Introduction

The pelagic sei whale (Balaenoptera borealis) has a cosmopolitan distribution and undertakes seasonal migrations between high-latitude summer foraging grounds and low-latitude winter breeding grounds (Mizroch et al. 1984). Sei whales were commercially hunted from the 1950s to 1980s after populations of the larger baleen whales were depleted by whaling (Mizroch et al. 1984; Prieto et al. 2012). The current population trends are unknown and the International Union for the Conservation of Nature (IUCN) estimated the current global abundance of sei whales at approximately 20% of pre-whaling levelsFootnote 1. Although the International Whaling Commission (IWC) placed a moratorium on commercial whaling in 1986, sei whales are still occasionally targeted under special permits for scientific whaling and aboriginal subsistence huntingFootnote 2.

In 1977, the IWC divided the global sei whale population into distinct ‘stocks’ for management purposes. The stock divisions were based upon the distribution of catches and sightings as well as mark-recapture data, which was the nature of the data available at the time (Donovan 1991). The Southern Hemisphere was divided into six stocks, following IWC management practice for other baleen whale species. Initially, three distinct stocks were proposed in the North Pacific, but these were subsequently combined into a single stock, due to absence of conclusive evidence for a three-stock hypothesis (Donovan 1991).

In the North Atlantic, sei whales were caught and sighted in eight main areas. However, the IWC did not presume these areas to represent different stocks and instead divided the North Atlantic sei whales into three stocks: the Nova Scotian, Iceland-Denmark Strait and Eastern North Atlantic stocks (Fig. 1). The possible presence of a fourth stock off Labrador north of the Nova Scotian stock boundary (Donovan 1991) was acknowledged, but this stock was never designated. After the cessation of commercial sei whaling, the overall research effort aimed specifically at sei whales was reduced and most efforts were directed towards the larger mysticetes (Prieto et al. 2012). As a result, the original stock boundaries for sei whales in the North Atlantic have remained unchanged, even though it is unclear whether they reflect an underlying ‘biological’ population structure (Donovan 1991).

Fig. 1
figure 1

Map with sampling locations in the North Atlantic and the current IWC stock boundaries

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As is the case for other cosmopolitan mysticete species, such as fin (Balaenoptera physalus) and humpback whales (Megaptera novaeangliae), each major ocean basin (i.e. the Southern, the Pacific and the Atlantic Ocean) likely represents a distinct stock or subspecies (e.g. Archer et al. 2013; Jackson et al. 2014), and perhaps even different species in the case of right whales (Eubalaena spp.; Rosenbaum et al. 2000). The sei whale’s annual migration cycle between low and high latitudes is similar to the annual migration pattern assumed for many mysticetes resulting in an anti-tropical temporal separation between populations in different hemispheres (Mizroch et al. 1984). In addition, the populations in the Atlantic and the Pacific are geographically separated by continental land masses.

Genetic analysis of sei whale materials began with an allozyme study by Wada and Numachi (1991) who compared the allozyme variation at 45 loci in sei whales sampled in the Southern Ocean and the North Pacific. The authors reported statistically significant differences in allele frequencies between the two hemispheres. A more recent study compared mitochondrial DNA (mtDNA) control region sequence variation in samples collected from sei whales in the two aforementioned ocean basins and the North Atlantic (Baker et al. 2004). The study revealed that North Atlantic sei whales were genetically distinct from their North Pacific and Southern Hemisphere conspecifics. In contrast to earlier findings by Wada and Numachi (1991), Baker et al. (2004) failed to detect a clear differentiation between the Southern Ocean and the North Pacific.

The population genetic structure of sei whales within each ocean basin remains poorly understood as well. No data or analyses of the sei whale population genetic structure within the Southern Ocean have been presented so far. In the cases of the North Pacific and North Atlantic populations, few analyses and data have been presented. Kanda et al. (2006) failed to detect any spatial or temporal heterogeneity in the genetic variation at both microsatellite loci and later mtDNA sequences (Kanda et al. 2009) in 790 North Pacific sei whales. Similarly, Daníelsdottír et al. (1991) did not detect any temporal heterogeneity at 40 allozyme loci genotyped in 101 sei whales caught off Iceland between 1985 and 1988. Population genetic structure across the North Atlantic basin has yet to be assessed.

Recent satellite tagging studies (Olsen et al. 2009; Prieto et al. 2014) have shed some light on possible sei whale migratory routes in the North Atlantic. Olsen et al. (2009) deployed a satellite radio transmitter on a sei whale off the Azores, which was tracked to the Labrador Sea, revealing that some sei whales traverse the entire North Atlantic during the spring migration. Prieto et al. (2014) later deployed satellite radio transmitters on seven sei whales off the Azores during their spring migration, which were all tracked to summer foraging grounds in the Labrador Sea. Signals from two transmitters were lost when the tagged whales were moving toward the Iceland-Denmark Strait. The trajectory of these two tagged whales suggests that sei whales can move among different high-latitude summer foraging grounds, but whether different sei whale breeding populations also utilize the same foraging grounds, remains unknown. The exact location of the low-latitude winter breeding grounds is unknown, although there may be a located ground off northwestern Africa (Ingebrigtsen 1929; Prieto et al. 2012, 2014).

