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The sequencing of a kidney sample (KW2013002) from a stranded Megaptera novaeangliae (Humpback whale) calf is the first chromosome-level reference genome for this species 1 . The calf, a 457 cm and 2,500 lbs male, was found stranded in Hawai'i Kai, HI, in 2013 and was marked as abandoned/orphaned. In 2023, 1 g of kidney was sequenced with PacBio long-read DNA sequencing, chromatin conformation capture (Hi-C), RNA sequencing, and mitochondrial sequencing to comprehensively characterize the genome and transcriptome of M. novaeangliae. Data validation includes a synteny analysis, mitochondrial annotation, and a comparison of BUSCO scores (scaffold v. reference genome and Balaenoptera musculus (Blue whale) v. M. novaeangliae). BUSCO analysis was performed on an M. novaeangliae scaffold-level assembly to determine genomic completeness of the reference genome, with a scaffold BUSCO score of 91.2% versus a score of 95.4%. Synteny analysis was performed using the B. musculus genome as comparison to determine chromosome-level coverage and structure. Further, a time-based phylogenetic tree was constructed using the sequenced data and publicly available genomes., (© 2024. The Author(s).)

The health of migratory eastern Australian humpback whales (Megaptera novaeangliae) can reflect the condition of their remote polar foraging environments. This study used gene expression (LEP, LEPR, ADIQ, AhR, TNF-α, HSP-70), blubber hormone concentrations (cortisol, testosterone), and photogrammetric body condition to assess this sentinel species during a period of unprecedented changes to anthropogenic activity and natural processes. The results revealed higher cortisol concentrations in 2020 compared to 2021, suggesting a decline in physiological stress between years. Additionally, metabolic transcripts LEPR, and AhR, which is also linked to xenobiotic metabolism, were upregulated during the 2020 southbound migration. These differences suggest that one or more environmental stressors were reduced between 2020 and 2021, with upregulated AhR possibly indicating a Southern Ocean pollutant declined between the years. This research confirms a Southern Ocean-wide decrease in whale stress during the study period and informs efforts to identify key stressors on Antarctic marine ecosystems., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Jacob MJ Linsky reports financial support was provided by Sea World Research and Rescue Foundation Inc. Jacob MJ Linsky reports financial support was provided by The Goodman Foundation (charity registered in Australia, details can be found at https://www.aegn.org.au/meet-our-member/john-and-meta-goodman/). Jacob MJ Linsky reports financial support was provided by Holsworth Wildlife Research Endowment. If there are other authors, they declare that they have no known competing financialinterests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Health information is essential for the conservation management of whale species. However, assessing the health of free-ranging whales is challenging as samples are primarily limited to skin and blubber tissue. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) offers a method to measure health from blubber RNA, providing insights into energetic status, stress and immune activity. To identify changes in health, natural differences in baseline gene expression linked to an individual's sex, reproductive status and life-history stage must first be quantified. This study aimed to establish baseline gene expression indices of health in migrating humpback whales (Megaptera novaeangliae). To do this, we developed an assay to quantify seven health-related gene transcripts (Leptin, Leptin Receptor, Adiponectin, Aryl Hydrocarbon Receptor, Tumour Necrosis Factor-α, Interleukin-6, Heat Shock Protein-70) and used Bayesian mixed effect models to assess differential baseline expression based on sex, lactation status and migration stage (northbound to and southbound from the annual breeding grounds). Results showed no significant contribution of sex to differential baseline expression. However, lactating individuals exhibited downregulated AhR and HSP-70 compared to non-lactating conspecifics. Additionally, southbound individuals demonstrated significantly upregulated HSP-70 and downregulated TNF-alpha, suggesting a relationship between these inflammation-linked transcripts and migratory fasting. Our results suggest that baseline differences due to migratory stage and lactation status should be considered in health applications of this assay. Future monitoring efforts can use our baseline measurements to better understand how gene expression is tied to population-level impacts, such as reduced prey availability or migratory stressors., (© 2023 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.)

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., (© The American Genetic Association. 2022.)

