Evolution of the Critically Endangered Green Sawfish Pristis zijsron (Rhinopristiformes, Pristidae), Inferred from the Whole Mitochondrial Genome.

The green sawfish Pristis zijsron (Bleeker, 1851), a species of sawfish in the family Pristidae (Rhinopristiformes), mainly inhabits the Indo-West Pacific region. In this study, the complete mitochondrial genome of the critically endangered green sawfish is first described. The length of the genome is 16,804 bp, with a nucleotide composition of 32.0% A, 24.8% C, 13.1% G, and 30.0% T. It contains 37 genes in the typical gene order of fish. Two start (GTG and ATG) and two stop (TAG and TAA/T-) codons are found in the thirteen protein-coding genes. The 22 tRNA genes range from 67 bp (tRNA-Ser) to 75 bp (tRNA-Leu). The ratio of nonsynonymous substitution (Ka) and synonymous substitution (Ks) indicates that the family Pristidae are suffering a purifying selection. The reconstruction of Bayesian inference and the maximum likelihood phylogenetic tree show the same topological structure, and the family Pristidae is a monophyletic group with strong posterior probability. Pristis zijsron and P. pectinata form a sister group in the terminal clade. And the divergence time of Rhinopristiformes show that P. zijsron and P. pectinata diverged as two separate species in about Paleogene 31.53 Mya. Complete mitochondrial genomes of all five sawfishes have been published and phylogenetic relationships have been analyzed. The results of our study will provide base molecular information for subsequent research (e.g., distribution, conservation, phylogenetics, etc.) on this endangered group.

Close-kin mark-recapture informs critically endangered terrestrial mammal status.

Reliable information on population size is fundamental to the management of threatened species. For wild species, mark-recapture methods are a cornerstone of abundance estimation. Here, we show the first application of the close-kin mark-recapture (CKMR) method to a terrestrial species of high conservation value; the Christmas Island flying-fox (CIFF). The CIFF is the island's last remaining native terrestrial mammal and was recently listed as critically endangered. CKMR is a powerful tool for estimating the demographic parameters central to CIFF management and circumvents the complications arising from the species' cryptic nature, mobility, and difficult-to-survey habitat. To this end, we used genetic data from 450 CIFFs captured between 2015 and 2019 to detect kin pairs. We implemented a novel CKMR model that estimates sex-specific abundance, trend, and mortality and accommodates observations from the kin-pair distribution of male reproductive skew and mate persistence. CKMR estimated CIFF total adult female abundance to be approximately 2050 individuals (95% CI (950, 4300)). We showed that on average only 23% of the adult male population contributed to annual reproduction and strong evidence for between-year mate fidelity, an observation not previously quantified for a Pteropus species in the wild. Critically, our population estimates provide the most robust understanding of the status of this critically endangered population, informing immediate and future conservation initiatives.

Implications of past and present genetic connectivity for management of the saltwater crocodile (Crocodylus porosus).

Effective management of protected species requires information on appropriate evolutionary and geographic population boundaries and knowledge of how the physical environment and life-history traits combine to shape the population structure and connectivity. Saltwater crocodiles (Crocodylus porosus) are the largest and most widely distributed of living crocodilians, extending from Sri Lanka to Southeast Asia and down to northern Australia. Given the long-distance movement capabilities reported for C. porosus, management units are hypothesised to be highly connected by migration. However, the magnitude, scale, and consistency of connection across managed populations are not fully understood. Here we used an efficient genotyping method that combines DArTseq and sequence capture to survey ≈ 3000 high-quality genome-wide single nucleotide polymorphisms from 1176 C. porosus sampled across nearly the entire range of the species in Queensland, Australia. We investigated historical and present-day connectivity patterns using fixation and diversity indices coupled with clustering methods and the spatial distribution of kin pairs. We inferred kinship using forward simulation coupled with a kinship estimation method that is robust to unspecified population structure. The results demonstrated that the C. porosus population has substantial genetic structure with six broad populations correlated with geographical location. The rate of gene flow was highly correlated with spatial distance, with greater differentiation along the east coast compared to the west. Kinship analyses revealed evidence of reproductive philopatry and limited dispersal, with approximately 90% of reported first and second-degree relatives showing a pairwise distance of <50 km between sampling locations. Given the limited dispersal, lack of suitable habitat, low densities of crocodiles and the high proportion of immature animals in the population, future management and conservation interventions should be considered at regional and state-wide scales.

