IntroductionEstimating the genetic diversity of cetaceans at sea, particularly abundant social delphinids, can be difficult with traditional biopsy sampling of individuals. Environmental DNA (eDNA) metabarcoding has been shown to be a powerful tool for the identification of species assemblages and estimation of genetic diversity, especially in aquatic environments.MethodsWe collected 126 samples of seawater from within the immediate vicinity of schools of dolphins during 15 encounters with the four most common delphinid taxa in the waters around Santa Catalina Island, California, USA: long-beaked common dolphins (Delphinus delphis bairdii, n = 8), short-beaked common dolphins (D. d. delphis, n = 3), common bottlenose dolphins (Tursiops truncatus, n = 2), and Risso's dolphins (Grampus griseus, n = 2). Next-generation sequencing was used to assign Amplicon Sequence Variants (ASVs) of mitochondrial DNA to species using GenBank and a region-specific reference database.ResultsA total of 240 ASVs were resolved for the four species. ASV richness and the effective number of ASVs, or true diversity, measured as Hill numbers of order 1, were consistent with known characteristics of the four species. Despite collecting up to 12 samples from a single group, a rarefaction analysis indicated that the population diversity was not fully represented for the more abundant species (genus Delphinus), but were closely approximated for G. griseus.DiscussionThis study demonstrates the application of eDNA for estimating population genetic diversity of abundant species and makes recommendations for improving future studies to better capture this diversity in wild delphinid populations. This provides a more solid foundation for studies using eDNA to monitor these species, which often include those in close proximity to anthropogenic threats.

Understanding how distinct populations interact demographically, ecologically and genetically at their boundaries allows us to understand patterns of connectivity and their conservation implications. Here, we build on previous research to evaluate the regional and local population structure of Aotearoa/New Zealand’s endemic and endangered Hector’s dolphin Cephalorhynchus hectori hectori , focusing on distinct areas representing population boundaries. We used DNA profiles comprising genetically identified sex, mitochondrial DNA (mtDNA) haplotype and microsatellite loci for 717 individuals throughout the species’ range, including 266 for which we generated new data. Population structure analyses of both mtDNA and microsatellite data confirmed previously described significant differentiation between the 3 main regions (East, West, and South Coast). There was a clear genetic boundary between the East and West Coast populations, consistent with a previously identified ecological boundary. The Queen Charlotte Sound subpopulation present adjacent to this boundary has very low genetic diversity, consistent with being at the end of a ‘stepping stone’ model of population structure. In contrast, we detected genetic admixture at the boundary between the South and East Coast populations, while confirming genetic differentiation amongst subpopulations in the region. Finally, a deep-water canyon was revealed to be a biogeographic break, with significant genetic differentiation to its north and south. Our results highlight how a single species can have different patterns of gene flow at the boundaries of genetically identifiable populations, likely driven by spatial differences in oceanography and resource distribution.

The species identification of whales, dolphins and porpoises at sea remains challenging, particularly for cryptic species like beaked whales (Family: Ziphiidae). Here we use environmental (e)DNA for identification of Sato’s beaked whale, Berardius minimus, at sea. This species was first described in 2019 and, to date, all but one genetic sample has been collected from beachcast carcasses. Sato’s beaked whales are difficult to distinguish at sea from the larger Baird’s beaked whales (Berardius bairdii), highlighting the need for reliable methods to identify this species in the field. Using high-throughput metabarcoding of the mtDNA control region, we confirmed visual identification of Sato’s beaked whales in six of the eight eDNA samples collected in the Nemuro Strait. One of the samples also provided a haplotype consistent with a killer whale. From the samples of Sato’s beaked whales, we recovered three of the five previously described haplotypes and resolved two novel variants. These haplotypes have the potential to further describe diversity and population structure in this elusive whale. Our results demonstrate the potential of eDNA metabarcoding as a non-invasive tool for detecting and assessing the genetic diversity of Sato’s beaked whales and other deep-diving cetaceans.