Monitoring aquatic species by identification of environmental DNA (eDNA) is becoming more common. To obtain quantitative eDNA datasets for individual species, organism-specific quantitative PCR (qPCR) assays are required. Here, we present detailed methodology of qPCR assay design and testing, including in silico, in vitro, and in vivo testing, and comment on the challenges associated with assay design and performance. We use the presented methodology to design assays for three important marine organisms common in the California Current Ecosystem (CCE): humpback whale (Megaptera novaeangliae), shortbelly rockfish (Sebastes jordani), and common murre (Uria aalge). All three assays have excellent sensitivity and high efficiencies ranging from 92% to 99%. However, specificities of the assays varied from species-specific in the case of common murre, genus-specific for the shortbelly rockfish assay, and broadly whale-specific for the humpback whale assay, which cross-amplified with other two other whale species, including one in a different family. All assays detected their associated targets in complex environmental water samples., Competing Interests: The authors have declared that no competing interests exist.

Fin whales (Balaenoptera physalus) and blue whales (B. musculus) are the two largest species on Earth and are widely distributed across the world's oceans. Hybrids between these species appear to be relatively widespread and have been reported in both the North Atlantic and North Pacific; they are also relatively common, and have been proposed to occur once in every thousand fin whales. However, despite known hybridization, fin and blue whales are not sibling species. Rather, the closest living relative of fin whales are humpback whales (Megaptera novaeangliae). To improve the quality of fin whale data available for analysis, we assembled and annotated a fin whale nuclear genome using in-silico mate pair libraries and previously published short-read data. Using this assembly and genomic data from a humpback, blue, and bowhead whale, we investigated whether signatures of introgression between the fin and blue whale could be found. We find no signatures of contemporary admixture in the fin and blue whale genomes, although our analyses support ancestral gene flow between the species until 2.4-1.3 Ma. We propose the following explanations for our findings; i) fin/blue whale hybridization does not occur in the populations our samples originate from, ii) contemporary hybrids are a recent phenomenon and the genetic consequences have yet to become widespread across populations, or iii) fin/blue whale hybrids are under large negative selection, preventing them from backcrossing and contributing to the parental gene pools., Competing Interests: The authors have declared that no competing interests exist.

Cetaceans are a clade of highly specialized aquatic mammals that include the largest animals that have ever lived. The largest whales can have ∼1,000× more cells than a human, with long lifespans, leaving them theoretically susceptible to cancer. However, large-bodied and long-lived animals do not suffer higher risks of cancer mortality than humans-an observation known as Peto's Paradox. To investigate the genomic bases of gigantism and other cetacean adaptations, we generated a de novo genome assembly for the humpback whale (Megaptera novaeangliae) and incorporated the genomes of ten cetacean species in a comparative analysis. We found further evidence that rorquals (family Balaenopteridae) radiated during the Miocene or earlier, and inferred that perturbations in abundance and/or the interocean connectivity of North Atlantic humpback whale populations likely occurred throughout the Pleistocene. Our comparative genomic results suggest that the evolution of cetacean gigantism was accompanied by strong selection on pathways that are directly linked to cancer. Large segmental duplications in whale genomes contained genes controlling the apoptotic pathway, and genes inferred to be under accelerated evolution and positive selection in cetaceans were enriched for biological processes such as cell cycle checkpoint, cell signaling, and proliferation. We also inferred positive selection on genes controlling the mammalian appendicular and cranial skeletal elements in the cetacean lineage, which are relevant to extensive anatomical changes during cetacean evolution. Genomic analyses shed light on the molecular mechanisms underlying cetacean traits, including gigantism, and will contribute to the development of future targets for human cancer therapies., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Mitochondrial DNA (mtDNA) differences between humpback whales on different feeding grounds can reflect the cultural transmission of migration destinations over generations, and therefore represent one of the very few cases of gene-culture coevolution identified in the animal kingdom. In Russian Pacific waters, photo-identification (photo-ID) studies have shown minimal interchange between whales feeding off the Commander Islands and those feeding in the Karaginsky Gulf, regions that are separated by only 500 km and have previously been lumped together as a single Russian feeding ground. Here, we assessed whether genetic differentiation exists between these 2 groups of humpback whales. We discovered a strong mtDNA differentiation between the 2 feeding sites (FST = 0.18, ΦST = 0.14, P < 0.001). In contrast, nuclear DNA (nuDNA) polymorphisms, determined at 8 microsatellite loci, did not reveal any differentiation. Comparing our mtDNA results with those from a previous ocean-basin study reinforced the differences between the 2 feeding sites. Humpback whales from the Commanders appeared most similar to those of the western Gulf of Alaska and the Aleutian feeding grounds, whereas Karaginsky differed from all other North Pacific feeding grounds. Comparison to breeding grounds suggests mixed origins for the 2 feeding sites; there are likely connections between Karaginsky and the Philippines and to a lesser extent to Okinawa, Japan, whereas the Commanders are linked to the Mexican breeding grounds. The mtDNA differentiation between the Commander Islands and Karaginsky Gulf suggests a case of gene-culture coevolution, correlated to fidelity to a specific feeding site within a particular feeding ground. From a conservation perspective, our findings emphasize the importance of considering these 2 feeding sites as separate management units.