From rivers to ocean basins: The role of ocean barriers and philopatry in the genetic structuring of a cosmopolitan coastal predator.

The Bull Shark (Carcharhinus leucas) faces varying levels of exploitation around the world due to its coastal distribution. Information regarding population connectivity is crucial to evaluate its conservation status and local fishing impacts. In this study, we sampled 922 putative Bull Sharks from 19 locations in the first global assessment of population structure of this cosmopolitan species. Using a recently developed DNA-capture approach (DArTcap), samples were genotyped for 3400 nuclear markers. Additionally, full mitochondrial genomes of 384 Indo-Pacific samples were sequenced. Reproductive isolation was found between and across ocean basins (eastern Pacific, western Atlantic, eastern Atlantic, Indo-West Pacific) with distinct island populations in Japan and Fiji. Bull Sharks appear to maintain gene flow using shallow coastal waters as dispersal corridors, whereas large oceanic distances and historical land-bridges act as barriers. Females tend to return to the same area for reproduction, making them more susceptible to local threats and an important focus for management actions. Given these behaviors, the exploitation of Bull Sharks from insular populations, such as Japan and Fiji, may instigate local decline that cannot readily be replenished by immigration, which can in turn affect ecosystem dynamics and functions. These data also supported the development of a genetic panel to ascertain the population of origin, which will be useful in monitoring the trade of fisheries products and assessing population-level impacts of this harvest.

Stepping up to genome scan allows stock differentiation in the worldwide distributed blue shark Prionace glauca.

The blue shark Prionace glauca is a top predator with one of the widest geographical distributions of any shark species. It is classified as Critically Endangered in the Mediterranean Sea, and Near Threatened globally. Previous genetic studies did not reject the null hypothesis of a single global population. The blue shark was proposed as a possible archetype of the "grey zone of population differentiation," coined to designate cases where population structure may be too recent or too faint to be detected using a limited set of markers. Here, blue shark samples collected throughout its global range were sequenced using a specific RAD method (DArTseq), which recovered 37,655 genome-wide single nucleotide polymorphisms (SNPs). Two main groups emerged, with Mediterranean Sea and northern Atlantic samples (Northern population) differentiated significantly from the Indo-west Pacific samples (Southern population). Significant pairwise FST values indicated further genetic differentiation within the Atlantic Ocean, and between the Atlantic Ocean and the Mediterranean Sea. Reconstruction of recent demographic history suggested divergence between Northern and Southern populations occurred about 500 generations ago and revealed a drastic reduction in effective population size from a large ancestral population. Our results illustrate the power of genome scans to detect population structure and reconstruct demographic history in highly migratory marine species. Given that the management plans of the blue shark (targeted or bycatch) fisheries currently assume panmictic regional stocks, we strongly recommend that the results presented here be considered in future stock assessments and conservation strategies.

Rapid assessment of adult abundance and demographic connectivity from juvenile kin pairs in a critically endangered species.

The viability of spatially structured populations depends on the abundance and connectivity between subpopulations of breeding adults. Yet, for many species, both are extremely difficult to assess. The speartooth shark is a critically endangered elasmobranch inhabiting tropical rivers with only three adults ever recorded in Australia. Close-kin mark-recapture models, informed by sibling pairs among 226 juveniles, were developed to estimate adult abundance and connectivity in two Australian river systems. Sixty-eight sibling pairs were found, and adult abundance was estimated at 892 for the Adelaide River and 1128 for the Alligator Rivers. We found strong evidence for female philopatry, with most females returning to the same river to pup. Adelaide River males appear largely philopatric, whereas Alligator Rivers males are highly connected to the Adelaide River. From only 4 years of sampling, our results demonstrate that juvenile-only kin pairs can inform simultaneous estimates of abundance and connectivity in a rare and threatened species.

