Demographic and physiological signals of reproductive events in 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 17ß-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.

Collecting baleen whale blow samples by drone: A minimally intrusive tool for conservation genetics.

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.

Using epigenetic clocks to investigate changes in the age structure of critically endangered Maui dolphins.

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. Maui 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 Maui 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 Maui 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.

A surplus no more? Variation in krill availability impacts reproductive rates of Antarctic baleen whales.

The krill surplus hypothesis of unlimited prey resources available for Antarctic predators due to commercial whaling in the 20th century has remained largely untested since the 1970s. Rapid warming of the Western Antarctic Peninsula (WAP) over the past 50 years has resulted in decreased seasonal ice cover and a reduction of krill. The latter is being exacerbated by a commercial krill fishery in the region. Despite this, humpback whale populations have increased but may be at a threshold for growth based on these human-induced changes. Understanding how climate-mediated variation in prey availability influences humpback whale population dynamics is critical for focused management and conservation actions. Using an 8-year dataset (2013-2020), we show that inter-annual humpback whale pregnancy rates, as determined from skin-blubber biopsy samples (n = 616), are positively correlated with krill availability and fluctuations in ice cover in the previous year. Pregnancy rates showed significant inter-annual variability, between 29% and 86%. Our results indicate that krill availability is in fact limiting and affecting reproductive rates, in contrast to the krill surplus hypothesis. This suggests that this population of humpback whales may be at a threshold for population growth due to prey limitations. As a result, continued warming and increased fishing along the WAP, which continue to reduce krill stocks, will likely impact this humpback whale population and other krill predators in the region. Humpback whales are sentinel species of ecosystem health, and changes in pregnancy rates can provide quantifiable signals of the impact of environmental change at the population level. Our findings must be considered paramount in developing new and more restrictive conservation and management plans for the Antarctic marine ecosystem and minimizing the negative impacts of human activities in the region.

An epigenetic clock to estimate the age of living beluga whales.

DNA methylation data facilitate the development of accurate molecular estimators of chronological age or "epigenetic clocks." We present a robust epigenetic clock for the beluga whale, Delphinapterus leucas, developed for an endangered population in Cook Inlet, Alaska, USA. We used a custom methylation array to measure methylation levels at 37,491 cytosine-guanine sites (CpGs) from skin samples of dead whales (n = 67) whose chronological ages were estimated based on tooth growth layer groups. Using these calibration data, a penalized regression model selected 23 CpGs, providing an R 2 = 0.92 for the training data; and an R 2 = 0.74 and median absolute age error = 2.9 years for the leave one out cross-validation. We applied the epigenetic clock to an independent dataset of 38 skin samples collected with a biopsy dart from living whales between 2016 and 2018. Age estimates ranged from 11 to 27 years. We also report sex correlations in CpG data and describe an approach of identifying the sex of an animal using DNA methylation. The epigenetic estimators of age and sex presented here have broad applications for conservation and management of Cook Inlet beluga whales and potentially other cetaceans.

High pregnancy rates in humpback whales (Megaptera novaeangliae) around the Western Antarctic Peninsula, evidence of a rapidly growing population.

Antarctic humpback whales are recovering from near extirpation from commercial whaling. To understand the dynamics of this recovery and establish a baseline to monitor impacts of a rapidly changing environment, we investigated sex ratios and pregnancy rates of females within the Western Antarctic Peninsula (WAP) feeding population. DNA profiling of 577 tissue samples (2010-2016) identified 239 males and 268 females. Blubber progesterone levels indicated 63.5% of the females biopsied were pregnant. This proportion varied significantly across years, from 36% in 2010 to 86% in 2014. A comparison of samples collected in summer versus fall showed significant increases in the proportion of females present (50% to 59%) and pregnant (59% to 72%), consistent with demographic variation in migratory timing. We also found evidence of annual reproduction among females; 54.5% of females accompanied by a calf were pregnant. These high pregnancy rates are consistent with a population recovering from past exploitation, but appear inconsistent with recent estimates of WAP humpback population growth. Thus, our results will help to better understand population growth potential and set a current baseline from which to determine the impact of climate change and variability on fecundity and reproductive rates.

Microsatellite markers reveal strong genetic structure in the endemic Chilean dolphin.

Understanding genetic differentiation and speciation processes in marine species with high dispersal capabilities is challenging. The Chilean dolphin, Cephalorhynchus eutropia, is the only endemic cetacean of Chile and is found in two different coastal habitats: a northern habitat with exposed coastlines, bays and estuaries from Valparaíso (33°02'S) to Chiloé (42°00'S), and a southern habitat with highly fragmented inshore coastline, channels and fjords between Chiloé and Navarino Island (55°14'S). With the aim of evaluating the potential existence of conservation units for this species, we analyzed the genetic diversity and population structure of the Chilean dolphin along its entire range. We genotyped 21 dinucleotide microsatellites for 53 skin samples collected between 1998 and 2012 (swab: n = 8, biopsy: n = 38, entanglement n = 7). Bayesian clustering and spatial model analyses identified two genetically distinct populations corresponding to the northern and southern habitats. Genetic diversity levels were similar in the two populations (He: 0.42 v/s 0.45 for southern and northern populations, respectively), while effective size population was higher in the southern area (Ne: 101 v/s 39). Genetic differentiation between these two populations was high and significant (FST = 0.15 and RST = 0.19), indicating little or no current gene flow. Because of the absence of evident geographical barriers between the northern and southern populations, we propose that genetic differentiation may reflect ecological adaptation to the different habitat conditions and resource uses. Therefore, the two genetic populations of this endemic and Near Threatened species should be considered as different conservation units with independent management strategies.