A clear understanding of population structure is essential for assessing conservation status and implementing management strategies. A small, non-migratory population of humpback whales in the Arabian Sea is classified as "Endangered" on the IUCN Red List of Threatened Species, an assessment constrained by a lack of data, including limited understanding of its relationship to other populations. We analysed 11 microsatellite markers and mitochondrial DNA sequences extracted from 67 Arabian Sea humpback whale tissue samples and compared them to equivalent datasets from the Southern Hemisphere and North Pacific. Results show that the Arabian Sea population is highly distinct; estimates of gene flow and divergence times suggest a Southern Indian Ocean origin but indicate that it has been isolated for approximately 70,000 years, remarkable for a species that is typically highly migratory. Genetic diversity values are significantly lower than those obtained for Southern Hemisphere populations and signatures of ancient and recent genetic bottlenecks were identified. Our findings suggest this is the world's most isolated humpback whale population, which, when combined with low population abundance estimates and anthropogenic threats, raises concern for its survival. We recommend an amendment of the status of the population to "Critically Endangered" on the IUCN Red List.

Humpback whales (Megaptera novaeangliae) annually undertake the longest migrations between seasonal feeding and breeding grounds of any mammal. Despite this dispersal potential, discontinuous seasonal distributions and migratory patterns suggest that humpbacks form discrete regional populations within each ocean. To better understand the worldwide population history of humpbacks, and the interplay of this species with the oceanic environment through geological time, we assembled mitochondrial DNA control region sequences representing approximately 2700 individuals (465 bp, 219 haplotypes) and eight nuclear intronic sequences representing approximately 70 individuals (3700 bp, 140 alleles) from the North Pacific, North Atlantic and Southern Hemisphere. Bayesian divergence time reconstructions date the origin of humpback mtDNA lineages to the Pleistocene (880 ka, 95% posterior intervals 550-1320 ka) and estimate radiation of current Northern Hemisphere lineages between 50 and 200 ka, indicating colonization of the northern oceans prior to the Last Glacial Maximum. Coalescent analyses reveal restricted gene flow between ocean basins, with long-term migration rates (individual migrants per generation) of less than 3.3 for mtDNA and less than 2 for nuclear genomic DNA. Genetic evidence suggests that humpbacks in the North Pacific, North Atlantic and Southern Hemisphere are on independent evolutionary trajectories, supporting taxonomic revision of M. novaeangliae to three subspecies., (© 2014 The Author(s) Published by the Royal Society. All rights reserved.)

Background: Telomeres, the protective cap of chromosomes, have emerged as powerful markers of biological age and life history in model and non-model species. The qPCR method for telomere length estimation is one of the most common methods for telomere length estimation, but has received recent critique for being too error-prone and yielding unreliable results. This critique coincides with an increasing awareness of the potentials and limitations of the qPCR technique in general and the proposal of a general set of guidelines (MIQE) for standardization of experimental, analytical, and reporting steps of qPCR. In order to evaluate the utility of the qPCR method for telomere length estimation in non-model species, we carried out four different qPCR assays directed at humpback whale telomeres, and subsequently performed a rigorous quality control to evaluate the performance of each assay., Results: Performance differed substantially among assays and only one assay was found useful for telomere length estimation in humpback whales. The most notable factors causing these inter-assay differences were primer design and choice of using singleplex or multiplex assays. Inferred amplification efficiencies differed by up to 40% depending on assay and quantification method, however this variation only affected telomere length estimates in the worst performing assays., Conclusion: Our results suggest that seemingly well performing qPCR assays may contain biases that will only be detected by extensive quality control. Moreover, we show that the qPCR method for telomere length estimation can be highly precise and accurate, and thus suitable for telomere measurement in non-model species, if effort is devoted to optimization at all experimental and analytical steps. We conclude by highlighting a set of quality controls which may serve for further standardization of the qPCR method for telomere length estimation, and discuss some of the factors that may cause variation in qPCR experiments.

