that either the A. s. pelewensis subspecies designations and/or its geographical range may require re-evaluation. Continued re-evaluation of evolutionary and taxonomic relationships is necessary when attempting to reconstruct and understand biogeographical histories, with important implications towards any attempts to implement conservation strategies.

Where east meets west: Phylogeography of the high Arctic North American brant goose.

Genetic variation in Arctic species is often influenced by vicariance during the Pleistocene, as ice sheets fragmented the landscape and displaced populations to low- and high-latitude refugia. The formation of secondary contact or suture zones during periods of ice sheet retraction has important consequences on genetic diversity by facilitating genetic connectivity between formerly isolated populations. Brant geese (Branta bernicla) are a maritime migratory waterfowl (Anseriformes) species that almost exclusively uses coastal habitats. Within North America, brant geese are characterized by two phenotypically distinct subspecies that utilize disjunct breeding and wintering areas in the northern Pacific and Atlantic. In the Western High Arctic of Canada, brant geese consist of individuals with an intermediate phenotype that are rarely observed nesting outside this region. We examined the genetic structure of brant geese populations from each subspecies and areas consisting of intermediate phenotypes using mitochondrial DNA (mtDNA) control region sequence data and microsatellite loci. We found a strong east-west partition in both marker types consistent with refugial populations. Within subspecies, structure was also observed at mtDNA while microsatellite data suggested the presence of only two distinct genetic clusters. The Western High Arctic (WHA) appears to be a secondary contact zone for both Atlantic and Pacific lineages as mtDNA and nuclear genotypes were assigned to both subspecies, and admixed individuals were observed in this region. The mtDNA sequence data outside WHA suggests no or very restricted intermixing between Atlantic and Pacific wintering populations which is consistent with published banding and telemetry data. Our study indicates that, although brant geese in the WHA are not a genetically distinct lineage, this region may act as a reservoir of genetic diversity and may be an area of high conservation value given the potential of low reproductive output in this species.

A renewed glance at the Palearctic golden eagle: Genetic variation in space and time.

Anthropogenic pressures on nature have been causing population declines for centuries. Intensified persecution of apex predators, like the golden eagle, resulted in population bottlenecks during the 19th and 20th centuries. To study population genetics and demographic history of the golden eagle throughout its distribution, we collected museum samples from previously underrepresented regions, such as Russia and Central Asia. We used 12 microsatellite loci and a fragment of the mitochondrial DNA control region to re-evaluate phylogeography of Eurasian golden eagles and study the impacts of the population bottleneck. Our results revealed a north-south genetic gradient, expressed by the difference between Mediterranean and Holarctic lineages, as well as genetically distinct Northern Europe and Central Asia and Caucasus regions. Furthermore, Northern Europe exhibited the lowest, whereas Central Asia and Caucasus had the highest genetic diversity. Although golden eagles maintained relatively high genetic diversity, we detected genetic signatures of the recent bottleneck, including reduced genetic diversity and a decline in the effective female population size around the year 1975. Our study improves the knowledge of the genetic composition of Eurasian golden eagles and highlights the importance of understanding their historical population dynamics in the face of ongoing and future conservation efforts.

Genetic structuring of Fishers in British Columbia, Canada: implications for population conservation and management

Abstract In the westernmost Canadian province of British Columbia, fishers (Pekania pennanti) occur in low-elevation forested ecosystems in the central and northern portions of the province, with several substantial mountain ranges and rivers bisecting this distribution. The effect of these geographic features on gene flow and population structuring is unknown and may contribute to fishers differentiating into 2 or more populations within the province. To better understand population structuring within the range of fishers in British Columbia, we collected tissue samples from 491 fishers from throughout the province and evaluated variation of neutral genetic markers using a 385-base pair section of the mitochondrial DNA control region and 13 microsatellite loci. Fishers appeared to be genetically structured as hierarchical stepping-stone populations where an upper hierarchical level of genetic partitioning resulted in 2 populations at the provincial scale, with 3 subpopulations occurring in the central interior region. We observed gene flow of 2 to 5 migrants per generation between the 2 upper populations, which was substantially less than the 90 migrants per generation estimated as needed to maintain genetic homogeneity. This resulted in differentiated upper populations (haplotype FST = 0.272, microsatellite FST = 0.049) characterized by relatively low Ne (Columbian population = 252, 95% CI = 185 to 332; Boreal population [British Columbia only] = 136, 95% CI = 92 to 234). The consequence of low gene flow and resultant population structuring is that the Columbian population likely receives relatively little genetic and demographic support from other populations and, combined with precipitous recent declines in its numbers, puts this population at heightened conservation risk. As a result, swift and effective actions are needed to conserve this vulnerable population of fishers.