Novel duplication remnant in the first complete mitogenome of Hemitriakis japanica and the unique phylogenetic position of family Triakidae.

In this study, we firstly determined the complete mitogenome of the Japanese topeshark (Hemitriakis japonica), which belong to the family Triakidae and was assessed as Endangered A2d on the IUCN Red List in 2021. The mitogenome is 17,301 bp long, has a high AT content (60.0%), and contains 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, a control region and specially a 594 bp-long non-coding region between Cytb gene and tRNA-Thr gene. The novel non-coding region share high sequence similarity with segments of the former and latter genes, so it was recognized as a duplication remnant. In addition, the Cytb gene and tRNA-Thr gene tandemly duplicated twice while accompanied by being deleted once at least. This is the first report of mitogenomic gene-arrangement in Triakidae. The phylogenetic trees were constructed using Bayesian inference (BI) and maximum likelihood (ML) methods based on the mitogenomic data of 51 shark species and two outgroups. In summary, basing on a novel type of gene rearrangements in houndshark mitogenome, the possibly rearranged process was analyzed and contributed further insight of shark mitogenomes evolution and phylogeny.

Sample size requirements for genetic studies on yellowfin tuna.

In population genetics, the amount of information for an analytical task is governed by the number of individuals sampled and the amount of genetic information measured on each of those individuals. In this work, we assessed the numbers of individual yellowfin tuna (Thunnus albacares) and genetic markers required for ocean-basin scale inferences. We assessed this for three distinct data analysis tasks that are often employed: testing for differences between genetic profiles; stock delineation, and; assignment of individuals to stocks. For all analytical tasks, we used real (not simulated) data from four sampling locations that span the tropical Pacific Ocean. Whilst spatially separated, the genetic differences between the sampling sites were not substantial, a maximum of approximately Fst = 0.02, which is quite typical of large pelagic fish. We repeatedly sub-sampled the data, mimicking a new survey, and performed the analyses. False positive rates were also assessed by re-sampling and randomly assigning fish to groups. Varying the sample sizes indicated that some analytical tasks, namely profile testing, required relatively few individuals per sampling location (n ≳ 10) and single nucleotide polymorphisms (SNPs, m ≳ 256). Stock delineation required more individuals per sampling location (n ≳ 25). Assignment of fish to sampling locations required substantially more individuals, more in fact than we had available (n > 50), although this sample size could be reduced to n ≳ 30 when individual fish were assumed to belong to one of the groups sampled. With these results, designers of molecular ecological surveys for yellowfin tuna, and users of information from them, can assess whether the information content is adequate for the required inferential task.

One panel to rule them all: DArTcap genotyping for population structure, historical demography, and kinship analyses, and its application to a threatened shark.

With recent advances in sequencing technology, genomic data are changing how important conservation management decisions are made. Applications such as Close-Kin Mark-Recapture demand large amounts of data to estimate population size and structure, and their full potential can only be realised through ongoing improvements in genotyping strategies. Here we introduce DArTcap, a cost-efficient method that combines DArTseq and sequence capture, and illustrate its use in a high resolution population analysis of Glyphis garricki, a rare, poorly known and threatened euryhaline shark. Clustering analyses and spatial distribution of kin pairs from four different regions across northern Australia and one in Papua New Guinea, representing its entire known range, revealed that each region hosts at least one distinct population. Further structuring is likely within Van Diemen Gulf, the region that included the most rivers sampled, suggesting additional population structuring would be found if other rivers were sampled. Coalescent analyses and spatially explicit modelling suggest that G. garricki experienced a recent range expansion during the opening of the Gulf of Carpentaria following the conclusion of the Last Glacial Maximum. The low migration rates between neighbouring populations of a species that is found only in restricted coastal and riverine habitats show the importance of managing each population separately, including careful monitoring of local and remote anthropogenic activities that may affect their environments. Overall we demonstrated how a carefully chosen SNP panel combined with DArTcap can provide highly accurate kinship inference and also support population structure and historical demography analyses, therefore maximising cost-effectiveness.

