Evolution of the recombination regulator PRDM9 in minke whales.

BACKGROUND: PRDM9 is a key regulator of meiotic recombination in most metazoans, responsible for reshuffling parental genomes. During meiosis, the PRDM9 protein recognizes and binds specific target motifs via its array of C2H2 zinc-fingers encoded by a rapidly evolving minisatellite. The gene coding for PRDM9 is the only speciation gene identified in vertebrates to date and shows high variation, particularly in the DNA-recognizing positions of the zinc-finger array, within and between species. Across all vertebrate genomes studied for PRDM9 evolution, only one genome lacks variability between repeat types - that of the North Pacific minke whale. This study aims to understand the evolution and diversity of Prdm9 in minke whales, which display the most unusual genome reference allele of Prdm9 so far discovered in mammals. RESULTS: Minke whales possess all the features characteristic of PRDM9-directed recombination, including complete KRAB, SSXRD and SET domains and a rapidly evolving array of C2H2-type-Zincfingers (ZnF) with evidence of rapid evolution, particularly at DNA-recognizing positions that evolve under positive diversifying selection. Seventeen novel PRDM9 variants were identified within the Antarctic minke whale species, plus a single distinct PRDM9 variant in Common minke whales - shared across North Atlantic and North Pacific minke whale subspecies boundaries. CONCLUSION: The PRDM9 ZnF array evolves rapidly, in minke whales, with at least one DNA-recognizing position under positive selection. Extensive PRDM9 diversity is observed, particularly in the Antarctic in minke whales. Common minke whales shared a specific Prdm9 allele across subspecies boundaries, suggesting incomplete speciation by the mechanisms associated with PRDM9 hybrid sterility.

Associations between organohalogen exposure and thyroid- and steroid-related gene responses in St. Lawrence Estuary belugas and minke whales.

Elevated concentrations of persistent organic pollutants (POPs) and emerging halogenated flame retardants (HFRs) have been reported in tissues of the endangered St. Lawrence Estuary (Canada) beluga population as well as in minke whales visiting that same feeding area. This study examined the linkages between blubber concentrations of POPs and emerging HFRs, and transcription in skin of genes involved in the regulation of thyroid and steroid axes in belugas and minke whales from the St. Lawrence Estuary. In belugas, concentrations of PCBs, OCs and hexabromobenzene (HBB) were positively correlated with the transcription of thyroid- and/or steroid-related genes, while Dec-604 CB concentrations were negatively associated with the transcription of glucocorticoid and thyroid genes. In minke whales, PBDE concentrations changed positively with Esrß transcript levels and HBB concentrations negatively with Nr3c1 transcripts. Present results suggest that several biological functions including reproduction and energetic metabolism may represent potential targets for organohalogens in these whales.

Whole genome resequencing reveals diagnostic markers for investigating global migration and hybridization between minke whale species.

BACKGROUND: In the marine environment, where there are few absolute physical barriers, contemporary contact between previously isolated species can occur across great distances, and in some cases, may be inter-oceanic. An example of this can be seen in the minke whale species complex. Antarctic minke whales are genetically and morphologically distinct from the common minke found in the north Atlantic and Pacific oceans, and the two species are estimated to have been isolated from each other for 5 million years or more. Recent atypical migrations from the southern to the northern hemisphere have been documented and fertile hybrids and back-crossed individuals between both species have also been identified. However, it is not known whether this represents a contemporary event, potentially driven by ecosystem changes in the Antarctic, or a sporadic occurrence happening over an evolutionary time-scale. We successfully used whole genome resequencing to identify a panel of diagnostic SNPs which now enable us address this evolutionary question. RESULTS: A large number of SNPs displaying fixed or nearly fixed allele frequency differences among the minke whale species were identified from the sequence data. Five panels of putatively diagnostic markers were established on a genotyping platform for validation of allele frequencies; two panels (26 and 24 SNPs) separating the two species of minke whale, and three panels (22, 23, and 24 SNPs) differentiating the three subspecies of common minke whale. The panels were validated against a set of reference samples, demonstrating the ability to accurately identify back-crossed whales up to three generations. CONCLUSIONS: This work has resulted in the development of a panel of novel diagnostic genetic markers to address inter-oceanic and global contact among the genetically isolated minke whale species and sub-species. These markers, including a globally relevant genetic reference data set for this species complex, are now openly available for researchers interested in identifying other potential whale hybrids in the world's oceans. The approach used here, combining whole genome resequencing and high-throughput genotyping, represents a universal approach to develop similar tools for other species and population complexes.