Entanglements in fishing gear are a significant threat to cetaceans worldwide and a concern for large whales in US waters. Yet, entanglement events are infrequently observed, and their lethal and sublethal impacts are likely underestimated. Photographic analysis of wrapping scars on whales shows promise to better assess entanglement. Here, we analyzed scars on 571 individual humpback whales photographed in Oregon, USA, waters (2005-2023). We scored 1533 photos of the tailstock and fluke regions for evidence of prior entanglement. We found that scarring prevalence varied by photo type, with perpendicular/forward tailstock photos showing the highest scarring prevalence (respectively 19.1% and 17.6% most likely caused by entanglements) compared to photos of the fluke underside that tended to miss likely entanglement events (57.4% false negatives). Depending on the scoring approach, 8.2 to 27.3% of sampled whales were likely entangled at least once in their lifetime. We found no significant spatial effect on scarring prevalence and a weak increasing (2016-2020), then decreasing (2020-2023), temporal trend. Males had a significantly higher scarring prevalence than females. Simulations of population trajectory and photographic sampling designs revealed that, even in an unrealistically optimistic scenario of new fishing regulations reducing the number of entanglements to zero, 165 ind. yr -1 , for 5 yr, would need to be sampled with good-quality tailstock photos to detect the decrease in scarring prevalence with 80% statistical power. Our findings enable recommendations for monitoring impacts of fishing gear interactions with humpback whales using US West Coast waters.

In 2024, an expedition was conducted off northwestern Baja California, M & eacute;xico, to find and identify the beaked whale species that produced the BW43 echolocation pulse previously recorded in this area and elsewhere in the North Pacific. There were five Mesoplodon sightings and 21 BW43 acoustic detections on both a towed array and drifting pole buoy recorders over the course of the survey. Three of the sightings had concurrent acoustic detections, and a biopsy sample and environmental DNA were also collected from one of the sightings. The genetic identification confirms that the Mesoplodon sighted and acoustically recorded was the ginkgo-toothed beaked whale (Mesoplodon ginkgodens), and the co-occurrence of these sightings with the BW43 acoustic detections definitively links the species and its echolocation pulse. This is the first time that genetically confirmed ginkgo-toothed beaked whales have been observed at sea and definitively linked to the BW43 pulse. This paper details the encounters, acoustic behavior, genetics, coloration, and external morphology of this species, including a comprehensive review of its distribution using historical sightings, strandings, and acoustic detection data from the North Pacific Ocean.

In coastal British Columbia, Canada, marine megafauna such as humpback whales (Megaptera novaeangliae) and fin whales (Balaenoptera physalus velifera) have been subject to a history of exploitation and near extirpation. While their populations have been in recovery, significant threats are posed to these vulnerable species by proposed natural resource ventures in this region, in addition to the compounding effects of anthropogenic climate change. Genetic tools play a vital role in informing conservation efforts, but the associated collection of tissue biopsy samples can be challenging for the investigators and disruptive to the ongoing behaviour of the targeted whales. Here, we evaluate a minimally intrusive approach based on collecting exhaled breath condensate, or respiratory 'blow' samples, from baleen whales using an unoccupied aerial system (UAS), within Gitga'at First Nation territory for conservation genetics. Minimal behavioural responses to the sampling technique were observed, with no response detected 87% of the time (of 112 UAS deployments). DNA from whale blow (n = 88 samples) was extracted, and DNA profiles consisting of 10 nuclear microsatellite loci, sex identification and mitochondrial (mt) DNA haplotypes were constructed. An average of 7.5 microsatellite loci per individual were successfully genotyped. The success rates for mtDNA and sex assignment were 80% and 89% respectively. Thus, this minimally intrusive sampling method can be used to describe genetic diversity and generate genetic profiles for individual identification. The results of this research demonstrate the potential of UAS-collected whale blow for conservation genetics from a remote location.