The sacred deer conflict of management after a 1000‐year history: Hunting in the name of conservation or loss of their genetic identity

AbstractHuman–wildlife conflict involves diverse stakeholders with conflicting values. Resolving such conflicts necessitates the development of management plans rooted in scientific knowledge and establishing of social consensus. In Nara City, situated within the Japanese Archipelago, wild sika deer (Cervus nippon) hold sacred significance due to religious reasons and have been protected for over a millennium, resulting in a distinct genetic identity. However, the escalating deer population has caused significant agricultural damage in the areas surrounding the sanctuary. Consequently, a debate has arisen regarding the advantages and disadvantages of implementing lethal measures to address individuals that might be considered sacred deer but are regarded as pests in the vicinity of the sanctuary. Here, we used mitochondrial DNA (mtDNA) control region and nuclear simple sequence repeat (SSR) markers to detect the origin of deer in the areas neighboring the sanctuary (management areas). As a result, two genetic populations of deer were detected in Nara City. In the sanctuary, we detected only one specific mtDNA haplotype (S4). On the other hand, seven haplotypes, including S4, were detected in the management area. SSR analysis also suggested that the sika deer in the management area may be an admixed population of multiple origins from the sanctuary and out of Nara City. Interbreeding populations may expand into the sanctuary, and unique genetic populations for more than 1000 years may disappear. This study suggests that ordinary deer could soon replace the deer revered and protected by the people of Nara. Additionally, the proximity of sanctuary deer to tourists worldwide and the interaction with wild deer in other areas pose a potential risk of spreading zoonotic diseases. Urgent decisions are required to determine whether to advocate for extermination in managed areas for ‘conservation’ purposes or risk losing the genetic identity of the sacred deer.

Evaluation of the Impact of Population Management on the Genetic Parameters of Selected Spiral-Horned Antelopes.

The rapid loss of biodiversity and the associated reduction and fragmentation of habitats means that ex situ populations have become an important part of species conservation. These populations, which are often established from a small number of founders, require careful management to avoid the negative effects of genetic drift and inbreeding. Although the inclusion of molecular data is recommended, their availability for captive breeding management remains limited. The aim of this study was to evaluate the relationship between the levels of genetic diversity in six spiral-horned antelope taxa bred under human care and their respective management strategies, conservation status, demography, and geographic origin, using 10 nuclear DNA microsatellite loci and mitochondrial control region DNA sequences. Our findings include associations between genetic diversity and management intensity but also with the diversity and contribution of wild populations to captive founders, with some populations apparently composed of animals from divergent wild lineages elevating captive genetic diversity. When population sizes are large, the potential advantages of maximizing genetic diversity in widely outcrossed populations may need careful consideration with respect to the potential disruption of adaptive diversity. Genetic data serve as a robust tool for managing captive populations, yet their interpretation necessitates a comprehensive understanding of species biology and history.

Study on the relationship between the dispersal of wild boar (Sus scrofa) and the associated variability of Cesium-137 concentrations in its muscle Post-Fukushima Daiichi Nuclear Power Plant accident

After the Fukushima Daiichi Nuclear Power Plant (FDNPP) accident in 2011, the wild boar (Sus scrofa) population within the Fukushima Evacuation Zone (FEZ) increased substantially in size and distribution. This growing population and their potential dispersal from the FEZ, where they are exposed to high levels of radionuclides, into the surrounding landscape underscores the need to better understand boar movement patterns in order to establish policies for managing shipping restrictions for boar meat and develop management strategies. In this study, we quantified the genetic population structure of boar in and around Fukushima prefecture using sequence data of the mitochondrial DNA control region and MIG-seq analysis using 348 boar samples to clarify boar dispersal patterns. Among boar samples, seven Asian haplotypes and one European haplotype were detected. The European haplotype originated from hybridization between domestic pigs and native boar in the evacuation zone after the accident and was detected in 15 samples across a broad geographic area. Our MIG-seq analysis revealed genetic structure of boar was significantly different between boar inhabiting the eastern (including FEZ. i.e., East clade) and western (i.e., West clade) regions in Fukushima prefecture. In addition, we investigated the relationships between boar dispersal and Cesium (Cs)-137 activity concentrations in boar muscle using MIG-seq genetic data in Nihonmatsu city, located in the central-northern region of Fukushima. High Cs-137 activity concentrations, exceeding 1000 Bq/kg, in boar muscle had a significantly high probability of belonging to the East clade within localized regions. Thus, our results provide evidence of the spatial scale of dispersal of individuals or offspring of boar from the FEZ. Results of this research also indicate that dispersal of individuals between areas with different Cs-137 contamination levels is one of the biggest factors contributing to variation in Cs-137 activity concentration in boar muscle within localized regions.

