Into the Blue: Exploring genetic mechanisms behind the evolution of baleen whales.

Marine ecosystems are ideal for studying evolutionary adaptations involved in lineage diversification due to few physical barriers and reduced opportunities for strict allopatry compared to terrestrial ecosystems. Cetaceans (whales, dolphins, and porpoises) are a diverse group of mammals that successfully adapted to various habitats within the aquatic environment around 50 million years ago. While the overall adaptive transition from terrestrial to fully aquatic species is relatively well understood, the radiation of modern whales is still unclear. Here high-quality genomes derived from previously published data were used to identify genomic regions that potentially underpinned the diversification of baleen whales (Balaenopteridae). A robust molecular phylogeny was reconstructed based on 10,159 single copy and complete genes for eight mysticetes, seven odontocetes and two cetacean outgroups. Analysis of positive selection across 3,150 genes revealed that balaenopterids have undergone numerous idiosyncratic and convergent genomic variations that may explain their diversification. Genes associated with aging, survival and homeostasis were enriched in all species. Additionally, positive selection on genes involved in the immune system were disclosed for the two largest species, blue and fin whales. Such genes can potentially be ascribed to their morphological evolution, allowing them to attain greater length and increased cell number. Further evidence is presented about gene regions that might have contributed to the extensive anatomical changes shown by cetaceans, including adaptation to distinct environments and diets. This study contributes to our understanding of the genomic basis of diversification in baleen whales and the molecular changes linked to their adaptive radiation, thereby enhancing our understanding of cetacean evolution.

Comparative genomic studies on the TGF-ß superfamily in blue whale.

TGF-ß supergene family has a wide range of physiological functions including cell adhesion, motility, proliferation, apoptosis, and differentiation. We systematically analyzed and characterized the TGF-ß gene superfamily from the whole blue whale (Balaenoptera musculus) genome, using comparative genomic and evolutionary analysis. We identified 30 TGF-ß genes and were split into two subgroups, BMP-like and TGF-like. All TGF-ß proteins demonstrating a basic nature, with the exception of BMP1, BMP2, BMP10, GDF2, MSTN, and NODAL modulator, had acidic characteristics. All the blue whale (B. musculus) TGF-ß proteins, excluding BMP1, are thermostable based on aliphatic index. The instability index showed all proteins except the NODAL modulator was unstable. TGF-ß proteins showed a hydrophilic character, with the exception of GDF1 and INHBC. Moreover, all the detected TGF-ß genes showed evolutionary conserved nature. A segmental duplication was indicated by TGF-ß gene family, and the Ka/Ks ratio showed that the duplicated gene pairs were subjected to selection pressure, indicating both purifying and positive selection pressure. Two possible recombination breakpoints were also predicted. This study provides insights into the genetic characterization and evolutionary aspects of the TGF-ß superfamily in blue whales (B. musculus).

A High-Quality Blue Whale Genome, Segmental Duplications, and Historical Demography.

The blue whale, Balaenoptera musculus, is the largest animal known to have ever existed, making it an important case study in longevity and resistance to cancer. To further this and other blue whale-related research, we report a reference-quality, long-read-based genome assembly of this fascinating species. We assembled the genome from PacBio long reads and utilized Illumina/10×, optical maps, and Hi-C data for scaffolding, polishing, and manual curation. We also provided long read RNA-seq data to facilitate the annotation of the assembly by NCBI and Ensembl. Additionally, we annotated both haplotypes using TOGA and measured the genome size by flow cytometry. We then compared the blue whale genome with other cetaceans and artiodactyls, including vaquita (Phocoena sinus), the world's smallest cetacean, to investigate blue whale's unique biological traits. We found a dramatic amplification of several genes in the blue whale genome resulting from a recent burst in segmental duplications, though the possible connection between this amplification and giant body size requires further study. We also discovered sites in the insulin-like growth factor-1 gene correlated with body size in cetaceans. Finally, using our assembly to examine the heterozygosity and historical demography of Pacific and Atlantic blue whale populations, we found that the genomes of both populations are highly heterozygous and that their genetic isolation dates to the last interglacial period. Taken together, these results indicate how a high-quality, annotated blue whale genome will serve as an important resource for biology, evolution, and conservation research.

Genomic analyses reveal an absence of contemporary introgressive admixture between fin whales and blue whales, despite known hybrids.

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.

Whole-genome sequencing of the blue whale and other rorquals finds signatures for introgressive gene flow.