Assessing the reproductive physiology via skin-blubber biopsy samples of recovering populations of marine mammals is critical for conservation and management. We used an extensive blubber archive and associated demographic data to assess the seasonal changes in three reproductive hormones for humpback whales on a southwest Pacific breeding ground. The field of marine mammal conservation has dramatically benefited from the rapid advancement of methods to assess the reproductive physiology of individuals and populations from steroid hormones isolated from minimally invasive skin-blubber biopsy samples. Historically, this vital information was only available from complete anatomical and physiological investigations of samples collected during commercial or indigenous whaling. Humpback whales (Megaptera novaeangliae) are a migratory, cosmopolitan species that reproduce in warm, low-latitude breeding grounds. New Caledonia is seasonally visited by a small breeding sub-stock of humpback whales, forming part of the endangered Oceania subpopulation. To better understand the demographic and seasonal patterns of reproductive physiology in humpback whales, we quantified baseline measurements of reproductive hormones (progesterone-P4, testosterone-T and 17 beta-estradiol-E2) using an extensive archive of skin-blubber biopsy samples collected from female humpback whales in New Caledonia waters between 2016 and 2019 (n = 194). We observed significant differences in the P4, T and E2 concentrations across different demographic groups of female humpback whales, and we described some of the first evidence of the endocrine patterns of estrous in live free-ranging baleen whales. This study is fundamental in its methodological approach to a wild species that has a global distribution, with seasonally distinct life histories. This information will assist in monitoring, managing and conserving this population as global ecological changes continue to occur unhindered.

Here, we describe variation in mtDNA haplotypes among 22 individual humpback whales off the Guerrero coast in southern Mexico and compare this to other wintering regions in the eastern North Pacific Ocean using published data. After correcting for different sample sizes, Guerrero humpback whales fit the cline in the eastern North Pacific in which A types are more abundant northwards, while E and F types are more abundant southwards. Whales observed around Guerrero in early winter exhibited a greater frequency of F types that are also observed earlier in winter at Bahía de Banderas in the mainland region, suggesting a seasonal passage of whales from Mexico to Central America. Four diversity indices were higher in different wintering regions between Nayarit and Guerrero. Departures from neutrality were observed in the bordering wintering grounds of the region. Four measures of differentiation by distance indicated boundaries between Nayarit and Southern Jalisco, and between Guerrero and Oaxaca, suggesting a transitional region between wintering regions of Mexico and Central America from 17.5° to 19.5°N.

The major histocompatibility complex (MHC) is a highly polymorphic gene family that is crucial in immunity, and its diversity can be effectively used as a fitness marker for populations. Despite this, MHC remains poorly characterised in non-model species (e.g., cetaceans: whales, dolphins and porpoises) as high gene copy number variation, especially in the fast-evolving class I region, makes analyses of genomic sequences difficult. To date, only small sections of class I and IIa genes have been used to assess functional diversity in cetacean populations. Here, we undertook a systematic characterisation of the MHC class I and IIa regions in available cetacean genomes. We extracted full-length gene sequences to design pan-cetacean primers that amplified the complete exon 2 from MHC class I and IIa genes in one combined sequencing panel. We validated this panel in 19 cetacean species and described 354 alleles for both classes. Furthermore, we identified likely assembly artefacts for many MHC class I assemblies based on the presence of class I genes in the amplicon data compared to missing genes from genomes. Finally, we investigated MHC diversity using the panel in 25 humpback and 30 southern right whales, including four paternity trios for humpback whales. This revealed copy-number variable class I haplotypes in humpback whales, which is likely a common phenomenon across cetaceans. These MHC alleles will form the basis for a cetacean branch of the Immuno-Polymorphism Database (IPD-MHC), a curated resource intended to aid in the systematic compilation of MHC alleles across several species, to support conservation initiatives.

Radiocarbon dating is arguably the most common method for dating Quaternary deposits. However, accurate age assignments using radiocarbon dating are dependent on knowing the radiocarbon reservoir. For the coastal waters across Antarctica, the radiocarbon reservoirs show significant variation, ranging from 700 to 6000 years depending on the material dated and the period in question. In this study, we examine the radiocarbon reservoir age for the shallow waters of the Southern Ocean using 23 whale bones salvaged from commercial whaling operations on or near the Western Antarctic Peninsula between 1904 and 1916. The species origin of the bones had been identified previously as humpback, fin, or blue whales using sequences of mitochondrial (mt)DNA. We find an average reservoir age of 1050 +/- 135 years for these 23 whale bones, with a <100-year difference in the reservoir age by species. A comparison between our results and other studies through the Holocene suggest that the Southern Ocean surface water radiocarbon reservoir age is of a similar magnitude across much of Antarctica and has not significantly changed for the last 14,000 years. Combining our new ages with existing data sets provides insight to the stability of the Southern Ocean marine radiocarbon reservoir age, enhancing our understanding of ocean ventilation and upwelling dynamics throughout the Holocene.