Introduced and extinct: neglected archival specimens shed new light on the historical biogeography of an iconic avian species in the Mediterranean.

Collection specimens provide valuable and often overlooked biological material that enables addressing relevant, long-unanswered questions in conservation biology, historical biogeography, and other research fields. Here, we use preserved specimens to analyze the historical distribution of the black francolin (Francolinus francolinus, Phasianidae), a case that has recently aroused the interest of archeozoologists and evolutionary biologists. The black francolin currently ranges from the Eastern Mediterranean and the Middle East to the Indian subcontinent, but, at least since the Middle Ages, it also had a circum-Mediterranean distribution. The species could have persisted in Greece and the Maghreb until the 19th century, even though this possibility had been questioned due to the absence of museum specimens and scant literary evidence. Nevertheless, we identified four 200-year-old stuffed black francolins-presumably the only ones still existing-from these areas and sequenced their mitochondrial DNA control region. Based on the comparison with conspecifics (n = 396) spanning the entirety of the historic and current species range, we found that the new samples pertain to previously identified genetic groups from either the Near East or the Indian subcontinent. While disproving the former occurrence of an allegedly native westernmost subspecies, these results point toward the role of the Crown of Aragon in the circum-Mediterranean expansion of the black francolin, including the Maghreb and Greece. Genetic evidence hints at the long-distance transport of these birds along the Silk Road, probably to be traded in the commerce centers of the Eastern Mediterranean.

Population structure and demographic history for year cohort dynamics of landlocked ayu Plecoglossus altivelis altivelis in dam reservoir of Japan.

Ayu Plecoglossus altivelis altivelis is a valuable osmeroid species for inland fishery in Japan. It is classified into two ecological forms of amphidromous migrating between rivers and sea and landlocked migrating between rivers and lakes or dam reservoirs. The number of dams and their reservoirs has remarkably increased in the twenty-first century under climate change, because of their respective roles in hydropower generation with negligible carbon emissions and in flood control. Dam reservoirs therefore become increasingly important as inland nursery grounds of ayu. In this study, we investigated the reproduction status of landlocked ayu migrating in the Haidzuka Dam reservoir and the Tabusa River in western Japan by molecular phylogenetic analysis based on population structure and demographic history for year cohort dynamics. A total of 849 individuals were collected monthly from October 2018 to September 2021 according to an annual life cycle of ayu. Nucleotide sequences of the partial mitochondrial DNA control region yielded 31 haplotypes, consisting of 4 shared haplotypes among the 2019, 2020 and 2021 cohorts and 27 unique haplotypes. The overall haplotype diversity and nucleotide diversity were calculated to be relatively low at 0.3503 ± 0.0206 and 0.0077 ± 0.0045, respectively, suggesting a founder event by dominant haplotypes. Star-shaped radiational haplotypes from dominant shared haplotypes on the median-joining network likely support a founder event. Although pairwise ФST values were determined to be very low among the year cohorts, only the 2019 cohort was found to have a significant difference from the 2020 and 2021 cohorts, for both of which Tajima's D values were also statistically significant. For the overall population, multimodal mismatch distribution and negative Tajima's D and Fu's Fs values in the neutrality test suggested population expansion or population subdivision. The native riverine population in the Tabusa River suffered habitat fragmentation and population bottleneck from dam construction, and therefore severe founder effect remained behind the artificially landlocked population with a low level of genetic diversity in the Haidzuka Dam reservoir.

Analysis of maternal genetic structure of mitochondrial DNA control region from Tai-Kadai-speaking Buyei population in southwestern China

BackgroundEven though the Buyei are a recognised ethnic group in southwestern China, there hasn’t been much work done on forensic population genetics, notably using mitochondrial DNA. The sequences and haplogroups of mitochondrial DNA control regions of the Buyei peoples were studied to provide support for the establishment of a reference database for forensic DNA analysis in East Asia.Methods and resultsThe mitochondrial DNA control region sequences of 200 Buyei individuals in Guizhou were investigated. The haplotype frequencies and haplogroup distribution of the Buyei nationality in Guizhou were calculated. At the same time, the paired Fst values of the study population and other populations around the world were computed, to explore their genetic polymorphism and population relationship. A total of 179 haplotypes were detected in the Buyei population, with frequencies of 0.005–0.015. All haplotypes were assigned to 89 different haplogroups. The haplotype diversity and random matching probability were 0.999283 and 0.0063, respectively. The paired Fst genetic distances and correlation p-values among the 54 populations revealed that the Guizhou Buyei was most closely related to the Henan Han and the Guizhou Miao, and closer to the Hazara population in Pakistan and the Chiang Mai population.ConclusionsThe study of mitochondrial DNA based on the maternal genetic structure of the Buyei nationality in Guizhou will benefit the establishment of an East Asian forensic DNA reference database and provide a reference for anthropological research in the future.