Tyrosinase-negative oculocutaneous albinism (OCA1A) is characterized by lifelong white hair and skin, a phenotype that has been described in most mammalian species worldwide. Tyrosinase is the key enzyme in melanin biosynthesis, and mutations in the tyrosinase gene result in OCA1A. We examined sequence variation at exon 1 of the tyrosinase gene in 66 humpback whale samples collected from the east coast of Australia, including an anomalously white humpback whale known as "Migaloo." We identified 3 novel variants, including a cytosine deletion that results in a premature stop codon in exon 1. The deletion truncates the tyrosinase protein including the putative catalytic domains that are essential for tyrosinase enzymatic activity. Migaloo was homozygous for this deletion, suggesting that the albino phenotype is a consequence of inactive tyrosinase caused by the frameshift in the tyrosinase gene.

Genetic identification of individuals is now commonplace, enabling the application of tagging methods to elusive species or species that cannot be tagged by traditional methods. A key aspect is determining the number of loci required to ensure that different individuals have non-matching multi-locus genotypes. Closely related individuals are of particular concern because of elevated matching probabilities caused by their recent co-ancestry. This issue may be addressed by increasing the number of loci to a level where full siblings (the relatedness category with the highest matching probability) are expected to have non-matching multi-locus genotypes. However, increasing the number of loci to meet this "full-sib criterion" greatly increases the laboratory effort, which in turn may increase the genotyping error rate resulting in an upward-biased mark-recapture estimate of abundance as recaptures are missed due to genotyping errors. We assessed the contribution of false matches from close relatives among 425 maternally related humpback whales, each genotyped at 20 microsatellite loci. We observed a very low (0.5-4%) contribution to falsely matching samples from pairs of first-order relatives (i.e., parent and offspring or full siblings). The main contribution to falsely matching individuals from close relatives originated from second-order relatives (e.g., half siblings), which was estimated at 9%. In our study, the total number of observed matches agreed well with expectations based upon the matching probability estimated for unrelated individuals, suggesting that the full-sib criterion is overly conservative, and would have required a 280% relative increase in effort. We suggest that, under most circumstances, the overall contribution to falsely matching samples from close relatives is likely to be low, and hence applying the full-sib criterion is unnecessary. In those cases where close relatives may present a significant issue, such as unrepresentative sampling, we propose three different genotyping strategies requiring only a modest increase in effort, which will greatly reduce the number of false matches due to the presence of related individuals.

For many biological investigations, groups of individuals are genetically sampled from several geographic locations. These sampling locations often do not reflect the genetic population structure. We describe a framework using marginal likelihoods to compare and order structured population models, such as testing whether the sampling locations belong to the same randomly mating population or comparing unidirectional and multidirectional gene flow models. In the context of inferences employing Markov chain Monte Carlo methods, the accuracy of the marginal likelihoods depends heavily on the approximation method used to calculate the marginal likelihood. Two methods, modified thermodynamic integration and a stabilized harmonic mean estimator, are compared. With finite Markov chain Monte Carlo run lengths, the harmonic mean estimator may not be consistent. Thermodynamic integration, in contrast, delivers considerably better estimates of the marginal likelihood. The choice of prior distributions does not influence the order and choice of the better models when the marginal likelihood is estimated using thermodynamic integration, whereas with the harmonic mean estimator the influence of the prior is pronounced and the order of the models changes. The approximation of marginal likelihood using thermodynamic integration in MIGRATE allows the evaluation of complex population genetic models, not only of whether sampling locations belong to a single panmictic population, but also of competing complex structured population models.