Cetaceans evolution: insights from the genome sequences of common minke whales.

BACKGROUND: Whales have captivated the human imagination for millennia. These incredible cetaceans are the only mammals that have adapted to life in the open oceans and have been a source of human food, fuel and tools around the globe. The transition from land to water has led to various aquatic specializations related to hairless skin and ability to regulate their body temperature in cold water. RESULTS: We present four common minke whale (Balaenoptera acutorostrata) genomes with depth of ×13 ~ ×17 coverage and perform resequencing technology without a reference sequence. Our results indicated the time to the most recent common ancestors of common minke whales to be about 2.3574 (95% HPD, 1.1521 - 3.9212) million years ago. Further, we found that genes associated with epilation and tooth-development showed signatures of positive selection, supporting the morphological uniqueness of whales. CONCLUSIONS: This whole-genome sequencing offers a chance to better understand the evolutionary journey of one of the largest mammals on earth.

Investigating population genetic structure in a highly mobile marine organism: the minke whale Balaenoptera acutorostrata acutorostrata in the North East Atlantic.

Inferring the number of genetically distinct populations and their levels of connectivity is of key importance for the sustainable management and conservation of wildlife. This represents an extra challenge in the marine environment where there are few physical barriers to gene-flow, and populations may overlap in time and space. Several studies have investigated the population genetic structure within the North Atlantic minke whale with contrasting results. In order to address this issue, we analyzed ten microsatellite loci and 331 bp of the mitochondrial D-loop on 2990 whales sampled in the North East Atlantic in the period 2004 and 2007-2011. The primary findings were: (1) No spatial or temporal genetic differentiations were observed for either class of genetic marker. (2) mtDNA identified three distinct mitochondrial lineages without any underlying geographical pattern. (3) Nuclear markers showed evidence of a single panmictic population in the NE Atlantic according STRUCTURE's highest average likelihood found at K = 1. (4) When K = 2 was accepted, based on the Evanno's test, whales were divided into two more or less equally sized groups that showed significant genetic differentiation between them but without any sign of underlying geographic pattern. However, mtDNA for these individuals did not corroborate the differentiation. (5) In order to further evaluate the potential for cryptic structuring, a set of 100 in silico generated panmictic populations was examined using the same procedures as above showing genetic differentiation between two artificially divided groups, similar to the aforementioned observations. This demonstrates that clustering methods may spuriously reveal cryptic genetic structure. Based upon these data, we find no evidence to support the existence of spatial or cryptic population genetic structure of minke whales within the NE Atlantic. However, in order to conclusively evaluate population structure within this highly mobile species, more markers will be required.

Minke whale genome and aquatic adaptation in cetaceans.

The shift from terrestrial to aquatic life by whales was a substantial evolutionary event. Here we report the whole-genome sequencing and de novo assembly of the minke whale genome, as well as the whole-genome sequences of three minke whales, a fin whale, a bottlenose dolphin and a finless porpoise. Our comparative genomic analysis identified an expansion in the whale lineage of gene families associated with stress-responsive proteins and anaerobic metabolism, whereas gene families related to body hair and sensory receptors were contracted. Our analysis also identified whale-specific mutations in genes encoding antioxidants and enzymes controlling blood pressure and salt concentration. Overall the whale-genome sequences exhibited distinct features that are associated with the physiological and morphological changes needed for life in an aquatic environment, marked by resistance to physiological stresses caused by a lack of oxygen, increased amounts of reactive oxygen species and high salt levels.

Hybrids between common and Antarctic minke whales are fertile and can back-cross.