Given the documented long seasonal migrations of sei whales in the North Atlantic (Olsen et al. 2009; Prieto et al. 2014) and wide summer ranges (see above), it is plausible that the genetic heterogeneity among North Atlantic sei whale summer grounds is low as in the case of the North Pacific sei whale (Kanda et al. 2006, 2009). Here we present the results of the first assessment of the population genetic structure of sei whales in three different locations in the North Atlantic, representing two of the three putative stocks; off Iceland, in the Gulf of Maine and in the Azores. Under the three-stock hypothesis, we expected Iceland and the Azores to be genetically similar, and different from the Gulf of Maine. However, Iceland and the Gulf of Maine most likely represent summer foraging grounds and the Azores a migratory corridor where whales from different winter breeding grounds potentially mix (Olsen et al. 2009; Prieto et al. 2014). We estimated the effective population size, divergence time and migration rates of sei whales in two different ocean basins: the North Atlantic and North Pacific Oceans. To this end, the genetic data on North Atlantic sei whales from the present study were combined with previously published genetic data collected from North Pacific sei whales (Kanda et al. 2006, 2009).

Materials and methods

Sample collection

The genetic data from the North Atlantic were obtained from tissue samples collected from sei whales caught during special-permit whaling operations off Iceland (1986–1988; n = 43), and as skin biopsies obtained from free-ranging sei whales using a crossbow and biopsy tips (Palsbøll et al. 1991) in the Gulf of Maine (1999, 2002–2004; n = 18) and the Azores (2005, 2008–2010; n = 26) (Fig. 1). Biopsy collection was conducted under national permits and according to national regulations. The laboratory methods described below pertain to the North Atlantic samples.

Data from previous studies

Genetic data from the North Pacific (collected 2002–2007) were obtained from previously published studies (n = 489; Kanda et al. 2006, 2009; Tamura et al. 2009). A single additional Antarctic sei whale mtDNA control region sequence was obtained from GenBank™ (accession number NC_006929.1; Sasaki et al. 2005).

DNA extraction and sexing

Total-cell DNA was extracted using the Qiagen DNeasy™ Blood and Tissue Kit (Qiagen Inc.) according to the manufacturer’s instructions. The extracted DNA was re-suspended in 1× TE buffer (10 mM Tris–HCl, 1 mM EDTA, pH 8.0). Samples were sexed using the ZFY/ZFX multiplexing system as described by Bérubé and Palsbøll (1996a, b).

Genotyping microsatellite loci

Eleven microsatellite loci were genotyped using the polymerase chain reaction (PCR; Mullis and Faloona 1987). The specific loci genotyped were: EV094 and EV037 (Valsecchi and Amos 1996), GATA028, GATA053, GATA098 (Palsbøll et al. 1997), GT011 (Bérubé et al. 1998), GT023 and GT211 (Bérubé et al. 2000) as well as AC082, CA232 and GT541 (Bérubé et al. 2005).

PCR amplifications of the above microsatellite loci were performed in 10 µL reaction volumes containing 1× Taq buffer (Fermentas Inc.), 3.2 mM dNTPs, 0.4 units Taq DNA polymerase (Fermentas Inc.) and 1 ng extracted DNA. The concentration of each PCR primer pair differed among loci. The concentrations of the forward and reverse primers were: 0.25 µM for locus EV094, GATA028, GATA053 and GATA098, and 0.50 µM for locus AC082, CA232, EV037, GT011, GT023, GT211 and GT541. The PCR amplifications were conducted using a MJ Research PTC-100™ (MJ Research Inc.) in the case of locus GATA028 and GT023, a MJ Research Dyad™ thermocycler (MJ Research Inc.) for locus AC082, CA232, EV037, EV094, GATA053, GATA098, GT011 and GT541, and a Stratagene Robocycler™ (Stratagene Inc.) for locus GT211. PCR cycling profiles were as described in the original publications of each locus.

The experimental conditions employed for the data generation of the North Pacific samples were described by Kanda et al. (2006, 2009). The microsatellite genotypes from the two datasets (the North Pacific and North Atlantic) were calibrated by re-genotyping the above microsatellite loci in 55 North Pacific samples.

Sequencing the mtDNA control region

The first 487 base pairs of the 3′ end of the mtDNA control region were amplified and the nucleotides sequenced. The fragment corresponds to positions 15,476–15,963 in the published sei whale mitochondrial genome (Árnason et al. 1993; Sasaki et al. 2005). The PCR primers used for the amplification were MT4F (Árnason et al. 1993) and Mn312-R (Palsbøll et al. 1995), as well as BP16071R (Drouot et al. 2004).