Accounting for kin sampling reveals genetic connectivity in Tasmanian and New Zealand school sharks, Galeorhinus galeus.

Fishing represents a major problem for conservation of chondrichthyans, with a quarter of all species being overexploited. School sharks, Galeorhinus galeus, are targeted by commercial fisheries in Australia and New Zealand. The Australian stock has been depleted to below 20% of its virgin biomass, and the species is recorded as Conservation Dependent within Australia. Individuals are known to move between both countries, but it is disputed whether the stocks are reproductively linked. Accurate and unbiased determination of stock and population connectivity is crucial to inform effective management. In this study, we assess the genetic composition and population connectivity between Australian and New Zealand school sharks using genome-wide SNPs, while accounting for non-random kin sampling. Between 2009 and 2013, 88 neonate and juvenile individuals from Tasmanian and New Zealand nurseries were collected and genotyped. Neutral loci were analyzed to detect fine-scale signals of reproductive connectivity. Seven full-sibling groups were identified and removed for unbiased analysis. Based on 6,587 neutral SNPs, pairwise genetic differentiation from Tasmanian and New Zealand neonates was non-significant (F ST = 0.0003, CI95 = [-0.0002, 0.0009], p = 0.1163; D est = 0.0006 ± 0.0002). This pattern was supported by clustering results. In conclusion, we show a significant effect of non-random sampling of kin and identify fine-scale reproductive connectivity between Australian and New Zealand school sharks. OPEN RESEARCH BADGES: This article has earned an Open Data Badge for making publicly available the digitally-shareable data necessary to reproduce the reported results. The data is available at https://doi.org/10.5061/dryad.pd8612j.

Robust estimates of a high Ne/N ratio in a top marine predator, southern bluefin tuna.

Genetic studies of several marine species with high fecundity have produced "tiny" estimates (≤10-3) of the ratio of effective population size (Ne) to adult census size (N), suggesting that even very large populations might be at genetic risk. A recent study using close-kin mark-recapture methods estimated adult abundance at N ≈ 2 × 106 for southern bluefin tuna (SBT), a highly fecund top predator that supports a lucrative (~$1 billion/year) fishery. We used the same genetic and life history data (almost 13,000 fish collected over 5 years) to generate genetic and demographic estimates of Ne per generation and Nb (effective number of breeders) per year and the Ne/N ratio. Demographic estimates, which accounted for age-specific vital rates, skip breeding, variation in fecundity at age, and persistent individual differences in reproductive success, suggest that Ne/N is >0.1 and perhaps about 0.5. The genetic estimates supported this conclusion. Simulations using true Ne = 5 × 105 (Ne/N = 0.25) produced results statistically consistent with the empirical genetic estimates, whereas simulations using Ne = 2 × 104 (Ne/N = 0.01) did not. Our results show that robust estimates of Ne and Ne/N can be obtained for large populations, provided sufficiently large numbers of individuals and genetic markers are used and temporal replication (here, 5 years of adult and juvenile samples) is sufficient to provide a distribution of estimates. The high estimated Ne/N ratio in SBT is encouraging and suggests that the species will not be compromised by a lack of genetic diversity in responding to environmental change and harvest.

Reliably discriminating stock structure with genetic markers: Mixture models with robust and fast computation.