BACKGROUND: Minke whales are separated into two genetically distinct species: the Antarctic minke whale found in the southern hemisphere, and the common minke whale which is cosmopolitan. The common minke whale is further divided into three allopatric sub-species found in the North Pacific, southern hemisphere, and the North Atlantic. Here, we aimed to identify the genetic ancestry of a pregnant female minke whale captured in the North Atlantic in 2010, and her fetus, using data from the mtDNA control region, 11 microsatellite loci and a sex determining marker. RESULTS: All statistical parameters demonstrated that the mother was a hybrid displaying maternal and paternal contribution from North Atlantic common and Antarctic minke whales respectively. Her female fetus displayed greater genetic similarity to North Atlantic common minke whales than herself, strongly suggesting that the hybrid mother had paired with a North Atlantic common minke whale. CONCLUSION: This study clearly demonstrates, for the first time, that hybrids between minke whale species may be fertile, and that they can back-cross. Whether contact between these species represents a contemporary event linked with documented recent changes in the Antarctic ecosystem, or has occurred at a low frequency over many years, remains open.

Genotyping errors in a calibrated DNA register: implications for identification of individuals.

BACKGROUND: The use of DNA methods for the identification and management of natural resources is gaining importance. In the future, it is likely that DNA registers will play an increasing role in this development. Microsatellite markers have been the primary tool in ecological, medical and forensic genetics for the past two decades. However, these markers are characterized by genotyping errors, and display challenges with calibration between laboratories and genotyping platforms. The Norwegian minke whale DNA register (NMDR) contains individual genetic profiles at ten microsatellite loci for 6737 individuals captured in the period 1997-2008. These analyses have been conducted in four separate laboratories for nearly a decade, and offer a unique opportunity to examine genotyping errors and their consequences in an individual based DNA register. We re-genotyped 240 samples, and, for the first time, applied a mixed regression model to look at potentially confounding effects on genotyping errors. RESULTS: The average genotyping error rate for the whole dataset was 0.013 per locus and 0.008 per allele. Errors were, however, not evenly distributed. A decreasing trend across time was apparent, along with a strong within-sample correlation, suggesting that error rates heavily depend on sample quality. In addition, some loci were more error prone than others. False allele size constituted 18 of 31 observed errors, and the remaining errors were ten false homozygotes (i.e., the true genotype was a heterozygote) and three false heterozygotes (i.e., the true genotype was a homozygote). CONCLUSIONS: To our knowledge, this study represents the first investigation of genotyping error rates in a wildlife DNA register, and the first application of mixed models to examine multiple effects of different factors influencing the genotyping quality. It was demonstrated that DNA registers accumulating data over time have the ability to maintain calibration and genotyping consistency, despite analyses being conducted on different genotyping platforms and in different laboratories. Although errors were detected, it is demonstrated that if the re-genotyping of individual samples is possible, these will have a minimal effect on the database's primary purpose, i.e., to perform individual identification.

Are Antarctic minke whales unusually abundant because of 20th century whaling?

Severe declines in megafauna worldwide illuminate the role of top predators in ecosystem structure. In the Antarctic, the Krill Surplus Hypothesis posits that the killing of more than 2 million large whales led to competitive release for smaller krill-eating species like the Antarctic minke whale. If true, the current size of the Antarctic minke whale population may be unusually high as an indirect result of whaling. Here, we estimate the long-term population size of the Antarctic minke whale prior to whaling by sequencing 11 nuclear genetic markers from 52 modern samples purchased in Japanese meat markets. We use coalescent simulations to explore the potential influence of population substructure and find that even though our samples are drawn from a limited geographic area, our estimate reflects ocean-wide genetic diversity. Using Bayesian estimates of the mutation rate and coalescent-based analyses of genetic diversity across loci, we calculate the long-term population size of the Antarctic minke whale to be 670,000 individuals (95% confidence interval: 374,000-1,150,000). Our estimate of long-term abundance is similar to, or greater than, contemporary abundance estimates, suggesting that managing Antarctic ecosystems under the assumption that Antarctic minke whales are unusually abundant is not warranted.

Estimating the number of whales entering trade using DNA profiling and capture-recapture analysis of market products.