Abstract Heteroplasmy in the mitochondrial genome offers a rare opportunity to track the evolution of a newly arising maternal lineage in populations of non-model species. Here, we identified a previously unreported mitochondrial DNA haplotype while assembling an integrated database of DNA profiles and photo-identification records from humpback whales in southeastern Alaska (SEAK). The haplotype, referred to as A8, was shared by only 2 individuals, a mature female with her female calf, and differed by only a single base pair from a common haplotype in the North Pacific, referred to as A−. To investigate the origins of the A8 haplotype, we reviewed n = 1,089 electropherograms (including replicate samples) of n = 710 individuals with A− haplotypes from an existing collection. From this review, we found 20 individuals with clear evidence of heteroplasmy for A−/A8 (parental/derived) haplotypes. Of these, 15 were encountered in SEAK, 4 were encountered on the Hawaiian breeding ground (the primary migratory destination for whales in SEAK), and 1 was encountered in the northern Gulf of Alaska. We used genotype exclusion and likelihood to identify one of the heteroplasmic females as the likely mother of the A8 cow and grandmother of the A8 calf, establishing the inheritance and germ-line fixation of the new haplotype from the parental heteroplasmy. The mutation leading to this heteroplasmy and the fixation of the A8 haplotype provide an opportunity to document the population dynamics and regional fidelity of a newly arising maternal lineage in a population recovering from exploitation.

In understanding the impact of commercial whaling, it is important to estimate the mixing of low latitude breeding populations on Antarctic feeding grounds, particularly the endangered humpback whale populations of Oceania. This paper estimates the degree of genetic differentiation among the putative populations of Oceania (New Caledonia, Tonga, the Cook Islands and French Polynesia) and Australia (western Australia and eastern Australia) using ten microsatellite loci and mtDNA, assesses the power of the data for a mixed-stock analysis, determines ways to improve statistical power for future studies and estimates the population composition of Antarctic samples collected in 2010 south of New Zealand and eastern Australia. A large proportion of individuals could not be assigned to a population of origin (> 52%) using a posterior probability threshold of > 0.90. The mixed-stock analysis simulations however, produced accurate results with humpback whales reapportioned to their population of origin above the 90% threshold for western Australia, New Caledonia and Oceania grouped using a combined mtDNA and microsatellite dataset. Removing the Cook Islands, considered a transient region for humpback whales, from the simulation analysis increased the ability to reapportion Tonga from 86% to 89% and French Polynesia from 89% to 92%. Breeding ground sample size was found to be a factor influencing the accuracy of population reapportionment whereas increasing the mixture or feeding ground sample size improved the precision of results. The mixed-stock analysis of our Antarctic samples revealed substantial contributions from both eastern Australia (53.2%, 6.8% SE) and New Caledonia (43.7%, 5.5% SE) [with Oceania contributing 46.8% (5.9% SE)] but not western Australia. Despite the need for more samples to improve estimates of population allocation, our study strengthens the emerging genetic and non-genetic evidence that Antarctic waters south of New Zealand and eastern Australia are used by humpback whales from both eastern Australia and the more vulnerable breeding population of New Caledonia, representing Oceania.