Delineating naturally occurring and self-sustaining subpopulations (stocks) of a species is an important task, especially for species harvested from the wild. Despite its central importance to natural resource management, analytical methods used to delineate stocks are often, and increasingly, borrowed from superficially similar analytical tasks in human genetics even though models specifically for stock identification have been previously developed. Unfortunately, the analytical tasks in resource management and human genetics are not identical-questions about humans are typically aimed at inferring ancestry (often referred to as "admixture") rather than breeding stocks. In this article, we argue, and show through simulation experiments and an analysis of yellowfin tuna data, that ancestral analysis methods are not always appropriate for stock delineation. In this work, we advocate a variant of a previously introduced and simpler model that identifies stocks directly. We also highlight that the computational aspects of the analysis, irrespective of the model, are difficult. We introduce some alternative computational methods and quantitatively compare these methods to each other and to established methods. We also present a method for quantifying uncertainty in model parameters and in assignment probabilities. In doing so, we demonstrate that point estimates can be misleading. One of the computational strategies presented here, based on an expectation-maximization algorithm with judiciously chosen starting values, is robust and has a modest computational cost.

The phylogenomic position of the Critically Endangered Largetooth Sawfish Pristis pristis (Rhinopristiformes, Pristidae), inferred from the complete mitochondrial genome.

The complete mitogenome of the Critically Endangered Largetooth Sawfish Pristis pristis (Rhinopristiformes, Pristidae) is presented in this study. The genome is 16,912 bp in length with a nucleotide base composition of 32.0% A, 26.5% C, 13.2% G, and 28.3% T, containing 37 genes typical of vertebrates. Two start (GTG and ATG) and two stop (TAG and TAA/T) codons are found in the protein-coding genes. The 22 tRNA genes range from 66 bp (tRNA-Ser2) to 75 bp (tRNA-Leu1). The tRNA-Pro gene is duplicated with an unknown sequence between the two copies. Bayesian phylogenetic reconstruction showed that P. pristis clusters with the Pristis clade with strong posterior probability (100%).

Inferring contemporary and historical genetic connectivity from juveniles.

Measuring population connectivity is a critical task in conservation biology. While genetic markers can provide reliable long-term historical estimates of population connectivity, scientists are still limited in their ability to determine contemporary patterns of gene flow, the most practical time frame for management. Here, we tackled this issue by developing a new approach that only requires juvenile sampling at a single time period. To demonstrate the usefulness of our method, we used the Speartooth shark (Glyphis glyphis), a critically endangered species of river shark found only in tropical northern Australia and southern Papua New Guinea. Contemporary adult and juvenile shark movements, estimated with the spatial distribution of kin pairs across and within three river systems, was contrasted with historical long-term connectivity patterns, estimated from mitogenomes and genome-wide SNP data. We found strong support for river fidelity in juveniles with the within-cohort relationship analysis. Male breeding movements were highlighted with the cross-cohort relationship analysis, and female reproductive philopatry to the river systems was revealed by the mitogenomic analysis. We show that accounting for juvenile river fidelity and female philopatry is important in population structure analysis and that targeted sampling in nurseries and juvenile aggregations should be included in the genomic toolbox of threatened species management.

Complete mitochondrial genome of the Endangered Narrow Sawfish Anoxypristis cuspidata (Rajiformes: Pristidae).

In this study, we describe the first complete mitochondrial sequence for the Endangered Narrow Sawfish Anoxypristis cuspidata. It is 17,243 bp in length and contains 13 protein-coding genes, two rRNA genes, 22 tRNA genes, and a control region with the common vertebrate mitogenomic organization. A total of 30 bp overlaps and 28 bp short intergenic spaces are located between all genes. The overall base composition is 32.7% A, 25.7% C, 12.9% G, and 28.6% T. Two start codons (ATG and GTG) and two stop codons (TAG and TAA/T) were used in all protein-coding genes. The origin of L-strand replication (OL) sequence (38 bp) formed a hairpin structure (13 bp stem and 12 bp loop) to initiate the replication of L-strand.

Complete mitochondrial genome of the Freshwater Whipray Himantura dalyensis.