Surveys of commercial markets combined with molecular taxonomy (i.e. molecular monitoring) provide a means to detect products from illegal, unregulated and/or unreported (IUU) exploitation, including the sale of fisheries bycatch and wild meat (bushmeat). Capture-recapture analyses of market products using DNA profiling have the potential to estimate the total number of individuals entering the market. However, these analyses are not directly analogous to those of living individuals because a 'market individual' does not die suddenly but, instead, remains available for a time in decreasing quantities, rather like the exponential decay of a radioactive isotope. Here we use mitochondrial DNA (mtDNA) sequences and microsatellite genotypes to individually identify products from North Pacific minke whales (Balaenoptera acutorostrata ssp.) purchased in 12 surveys of markets in the Republic of (South) Korea from 1999 to 2003. By applying a novel capture-recapture model with a decay rate parameter to the 205 unique DNA profiles found among 289 products, we estimated that the total number of whales entering trade across the five-year survey period was 827 (SE, 164; CV, 0.20) and that the average 'half-life' of products from an individual whale on the market was 1.82 months (SE, 0.24; CV, 0.13). Our estimate of whales in trade (reflecting the true numbers killed) was significantly greater than the officially reported bycatch of 458 whales for this period. This unregulated exploitation has serious implications for the survival of this genetically distinct coastal population. Although our capture-recapture model was developed for specific application to the Korean whale-meat markets, the exponential decay function could be modified to improve the estimates of trade in other wildmeat or fisheries markets or abundance of living populations by noninvasive genotyping.

Identification and hepatic expression profiles of cytochrome P450 1-4 isozymes in common minke whales (Balaenoptera acutorostrata).

Full-length cDNA sequences of cytochrome P450 (CYP) 2C78, 2E1, 3A72, 4A35 and 4V6 isozymes were isolated from a hepatic cDNA library of common minke whale (Balaenoptera acutorostrata). The deduced amino acid sequences of minke whale CYP2C78, 2E1, 3A72, 4A35 and 4V6 showed high identities with cattle CYP2C86 (83%), pig CYP2E1 (85%), sheep CYP3A24 (82%), pig CYP4A21 (80%), and human CYP4V2 (76%), respectively. To investigate whether or not these CYP expression levels are altered by contamination of organochlorine contaminants (OCs), mRNA levels of these CYPs in the liver of common minke whale were measured using a quantitative real-time RT-PCR method, and the quantified mRNA levels were employed for the statistical analysis with the residue levels of OCs including PCBs, DDTs (p,p'-DDT, p,p'-DDD and p,p'-DDE), chlordanes (cis-chlordane, trans-chlordane, cis-nonachlor, trans-nonachlor and oxychlordane), HCHs (alpha-, beta- and gamma-isomers) and hexachlorobenzene that have already been reported elsewhere. Spearman's rank correlation analyses showed no significant correlation between CYP expression levels and each OC level in the common minke whale liver, implying that these environmental chemicals have no potential to alter the expression levels of these CYPs or the residue levels encountered in the whale livers may not reach their transcriptional regulation levels. This suggests that the expression of individual CYPs in the whale liver may be at basal level. Relationships among hepatic mRNA expression levels of these CYP2-4 isozymes together with CYP1A1 and CYP1A2 were also examined. Significant positive correlations were detected among mRNA expression levels of individual CYP isozymes in most cases. These associations indicate that the transcriptional regulation of these CYPs examined in this study may be reciprocally related. CYP1A1 levels showed a positive correlation with CYP1A2 levels (r=0.64, p<0.01) indicating that both CYP isozymes were regulated by aryl hydrocarbon receptor activated by endogenous ligands. A strong positive correlation between CYP2C78 and 3A72 (r=0.90, p<0.001) suggests that expression of these CYP isozymes may be under a regulation mechanism of cross-talk in which specific nuclear receptors such as constitutive androstane receptor and pregnane X receptor are involved. The present study indicates that minke whale from the North Pacific may be a model species to investigate the mechanism of basal regulation of these CYPs.

Radiation and speciation of pelagic organisms during periods of global warming: the case of the common minke whale, Balaenoptera acutorostrata.