Mitochondrial point heteroplasmy: insights from deep-sequencing of human replicate samples

BackgroundHuman mitochondrial heteroplasmy is an extensively investigated phenomenon in the context of medical diagnostics, forensic identification and molecular evolution. However, technical limitations of high-throughput sequencing hinder reliable determination of point heteroplasmies (PHPs) with minor allele frequencies (MAFs) within the noise threshold.ResultsTo investigate the PHP landscape at an MAF threshold down to 0.1%, we sequenced whole mitochondrial genomes at approximately 7.700x coverage, in multiple technical and biological replicates of longitudinal blood and buccal swab samples from 11 human donors (159 libraries in total). The results obtained by two independent sequencing platforms and bioinformatics pipelines indicate distinctive PHP patterns below and above the 1% MAF cut-off. We found a high inter-individual prevalence of low-level PHPs (MAF < 1%) at polymorphic positions of the mitochondrial DNA control region (CR), their tissue preference, and a tissue-specific minor allele linkage. We also established the position-dependent potential of minor allele expansion in PHPs, and short-term PHP instability in a mitotically active tissue. We demonstrate that the increase in sensitivity of PHP detection to minor allele frequencies below 1% within a robust experimental and analytical pipeline, provides new information with potential applicative value.ConclusionsOur findings reliably show different mutational loads between tissues at sub-1% allele frequencies, which may serve as an informative medical biomarker of time-dependent, tissue-specific mutational burden, or help discriminate forensically relevant tissues in a single person, close maternal relatives or unrelated individuals of similar phylogenetic background.

Genetic structure and diversity of green turtle (Chelonia mydas) in the Gulf of Thailand.

The International Union for the Conservation of Nature and Natural Resources lists the green turtle as endangered. Green turtle nesting behavior in the Gulf of Thailand has decreased to <50% of the 1995 level. The population structure of green turtles in the Gulf of Thailand has not yet been studied. This study aimed to characterize the genetic diversity of green turtles in the Gulf of Thailand based on comparisons of mitochondrial DNA (mtDNA) control region with sequences of Indo-Pacific management units (MUs) and rookeries, to investigate population structures, and to explore phylogeographic relationships. Blood samples (1 mL each) from 91 stranded green turtles were collected from four parts of the Gulf of Thailand (eastern, upper, central, and lower). The control mtDNA region was amplified by polymerase chain reaction using LCM15382 and H950 primer. The obtained 384-bp or 770-bp sequences were analyzed for haplotype, clade, and haplotype and nucleotide diversities and were used to construct a phylogenetic tree and haplotype network diagram, respectively. In addition, we analyzed genetic differentiation within and among populations of green turtles in the Gulf of Thailand and between green turtles in the Gulf of Thailand and other Indo-Pacific MUs and rookeries. In total, 12 (based on 384 bp) or 13 (based on 770 bp) haplotypes and two clades (clades VII and VIII) were identified, with nine or 10 haplotypes belonging to clade VIII and three haplotypes belonging to clade VII. Of the new haplotypes, four or five were identified and classified as clade VII (two haplotypes, for both fragment lengths) and clade VIII (two or three haplotypes, for 384 bp or 770 bp fragments, respectively). The overall haplotype and nucleotide diversity of green turtles in the Gulf of Thailand were high (0.755 ± 0.039 and 0.01146 ± 0.00248, respectively). Based on the analysis of molecular variance, green turtles in the Gulf of Thailand could be divided into two subpopulations (UC-Eastern Gulf of Thailand [UC-EGT] and lower Gulf of Thailand [LGT]). Comparisons with other MUs and rookeries in the Indo-Pacific showed that UC-EGT was not genetically different from the Peninsular Malaysia and Eastern Taiwan (Lanyu) MUs and the Terrangganu and Mersing rookeries, and LGT were not genetically different from Peninsular Malaysia, Sipadan, Brunei Bay, Eastern Taiwan (Lanyu), Scott Reef and Browse Island, and Gulf of Carpentaria MUs and the Perak, Perhentain Island, Redang, Pahang, and Vietnam rookeries. To the best of our knowledge, this is the first report to identify the haplotypes and clades of green turtles in the Gulf of Thailand and to show that the populations in the Gulf of Thailand not only present high genetic diversity but also have haplotypic endemism. Longer mtDNA fragments (770 bp) increased the resolution of the stock structure. Clade VII is a unique clade not only for Japan but also for Thailand and Malaysia, and CmP82 is a unique haplotype for both the Gulf of Thailand and Malaysia. Conservation and management of these populations are important to preserve the genetic diversity, biological diversity, and evolutionary potential of green turtles in the Gulf of Thailand.