Circumpolar diversity and geographic differentiation of mtDNA in the critically endangered Antarctic blue whale (Balaenoptera musculus intermedia).

The Antarctic blue whale (Balaenoptera musculus intermedia) was hunted to near extinction between 1904 and 1972, declining from an estimated initial abundance of more than 250,000 to fewer than 400. Here, we describe mtDNA control region diversity and geographic differentiation in the surviving population of the Antarctic blue whale, using 218 biopsy samples collected under the auspices of the International Whaling Commission (IWC) during research cruises from 1990-2009. Microsatellite genotypes and mtDNA sequences identified 166 individuals among the 218 samples and documented movement of a small number of individuals, including a female that traveled at least 6,650 km or 131° longitude over four years. mtDNA sequences from the 166 individuals were aligned with published sequences from 17 additional individuals, resolving 52 unique haplotypes from a consensus length of 410 bp. From this minimum census, a rarefaction analysis predicted that only 72 haplotypes (95% CL, 64, 86) have survived in the contemporary population of Antarctic blue whales. However, haplotype diversity was relatively high (0.968±0.004), perhaps as a result of the longevity of blue whales and the relatively recent timing of the bottleneck. Despite the potential for circumpolar dispersal, we found significant differentiation in mtDNA diversity (F(ST) = 0.032, p<0.005) and microsatellite alleles (F(ST) = 0.005, p<0.05) among the six Antarctic Areas historically used by the IWC for management of blue whales.

Contamination levels of mercury and cadmium in melon-headed whales (Peponocephala electra) from a mass stranding on the Japanese coast.

Total mercury (T-Hg), methyl mercury (M-Hg), cadmium (Cd), selenium (Se), zinc (Zn) and copper (Cu) concentrations in the organs of melon-headed whales from a mass stranding on the Japanese coast were analyzed. The mean concentration of T-Hg in the liver (126+/-97 microg/wet g, n=13) was markedly higher than those in kidney (6.34+/-2.36 microg/wet g, n=12) and muscle (4.90+/-2.33 microg/wet g, n=15). In contrast, the mean concentration of M-Hg in the liver (9.08+/-2.24 microg/wet g) was similar to those in the kidney (3.47+/-0.91 microg/wet g) and muscle (3.78+/-1.53 microg/wet g). The mean percentage of M-Hg in the T-Hg found in the liver (13.1+/-10.3) was significantly lower than those in the kidney (58.3+/-15.0) and muscle (78.9+/-8.4). The molar ratio of T-Hg to Se in the liver was effectively 1.0, but those in the kidney and muscle were markedly lower. Conversely, the mean concentration of Cd was markedly higher in the kidney (24.4+/-7.4 microg/wet g) than in the liver (7.24+/-2.08 microg/wet g) and muscle (less than 0.05 microg/wet g). These results suggest that the formation of Hg-Se compounds mainly occurs in the liver after the demethylation of M-Hg, and Cd preferentially accumulates in the kidney of melon-headed whales.

Worldwide structure of mtDNA diversity among Cuvier's beaked whales (Ziphius cavirostris): implications for threatened populations.

We present the first description of phylogeographic structure among Cuvier's beaked whales (Ziphius cavirostris) worldwide using mitochondrial DNA (mtDNA) control region sequences obtained from strandings (n = 70), incidental fisheries takes (n = 11), biopsy (n = 1), and whale-meat markets (n = 5). Over a 290-base pair fragment, 23 variable sites defined 33 unique haplotypes among the total of 87 samples. Nucleotide diversity at the control region was relatively low (pi = 1.27%+/- 0.723%) compared to wide-ranging baleen whales, but higher than strongly matrifocal sperm, pilot and killer whales. Phylogenetic reconstruction using maximum likelihood revealed four distinct haplotype groups, each of which displayed strong frequency differences among ocean basins, but no reciprocal monophyly or fixed character differences. Consistent with this phylogeographic pattern, an analysis of molecular variance showed high levels of differentiation among ocean basins (F(ST) = 0.14, Phi ST = 0.42; P < 0.001). Estimated rates of female migration among ocean basins were low (generally < or = 2 individuals per generation). Regional sample sizes were too small to detect subdivisions within oceans except in the North Atlantic, where the Mediterranean Sea (n = 12) was highly differentiated due to the presence of two private haplotypes. One market product purchased in South Korea grouped with other haplotypes found only in the North Atlantic, suggesting a violation of current agreements banning international trade in cetacean species. Together, these results demonstrate a high degree of isolation and low maternal gene flow among oceanic, and in some cases, regional populations of Cuvier's beaked whales. This has important implications for understanding the threats of human impact, including fisheries by-catch, direct hunting, and disturbance or mortality from anthropogenic sound.