How do populations of highly mobile species inhabiting open environments become reproductively isolated and evolve into new species? We test the hypothesis that elevated ocean-surface temperatures can facilitate allopatry among pelagic populations and thus promote speciation. Oceanographic modelling has shown that increasing surface temperatures cause localization and reduction of upwelling, leading to fragmentation of feeding areas critical to pelagic species. We test our hypothesis by genetic analyses of populations of two closely related baleen whales, the Antarctic minke whale (Balaenoptera bonaerensis) and common minke whale (Balaenoptera acutorostrata) whose current distributions and migration patterns extent are largely determined by areas of consistent upwelling with high primary production. Phylogeographic and population genetic analyses of mitochondrial DNA control-region nucleotide sequences collected from 467 whales sampled in four different ocean basins were employed to infer the evolutionary relationship among populations of B. acutorostrata by rooting an intraspecific phylogeny with a population of B. bonaerensis. Our findings suggest that the two species diverged in the Southern Hemisphere less than 5 million years ago (Ma). This estimate places the speciation event during a period of extended global warming in the Pliocene. We propose that elevated ocean temperatures in the period facilitated allopatric speciation by disrupting the continuous belt of upwelling maintained by the Antarctic Circumpolar Current. Our analyses revealed that the current populations of B. acutorostrata likely diverged after the Pliocene some 1.5 Ma when global temperatures had decreased and presumably coinciding with the re-establishment of the polar-equatorial temperature gradient that ultimately drives upwelling. In most population samples, we detected genetic signatures of exponential population expansions, consistent with the notion of increasing carrying capacity after the Pliocene. Our hypothesis that prolonged periods of global warming facilitate speciation in pelagic marine species that depend on upwelling should be tested by comparative analyses in other pelagic species.

DNA registers of legally obtained wildlife and derived products as means to identify illegal takes.

The exploitation and sale of wildlife species that are endangered in only part of their range present regulators with the critical challenge of separating legal from illegal takes. Wildlife DNA registers created from tissue samples of legally obtained individual wildlife specimens can address this problem by allowing managers to identify unregistered (presumably illegally obtained) specimens. We tested the effectiveness of the only current, fully operational wildlife DNA register of individual genetic profiles collected from legally caught minke whales (Balaenoptera acutorostrata). Twenty minke whale tissue samples collected at markets in Norway and 2 additional samples collected from beached minke whales in Denmark were genotyped at 12 loci used by the Norwegian minke whale DNA register Genetic profiles of these samples then were compared against the 2676 individual profiles deposited in the Norwegian register The high number of genetic markers used to identify individuals in our study allowed consistent matching of sample and reference profiles despite an overall error rate (due to experimental and interlaboratory data standardization) estimated at 0.015 per locus. Of the 22 test samples only the 2 Danish samples failed to match an existing profile in the Norwegian minke whale DNA register Our results show that the basic principle of wildlife DNA registers can work in a real-life situation. The strength of wildlife DNA registers lies in their ability to unambiguously identify unregistered specimens with the aid of sensitive genetic methods that enable analysis of highly processed or degraded tissue samples. Our study also highlights a number of methodological problems such as laboratory errors and interlaboratory data standardization, which need be addressed to ensure a successful implementation of wildlife DNA registers.

Molecular cloning and mRNA expression of cytochrome P4501A1 and 1A2 in the liver of common minke whales (Balaenoptera acutorostrata).

This study presents full-length cDNA sequences of CYP1A1 and 1A2, in common minke whale (Balaenoptera acutorostrata) from the North Pacific. Both CYP1A1 and CYP1A2 cDNAs had an open reading frame of 516 amino acid residues, and predicted molecular masses were 58.3 kDa and 58.1 kDa, respectively. The deduced full-length amino acid sequence of CYP1A1 revealed higher identities with those of sheep (86%) and pig (87%), and that of CYP1A2 was most closely related to human (82%) and monkey CYP1A2 (82%) among species from which CYP1A2 has been isolated so far. Differences in certain conserved and functional amino acid residues of CYP1A1 and 1A2 between common minke whale and other mammalian species indicate the possibility of their specific metabolic function. Concentrations of organochlorine compounds (OCs) including PCBs and DDTs analyzed in common minke whale liver showed no significant correlation with hepatic mRNA expression levels of CYP1A1 and CYP1A2, indicating no induction of these enzymes by such OCs.