The 20th century commercial whaling industry severely reduced populations of great whales throughout the Southern Hemisphere. The effect of this exploitation on genetic diversity and population structure remains largely undescribed. Here, we compare pre- and post-whaling diversity of mitochondrial DNA (mtDNA) control region sequences for 3 great whales in the South Atlantic, such as the blue, humpback, and fin whale. Pre-whaling diversity is described from mtDNA extracted from bones collected near abandoned whaling stations, primarily from the South Atlantic island of South Georgia. These bones are known to represent the first stage of 20th century whaling and thus pre-whaling diversity of these populations. Post-whaling diversity is described from previously published studies reporting large-scale sampling of living whales in the Southern Hemisphere. Despite relatively high levels of surviving genetic diversity in the post-whaling populations, we found evidence of a probable loss of mtDNA lineages in all 3 species. This is evidenced by the detection of a large number of haplotypes found in the pre-whaling samples that are not present in the post-whaling samples. A rarefaction analysis further supports a loss of haplotypes in the South Atlantic humpback and Antarctic blue whale populations. The bones from former whaling stations in the South Atlantic represent a remarkable molecular archive for further investigation of the decline and ongoing recovery in the great whales of the Southern Hemisphere.

In many organisms, especially those of conservation concern, traditional lines of evidence for taxonomic delineation, such as morphological data, are often difficult to obtain. In these cases, genetic data are often the only source of information available for taxonomic studies. In particular, population surveys of mitochondrial genomes offer increased resolution and precision in support of taxonomic decisions relative to conventional use of the control region or other gene fragments of the mitochondrial genome. To improve quantitative guidelines for taxonomic decisions in cetaceans, we build on a previous effort targeting the control region and evaluate, for whole mitogenome sequences, a suite of divergence and diagnosability estimates for pairs of recognized cetacean populations, subspecies, and species. From this overview, we recommend new guidelines based on complete mitogenomes, combined with other types of evidence for isolation and divergence, which will improve resolution for taxonomic decisions, especially in the face of small sample sizes or low levels of genetic diversity. We further use simulated data to assist interpretations of divergence in the context of varying forms of historical demography, culture, and ecology.

Fin whales Balaenoptera physalus were hunted unsustainably across the globe in the 19th and 20th centuries, leading to vast reductions in population size. Whaling catch records indicate the importance of the Southern Ocean for this species; approximately 730,000 fin whales were harvested during the 20th century in the Southern Hemisphere (SH) alone, 94% of which were at high latitudes. Genetic samples from contemporary whales can provide a window to past population size changes, but the challenges of sampling in remote Antarctic waters limit the availability of data. Here, we take advantage of historical samples in the form of bones and baleen available from ex-whaling stations and museums to assess the pre-whaling diversity of this once abundant species. We sequenced 27 historical mitogenomes and 50 historical mitochondrial control region sequences of fin whales to gain insight into the population structure and genetic diversity of Southern Hemisphere fin whales (SHFWs) before and after the whaling. Our data, both independently and when combined with mitogenomes from the literature, suggest SHFWs are highly diverse and may represent a single panmictic population that is genetically differentiated from Northern Hemisphere populations. These are the first historic mitogenomes available for SHFWs, providing a unique time series of genetic data for this species.

Detection and identification of species, subspecies or stocks of whales, dolphins and porpoises at sea remain challenging, particularly for cryptic or elusive species like beaked whales (Family: Ziphiidae). Here we investigated the potential for using an acoustically assisted sampling design to collect environmental (e)DNA from beaked whales on the U.S. Navy's Atlantic Undersea Test and Evaluation Center (AUTEC) in The Bahamas. During 12 days of August 2019, we conducted 9 small-boat surveys and collected 56 samples of seawater (paired subsamples of 1L each, including controls) using both a spatial collection design in the absence of visual confirmation of whales, and a serial collection design in the proximity of whales at the surface. There were 7 sightings of whales, including 11 Blainville's beaked whales (Mesoplodon densirostris). All whales were located initially with the assistance of information from a bottom-mounted acoustic array available on the AUTEC range. Quantification by droplet digital (dd)PCR from the four spatial design collections showed no samples of eDNA above the threshold of detection and none of these 20 samples yielded amplicons for conventional or next-generation sequencing. Quantification of the 31 samples from four serial collections identified 11 likely positive detections. eDNA barcoding by conventional sequencing and eDNA metabarcoding by next-generation sequencing confirmed species identification for 9 samples from three of the four serial collections. We further resolved five intra-specific variants (i.e., haplotypes), two of which showed an exact match to previously published haplotypes and three that have not been reported previously to the international repository, GenBank. A minimum spanning network of the five eDNA haplotypes, with all other published haplotypes of Blainville's beaked whales, suggested the potential for further resolution of differences between oceanic populations.