Reconstructing the evolution of baleen whales (Mysticeti) has been problematic because morphological and genetic analyses have produced different scenarios. This might be caused by genomic admixture that may have taken place among some rorquals. We present the genomes of six whales, including the blue whale (Balaenoptera musculus), to reconstruct a species tree of baleen whales and to identify phylogenetic conflicts. Evolutionary multilocus analyses of 34,192 genome fragments reveal a fast radiation of rorquals at 10.5 to 7.5 million years ago coinciding with oceanic circulation shifts. The evolutionarily enigmatic gray whale (Eschrichtius robustus) is placed among rorquals, and the blue whale genome shows a high degree of heterozygosity. The nearly equal frequency of conflicting gene trees suggests that speciation of rorqual evolution occurred under gene flow, which is best depicted by evolutionary networks. Especially in marine environments, sympatric speciation might be common; our results raise questions about how genetic divergence can be established.

Genotyping-by-sequencing for estimating relatedness in nonmodel organisms: Avoiding the trap of precise bias.

There has been remarkably little attention to using the high resolution provided by genotyping-by-sequencing (i.e., RADseq and similar methods) for assessing relatedness in wildlife populations. A major hurdle is the genotyping error, especially allelic dropout, often found in this type of data that could lead to downward-biased, yet precise, estimates of relatedness. Here, we assess the applicability of genotyping-by-sequencing for relatedness inferences given its relatively high genotyping error rate. Individuals of known relatedness were simulated under genotyping error, allelic dropout and missing data scenarios based on an empirical ddRAD data set, and their true relatedness was compared to that estimated by seven relatedness estimators. We found that an estimator chosen through such analyses can circumvent the influence of genotyping error, with the estimator of Ritland (Genetics Research, 67, 175) shown to be unaffected by allelic dropout and to be the most accurate when there is genotyping error. We also found that the choice of estimator should not rely solely on the strength of correlation between estimated and true relatedness as a strong correlation does not necessarily mean estimates are close to true relatedness. We also demonstrated how even a large SNP data set with genotyping error (allelic dropout or otherwise) or missing data still performs better than a perfectly genotyped microsatellite data set of tens of markers. The simulation-based approach used here can be easily implemented by others on their own genotyping-by-sequencing data sets to confirm the most appropriate and powerful estimator for their data.

Genetic variation in blue whales in the eastern pacific: implication for taxonomy and use of common wintering grounds.

Many aspects of blue whale biology are poorly understood. Some of the gaps in our knowledge, such as those regarding their basic taxonomy and seasonal movements, directly affect our ability to monitor and manage blue whale populations. As a step towards filling in some of these gaps, microsatellite and mtDNA sequence analyses were conducted on blue whale samples from the Southern Hemisphere, the eastern tropical Pacific (ETP) and the northeast Pacific. The results indicate that the ETP is differentially used by blue whales from the northern and southern eastern Pacific, with the former showing stronger affinity to the region off Central America known as the Costa Rican Dome, and the latter favouring the waters of Peru and Ecuador. Although the pattern of genetic variation throughout the Southern Hemisphere is compatible with the recently proposed subspecies status of Chilean blue whales, some discrepancies remain between catch lengths and lengths from aerial photography, and not all blue whales in Chilean waters can be assumed to be of this type. Also, the range of the proposed Chilean subspecies, which extends to the Galapagos region of the ETP, at least seasonally, perhaps should include the Costa Rican Dome and the eastern North Pacific as well.

Genetic Variation at Exon 2 of the MHC Class II DQB Locus in Blue Whale (Balaenoptera musculus) from the Gulf of California.

The genes of the Major Histocompatibility Complex (MHC) play an important role in the vertebrate immune response and are among the most polymorphic genes known in vertebrates. In some marine mammals, MHC genes have been shown to be characterized by low levels of polymorphism compared to terrestrial taxa; this reduction in variation is often explained as a result of lower pathogen pressures in marine habitats. To determine if this same reduction in variation applies to the migratory population of blue whales (Balaenoptera musculus) that occurs in the Gulf of California, we genotyped a 172 bp fragment of exon 2 of the MHC Class II DQB locus for 80 members of this population. Twenty-two putatively functional DQB allotypes were identified, all of which were homologous with DQB sequences from other cetacean species. Up to 5 putative alleles per individual were identified, suggesting that gene duplication has occurred at this locus. Rates of non-synonymous to synonymous substitutions (ω) and maximum likelihood analyses of models of nucleotide variation provided potential evidence of ongoing positive selection at this exon. Phylogenetic analyses of DQB alleles from B. musculus and 16 other species of cetaceans revealed trans-specific conservation of MHC variants, suggesting that selection has acted on this locus over prolonged periods of time. Collectively our findings reveal that immunogenic variation in blue whales is comparable to that in terrestrial mammals, thereby providing no evidence that marine taxa are subject to reduced pathogen-induced selective pressures.

Low genetic diversity in pygmy blue whales is due to climate-induced diversification rather than anthropogenic impacts.