The Southwestern Atlantic Ocean humpback whales wintering ground (breeding stock A) are distributed along the Brazilian coast (5-23 degrees S), and their main mating and calving ground is in the Abrolhos Bank. We investigated genetic diversity, population structure, and relatedness of individuals sampled from the entire Southwest Atlantic humpback whale population. A total of 275 individuals sampled from 2 subregions (Abrolhos Bank, n = 229 and Praia do Forte, n = 46) were screened for 9 microsatellite loci. This population showed a high level of allelic diversity (A = 12.1) and a high mean observed heterozygosity (H(O) = 0.733). No signal of significant genetic bottleneck was detected in accordance with the mitochondrial DNA data. We find no evidence of temporal (between years) genetic structure as well as no genetic differentiation between whales from the 2 subregions of the Brazilian breeding ground. We observed that the proportion of males and females in this population was approximately 1:1, which differs from the male-biased sex ratio observed in other breeding grounds. The data obtained through this study provided no evidence of kinship associations within social groups. Finally, a female sampled off South Georgia Islands showed a putative parent-offspring relationship with a female off Abrolhos Bank, supporting the migratory link between these 2 areas.

Although humpback whales are among the best-studied of the large whales, population boundaries in the Southern Hemisphere (SH) have remained largely untested. We assess population structure of SH humpback whales using 1,527 samples collected from whales at fourteen sampling sites within the Southwestern and Southeastern Atlantic, the Southwestern Indian Ocean, and Northern Indian Ocean (Breeding Stocks A, B, C and X, respectively). Evaluation of mtDNA population structure and migration rates was carried out under different statistical frameworks. Using all genetic evidence, the results suggest significant degrees of population structure between all ocean basins, with the Southwestern and Northern Indian Ocean most differentiated from each other. Effective migration rates were highest between the Southeastern Atlantic and the Southwestern Indian Ocean, followed by rates within the Southeastern Atlantic, and the lowest between the Southwestern and Northern Indian Ocean. At finer scales, very low gene flow was detected between the two neighbouring sub-regions in the Southeastern Atlantic, compared to high gene flow for whales within the Southwestern Indian Ocean. Our genetic results support the current management designations proposed by the International Whaling Commission of Breeding Stocks A, B, C, and X as four strongly structured populations. The population structure patterns found in this study are likely to have been influenced by a combination of long-term maternally directed fidelity of migratory destinations, along with other ecological and oceanographic features in the region.

Group formation in humpback whales has been described in relation to different components of the migratory cycle, yet it is debated whether such groups represent real social bonding or ephemeral aggregations. Cooperative behaviours are exhibited during feeding activities, and it has been suggested that males may cooperate during competition for mates. Since most cooperative behaviours are expected to originate among kin, genetic relatedness represents a critical variable in the understanding of any social phenomenon, especially when cooperation cannot be confirmed unequivocally. Using an approach combining multi-locus microsatellite genotyping and several genetic relatedness estimators, we analyzed whale associations for two different wintering grounds in the Southern Hemisphere. The analyses included 648 whales sampled from 292 groups off the coast of Gabon and Northeast Madagascar, and screened for eleven microsatellite loci. Through simulations, we assessed the performance of three pairwise relatedness estimators. The individuals were molecularly sexed and their associations were investigated in the context of sex and group type. No significant association among relatives was found with the exception of mother-offspring pairs, supporting previous indications of extended maternal care. The analysis from the Gabon population also suggests that related males may avoid each other during competitive activities. Our results demonstrate that if cooperative behaviours occur on wintering grounds they are not favoured by kin selection.

Humpback whales seasonally migrate long distances between tropical and polar regions. However, inter-oceanic exchange is rare and difficult to document. Using skin biopsy samples collected in the Indian Ocean and in the South Atlantic Ocean, and a genetic capture-recapture approach based on microsatellite genotyping, we were able to reveal the first direct genetic evidence of the inter-oceanic migration of a male humpback whale. This exceptional migration to wintering grounds of two different ocean basins questions traditional notions of fidelity to an ocean basin, and demonstrates how the behaviour of highly mobile species may be elucidated from combining genetics with long-term field studies. Our finding has implications for management of humpback whale populations, as well as for hypotheses concerning cultural transmission of behaviour.