The complete mitochondrial genome of the Freshwater Whipray Himantura dalyensis is presented in this study. It is 17,693 bp in length and contains 37 genes in typical gene order and transcriptional orientation observed in vertebrates. There were a total of 86 bp short intergenic spacers and 22 bp overlaps in the genome. The overall base composition was 31.4% A, 25.5% C, 13.2% G and 29.9% T. Two start codons (GTG and ATG) and two stop codons (TAG and TAA/T) were found in 13 protein-coding genes. The length of 22 tRNA genes ranged from 68 (tRNA-Cys and tRNA-Ser2) to 75 bp (tRNA-Leu1). The origin of L-strand replication (OL) was found between the tRNA-Asn and tRNA-Cys genes. The base composition of the control region (1940 bp) was similar to the whole mitogenome.

Complete mitochondrial genome of the Pigeye Shark Carcharhinus amboinensis (Carcharhiniformes: Carcharhinidae).

In this manuscript we describe the first complete mitochondrial sequence for the Data Deficient Pigeye Shark Carcharhinus amboinensis. The mitogenome is 16,704 bp long and consists of 1 control region, 2 rRNA genes, 22 tRNA genes and 13 protein-coding genes with an overall base composition of 31.6% A, 24.9% C, 13.1% G and 30.4% T. The gene arrangement pattern and transcriptional direction were typical for a vertebrate species. The tRNA-Ser2 lacks the dihydrouridine arm and forms a simple loop, therefore it cannot be folded into the typical cloverleaf secondary structures like other tRNAs.

The phylogenomic position of the Winghead Shark Eusphyra blochii (Carcharhiniformes, Sphyrnidae) inferred from the mitochondrial genome.

The complete mitogenome of the Winghead Shark Eusphyra blochii (Carcharhiniformes: Sphyrnidae) is determined in this study, which is 16,727 bp with a nucleotide base composition: 31.6% A, 25.7% C, 13.0% G and 29.7% T, containing 37 genes with the typical gene arrangement pattern and translate orientation in vertebrates. Two start codons (ATG and GTG) and two stop codons (TAG and TAA/T) are found in the protein-coding genes. The 22 tRNA genes range from 67 bp (tRNA-Cys and tRNA-Ser2) to 75 bp (tRNA-Leu1). The phylogenetic position showed that E. blochii clustered with the Sphyrna clade with strong posterior probability (100%).

Complete mitochondrial genome of the Critically Endangered Smalltooth Sawfish Pristis pectinata (Rajiformes: Pristidae).

In this study we describe the first complete mitochondrial sequence of the Critically Endangered Smalltooth Sawfish Pristis pectinata. It is 16,802 bp in length and contains all 37 genes found in typical vertebrate mitogenomes. The nucleotide composition of the coding strand is 31.1% A, 26.0% C, 13.1% G and 28.9% T. There are 29 bp overlaps and 38 short intergenic spaces dispersed in the mitogenome. Two start codons (ATG and GTG) and two stop codons (TAG and TAA/T) were found in the protein-coding genes. The length of the 22 tRNA genes range from 67 bp (tRNA(Ser2)) to 75 bp (tRNA(Leu1)). The control region is 1102 bp in length with high A + T (62.0%) and poor G (13.5%) content.

Complete mitogenome of the Graceful Shark Carcharhinus amblyrhynchoides (Carcharhiniformes: Carcharhinidae).

In this manuscript we describe the first complete mitochondrial sequence for the Near Threatened Graceful Shark Carcharhinus amblyrhynchoides. It is 16,705 bp in length, consists of two rRNA genes, 22 tRNA genes, 13 protein-coding genes and one control region with the typical gene arrangement pattern and translate orientation in vertebrates. The overall base composition is 31.4% A, 25.1% C, 13.2% G and 30.3% T. The shortest tRNA-Ser2 cannot fold into a clover-leaf secondary structure due to the lack of the dihydrouridine arm.