Unusually low genetic diversity can be a warning of an urgent need to mitigate causative anthropogenic activities. However, current low levels of genetic diversity in a population could also be due to natural historical events, including recent evolutionary divergence, or long-term persistence at a small population size. Here, we determine whether the relatively low genetic diversity of pygmy blue whales (Balaenoptera musculus brevicauda) in Australia is due to natural causes or overexploitation. We apply recently developed analytical approaches in the largest genetic dataset ever compiled to study blue whales (297 samples collected after whaling and representing lineages from Australia, Antarctica and Chile). We find that low levels of genetic diversity in Australia are due to a natural founder event from Antarctic blue whales (Balaenoptera musculus intermedia) that occurred around the Last Glacial Maximum, followed by evolutionary divergence. Historical climate change has therefore driven the evolution of blue whales into genetically, phenotypically and behaviourally distinct lineages that will likely be influenced by future climate change.

Blue whale population structure along the eastern South Pacific Ocean: evidence of more than one population.

Blue whales (Balaenoptera musculus) were among the most intensively exploited species of whales in the world. As a consequence of this intense exploitation, blue whale sightings off the coast of Chile were uncommon by the end of the 20th century. In 2004, a feeding and nursing ground was reported in southern Chile (SCh). With the aim to investigate the genetic identity and relationship of these Chilean blue whales to those in other Southern Hemisphere areas, 60 biopsy samples were collected from blue whales in SCh between 2003 and 2009. These samples were genotyped at seven microsatellite loci and the mitochondrial control region was sequenced, allowing us to identify 52 individuals. To investigate the genetic identity of this suspected remnant population, we compared these 52 individuals to blue whales from Antarctica (ANT, n = 96), Northern Chile (NCh, n = 19) and the eastern tropical Pacific (ETP, n = 31). No significant differentiation in haplotype frequencies (mtDNA) or among genotypes (nDNA) was found between SCh, NCh and ETP, while significant differences were found between those three areas and Antarctica for both the mitochondrial and microsatellite analyses. Our results suggest at least two breeding population units or subspecies exist, which is also supported by other lines of evidence such as morphometrics and acoustics. The lack of differences detected between SCh/NCh/ETP areas supports the hypothesis that eastern South Pacific blue whales are using the ETP area as a possible breeding area. Considering the small population sizes previously reported for the SCh area, additional conservation measures and monitoring of this population should be developed and prioritized.

Population differentiation of 2 forms of Bryde's whales in the Indian and Pacific Oceans.

Accurate identification of units for conservation is particularly challenging for marine species as obvious barriers to gene flow are generally lacking. Bryde's whales (Balaenoptera spp.) are subject to multiple human-mediated stressors, including fisheries bycatch, ship strikes, and scientific whaling by Japan. For effective management, a clear understanding of how populations of each Bryde's whale species/subspecies are genetically structured across their range is required. We conducted a population-level analysis of mtDNA control region sequences with 56 new samples from Oman, Maldives, and Bangladesh, plus published sequences from off Java and the Northwest Pacific. Nine diagnostic characters in the mitochondrial control region and a maximum parsimony phylogenetic analysis identified 2 genetically recognized subspecies of Bryde's whale: the larger, offshore form, Balaenoptera edeni brydei, and the smaller, coastal form, Balaenoptera edeni edeni. Genetic diversity and differentiation indices, combined with a reconstructed maximum parsimony haplotype network, indicate strong differences in the genetic diversity and population structure within each subspecies. Discrete population units are identified for B. e. brydei in the Maldives, Java, and the Northwest Pacific and for B. e. edeni between the Northern Indian Ocean (Oman and Bangladesh) and the coastal waters of Japan.

Fine-scale population structure of blue whale wintering aggregations in the Gulf of California.

Population differentiation in environments without well-defined geographical barriers represents a challenge for wildlife management. Based on a comprehensive database of individual sighting records (1988-2009) of blue whales from the winter/calving Gulf of California, we assessed the fine-scale genetic and spatial structure of the population using individual-based approaches. Skin samples of 187 individuals were analyzed for nine microsatellite loci. A single population with no divergence among years and months and no isolation by distance (Rxy = 0.1-0.001, p>0.05) were found. We ran two bayesian clustering methods using Structure and Geneland softwares in two different ways: 1) a general analysis including all individuals in which a single cluster was identified with both softwares; 2) a specific analysis of females only in which two main clusters (Loreto Bay and northern areas, and San Jose-La Paz Bay area) were revealed by Geneland program. This study provides information indicating that blue whales wintering in the Gulf of California are part of a single population unit and showed a fine-scale structure among females, possibly associated with their high site fidelity, particularly when attending calves. It is likely that the loss of genetic variation is minimized by male mediated gene flow, which may reduce the genetic drift effect. Opportunities for kin selection may also influence calf survival and, in consequence, have a positive impact on population demography in this small and endangered population.