Genetic investigation of Cuvier's beaked whale, Ziphius cavirostris, along the coast of Türkiye and Northern Cyprus, based on mtDNA sequences

Abstract The Cuvier's beaked whale, Ziphius cavirostris, is a cosmopolitan species and the only beaked whale species commonly found in the Mediterranean Sea. Five strandings of Cuvier's beaked whale were reported along the Aegean/Mediterranean Seas coasts of Türkiye and northern coast of Cyprus in 2016–2017. Mitochondrial DNA (mtDNA) control region (430–444 bp) and cytochrome b (cytb) (382–424 bp) sequences each revealed two different haplotypes (four out of five individuals had the same haplotype for each locus) on these stranded animals. The control region haplotypes were identical to two previously identified haplotypes from the Ionian (Greece) and Adriatic (Croatia) Seas. Only one of the cytb haplotypes had previously been described from the Adriatic Sea (Italy) and the other one was detected for the first time. In a comparison of these haplotypes with Cuvier's beaked whale haplotypes previously reported from outside the Mediterranean Sea, the Mediterranean subpopulation shows genetic differentiation based upon the presence of two unique haplotypes. Additional mtDNA sequences from the Mediterranean Sea are needed for a better understanding of the genetic population structure of this species and to elaborate more concrete conservation measures.

Assessment of genetic diversity, population structure and wolf-dog hybridisation in the Eastern Romanian Carpathian wolf population

The Carpathian Mountains have been constantly inhabited by grey wolves and present one of the largest distribution areas in Europe, comprising between 2300 and 2700 individuals in Romania. To date, however, relatively little is known about the Romanian wolf population. We aimed to provide a first assessment of genetic diversity, population structure and wolf-dog hybridisation based on 444 mostly non-invasively collected samples in the Eastern Romanian Carpathians. Pack reconstruction and analysis of population genetic parameters were performed with mitochondrial DNA control-region sequencing and microsatellite genotyping. We found relatively high levels of genetic diversity, which is similar to values found in previous studies on Carpathian wolves from Poland and Slovakia, as well as to the long-lasting Dinaric-Balkan wolf population. We found no significant population structure in our study region, suggesting effective dispersal and admixture. Analysis of wolf-dog hybridisation using a Single Nucleotide Polymorphism panel optimised for hybrid detection revealed low rates of admixture between wolves and domestic dogs. Our results provide evidence for the existence of a genetically viable wolf population in the Romanian Carpathians. The genetic data obtained in this study may serve as valuable baseline information for the elaboration of monitoring standards and management plans for wolves in Romania.

Comparison of DNA Extraction Methods for the Detection of Canned Tuna Species with DNA Mini-Barcoding

Tuna is susceptible to species mislabeling due to its high demand, quick rate of production, and wide range of price points. DNA barcoding, a sequencing-based technique, allows for the detection of species mislabeling by targeting a standardized region of DNA. A mitochondrial control region (CR) DNA barcode has been found to be capable of species discrimination for tuna, but it is challenging to recover the entire DNA fragment from canned tuna. While a short fragment of CR, referred to as a “mini-barcode,” has shown some success with canned tuna species identification, more research is needed to improve identification rates. The objective of this study was to determine the optimal DNA extraction method for species identification of canned tuna using CR mini-barcoding. Four commercial DNA extraction kits were compared using a sample set of 24 different cans of tuna labeled as albacore, light tuna, skipjack, or yellowfin. All samples were tested in duplicate. The greatest success was found using the Qiagen DNeasy Blood and Tissue Kit and the Qiagen DNeasy Mericon Food Kit, which resulted in species identification for 42% of the samples. In comparison, the MP Biomedicals FastPrep-24 + Machery-Nagel NucleoSpin Tissue Kit resulted in species identification for 30% of the samples and the Qiagen DNeasy Blood and Tissue Kit + PowerClean Pro Cleanup Kit resulted in species identification for 21% of the samples. Overall, the top-performing DNA extraction methods for use with CR mini-barcoding of canned tuna products were determined to be the DNeasy Blood and Tissue Kit and the DNeasy Mericon Food Kit.