Aging, often considered a result of random cellular damage, can be accurately estimated using DNA methylation profiles, the foundation of pan-tissue epigenetic clocks. Here, we demonstrate the development of universal pan-mammalian clocks, using 11,754 methylation arrays from our Mammalian Methylation Consortium, which encompass 59 tissue types across 185 mammalian species. These predictive models estimate mammalian tissue age with high accuracy ( r > 0.96). Age deviations correlate with human mortality risk, mouse somatotropic axis mutations and caloric restriction. We identified specific cytosines with methylation levels that change with age across numerous species. These sites, highly enriched in polycomb repressive complex 2-binding locations, are near genes implicated in mammalian development, cancer, obesity and longevity. Our findings offer new evidence suggesting that aging is evolutionarily conserved and intertwined with developmental processes across all mammals.

To investigate movements of humpback whales among breeding and migratory areas of Oceania in the South Pacific Ocean, comparisons of individually identified whales were undertaken using catalogues from New Caledonia, Tonga, New Zealand, the Cook Islands and French Polynesia. These locations probably represent wintering grounds or migratory areas for the Group V and VI stocks, as recognised by the International Whaling Commission for management purposes. Comparisons were also made to small samples of photos from Colombia, Ecuador and the Antarctic Peninsula, representing wintering and feeding grounds of the Group I stock. Overall, the combined catalogues contained photographs of 912 individual whales, 767 of which were from Oceania. Twelve fluke matches were made, indicating movement between the following areas: New Caledonia and New Zealand (2); New Caledonia and Tonga (6, plus one made by dorsal fin); Tonga and the Cook Islands (2); the Cook Islands and French Polynesia (1, plus one made by dorsal fin); and between Ecuador and the Antarctic Peninsula (1). These results add to previously known connections between eastern Australia and the westerly component of Oceania (New Caledonia, Tonga and New Zealand). The data also suggest little movement between Oceania and Area I (western South America and the Antarctic Peninsula), although sample sizes for the latter region were too small to conclude this with certainty. The documented movement of some whales among portions of Oceania indicates that stock assessments based on combining regional estimates of abundance are likely to be positively biased, although this may be countered by problems of heterogeneity in sampling effort and whale distribution. In contrast with the recovery exhibited in Area IV and in the western portion of Area V, humpback whale abundance appears to remain low in Oceania, presumably because of overexploitation in the feeding grounds of Area VI and the easterly component of Area V.

Abstract The age of an individual is an essential demographic parameter but is difficult to estimate without long‐term monitoring or invasive sampling. Epigenetic approaches are increasingly used to age organisms, including nonmodel organisms such as cetaceans. Māui dolphins (Cephalorhynchus hectori maui) are a critically endangered subspecies endemic to Aotearoa New Zealand, and the age structure of this population is important for informing conservation. Here we present an epigenetic clock for aging Māui and Hector's dolphins (C. h. hectori) developed from methylation data using DNA from tooth aged individuals (n = 48). Based on this training data set, the optimal model required only eight methylation sites, provided an age correlation of .95, and had a median absolute age error of 1.54 years. A leave‐one‐out cross‐validation analysis with the same parameters resulted in an age correlation of .87 and median absolute age error of 2.09 years. To improve age estimation, we included previously published beluga whale (Delphinapterus leucas) data to develop a joint beluga/dolphin clock, resulting in a clock with comparable performance and improved estimation of older individuals. Application of the models to DNA from skin biopsy samples of living Māui dolphins revealed a shift from a median age of 8–9 years to a younger population aged 7–8 years 10 years later. These models could be applied to other dolphin species and demonstrate the ability to construct a clock even when the number of known age samples is limited, removing this impediment to estimating demographic parameters vital to the conservation of critically endangered species.