Hybridization of Southern Hemisphere blue whale subspecies and a sympatric area off Antarctica: impacts of whaling or climate change?

Understanding the degree of genetic exchange between subspecies and populations is vital for the appropriate management of endangered species. Blue whales (Balaenoptera musculus) have two recognized Southern Hemisphere subspecies that show differences in geographic distribution, morphology, vocalizations and genetics. During the austral summer feeding season, the Antarctic blue whale (B. m. intermedia) is found in polar waters and the pygmy blue whale (B. m. brevicauda) in temperate waters. Here, we genetically analyzed samples collected during the feeding season to report on several cases of hybridization between the two recognized blue whale Southern Hemisphere subspecies in a previously unconfirmed sympatric area off Antarctica. This means the pygmy blue whales using waters off Antarctica may migrate and then breed during the austral winter with the Antarctic subspecies. Alternatively, the subspecies may interbreed off Antarctica outside the expected austral winter breeding season. The genetically estimated recent migration rates from the pygmy to Antarctic subspecies were greater than estimates of evolutionary migration rates and previous estimates based on morphology of whaling catches. This discrepancy may be due to differences in the methods or an increase in the proportion of pygmy blue whales off Antarctica within the last four decades. Potential causes for the latter are whaling, anthropogenic climate change or a combination of these and may have led to hybridization between the subspecies. Our findings challenge the current knowledge about the breeding behaviour of the world's largest animal and provide key information that can be incorporated into management and conservation practices for this endangered species.

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.

Recovering population parameters from a single gene genealogy: an unbiased estimator of the growth rate.

We show that the number of lineages ancestral to a sample, as a function of time back into the past, which we call the number of lineages as a function of time (NLFT), is a nearly deterministic property of large-sample gene genealogies. We obtain analytic expressions for the NLFT for both constant-sized and exponentially growing populations. The low level of stochastic variation associated with the NLFT of a large sample suggests using the NLFT to make estimates of population parameters. Based on this, we develop a new computational method of inferring the size and growth rate of a population from a large sample of DNA sequences at a single locus. We apply our method first to a sample of 1,212 mitochondrial DNA (mtDNA) sequences from China, confirming a pattern of recent population growth previously identified using other techniques, but with much smaller confidence intervals for past population sizes due to the low variation of the NLFT. We further analyze a set of 63 mtDNA sequences from blue whales (BWs), concluding that the population grew in the past. This calls for reevaluation of previous studies that were based on the assumption that the BW population was fixed.

Balaenoptera omurai is a newly discovered baleen whale that represents an ancient evolutionary lineage.

Balaenoptera omurai, formerly classified as a small form of Bryde's whale, was recently reclassified as a new baleen whale species of the family Balaenopteridae. Although researchers have investigated the evolutionary history of Balaenopteridae and their relatives using molecular phylogenetic methods, the taxonomy of the ordinary Bryde's whale (Balaenoptera brydei) and small-form Bryde's whales (Balaenoptera edeni and B. omurai) remains unclear. We have used complete mtDNA sequences and short interspersed repetitive element (SINE) insertion patterns to construct the evolutionary history of both B. omurai and the taxonomically redefined species, B. edeni. The combined results demonstrate that B. omurai forms a monophyletic lineage with B. musculus, B. brydei, B. edeni and B. borealis and that B. omurai and B. musculus successively diverged from their common ancestor. In addition, we also showed that B. edeni constitutes a sister taxon to B. brydei. Our data suggest that B. omurai evolved as an ancient independent lineage that diverged much earlier than B. borealis, B. brydei and B. edeni, which were previously believed to be closely related to B. omurai.

Genetic characteristics of western North Pacific sei whales, Balaenoptera borealis, as revealed by microsatellites.

Genetic characteristics of sei whales, Balaenoptera borealis, inhabiting the western North Pacific were analyzed at 17 microsatellite loci in a total of 89 whales obtained from the area between 37 degrees N-45 degrees N and 147 degrees E-166 degrees E in 2002 (N=39) and 2003 (N=50). All the loci analyzed were polymorphic over the samples, some of the loci had more than 10 alleles, indicating a high level of genetic variation within samples. No significant deviation from the expected Hardy-Weinberg genotypic proportion was observed at the 17 loci in the samples. No evidence of genetic heterogeneity in allele frequencies was observed between sexes within samples as well as between the two temporally different samples, indicating a single population of sei whales inhabiting the western North Pacific. We finally tested and demonstrated that the population appeared not to suffer from genetic bottleneck as a result of population decline from past commercial whaling.