For the North Atlantic samples, PCR amplification was performed in a final volume at 15µL containing: 1 µM of each PCR primer, 1× Taq DNA polymerase buffer (Fermentas Inc.), 3.2 mM dNTPs, 0.09 units Taq DNA polymerase (Fermentas Inc.), and 1 ng of extracted DNA. The PCR amplifications were conducted using an MJ Research PTC-100™ thermocycler (MJ Research Inc.) and occurred in 25 reaction cycles, each consisting of a denaturing step of 30 s at 94 °C, a 30 s annealing step at 54 °C and a 120 s extension step at 72 °C. These 25 cycles were preceded by a single 120 s denaturing step at 94 °C.

Unincorporated ddNTPs and PCR primers were removed using the Shrimp Alkaline Phosphate/Exo-I protocol described by Werle et al. (1994). Cycle-sequencing of the PCR products obtained by the above described amplifications was performed using the BigDye Terminator™ ver. 3.1 Cycle Sequencing Kit (Applied Biosystems Inc.) following the manufacturer’s instructions, in both directions using the same primers as used for the initial PCR amplification. The cycle-sequencing products were purified by ethanol/sodium acetate precipitation (Sambrook and Russell 2001). The order of labeled sequencing fragments was resolved by capillary electrophoresis on an ABI 3730 DNA Genetic Analyzer™ (Applied Biosystems Inc.).

Analysis of microsatellite genotypes

Quality control and levels of polymorphism

Microsatellite alleles were visually checked and sized using GENEMAPPER™ (ver. 4.0, Applied Biosystems Inc.). All 87 samples were re-typed once at all 11 loci to estimate a genotyping ‘inconsistency rate’ per genotype. We estimated the number of alleles (A), the expected (HE), the observed heterozygosity (HO), and the probability of identity (I; Paetkau et al. 1995). I was subsequently employed to detect duplicate samples from the same individuals. HE and HO were estimated using ARLEQUIN (ver. 3.5.2.2, Excoffier and Lischer 2010) and I was estimated using GENALEX (ver. 6.5, Peakall and Smouse 2006, 2012). The 95% confidence interval for the mean HE and HO was estimated by bootstrapping over loci (10,000 replicates) using the R package POPGENKIT (Paquette 2012) in R (ver. 3.2.5, R Development Core Team 2016).

Controlling procedure for multiple comparisons

The false discovery rate correction developed by Benjamini and Hochberg (FDR; Benjamini and Hochberg 1995) was applied in all instances when multiple simultaneous tests were conducted, using a critical alpha-value at 0.05.

Assessing deviations from Hardy–Weinberg expectations and linkage disequilibrium

Deviations from the expected Hardy–Weinberg genotype proportions and linkage disequilibrium were assessed using Fisher’s exact test (Fisher 1935) implemented in GENEPOP (ver. 4.1.4, Raymond and Rousset 1995; Rousset 2008) using the default analysis parameters and a complete enumeration whenever possible.

Homogeneity tests and genetic divergence

The degree of genetic differentiation was estimated as ϴ (Weir and Cockerham 1984). The probability of ϴ being equal to or larger than the observed value of ϴ under the null hypothesis of a panmictic population was estimated from 10,000 permutations (without replacement) as implemented in ARLEQUIN (ver. 3.5.2.2, Excoffier and Lischer 2010). The 95% confidence intervals of the observed estimates were obtained from 10,000 bootstrap replicates as implemented in the package DIVERSITY (Keenan et al. 2013) in R (ver. 3.2.5, R Development Core Team 2016).

Bayesian clustering

The software STRUCTURE (ver. 2.3.4, Pritchard et al. 2000; Falush et al. 2007) was employed to assess possible cryptic population genetic structure. We followed the recommendation by Wang (2017). In each assessment, we employed the admixture and the ‘F’ model, the sample location as a prior, and 100,000 burn-in Markov chains, followed by 200,000 Markov chains. Fifteen replicates were conducted per value of K, ranging from one to five. Lambda was inferred per ‘population’. The remaining estimation parameters were the software default values. The output was summarized using the program CLUMPAK (Kopelman et al. 2015). The most probable value of K was determined from the posterior mean likelihood values (Pritchard et al. 2000).

Analysis of mtDNA control region sequences

Levels of polymorphism

The chromatograms of the mtDNA control region sequences were visually checked using CHROMAS™ (ver. 2.13, Technelysium Inc.) and sequences were aligned using CLUSTALW (ver. 1, Thompson et al. 1994) with default parameter settings as implemented in MEGA (ver. 6.06, Tamura et al. 2013). DNASP (ver. 5.10, Librado and Rozas 2009) was employed to estimate the haplotype (HD) and nucleotide diversity (π; Nei 1987). Coalescent simulations (Hudson 1990, implemented in DNASP) were employed to estimate the 95% confidence interval for both HD and π from 10,000 replicates.

Estimation of mtDNA haplotype sequence genealogy

Nucleotide positions subject to alignment gaps were deleted from the entire dataset. The genetic distances among the haplotypes were estimated and visualized using MEGA (ver. 6.06, Tamura et al. 2013). Genetic distances were estimated using Kimura’s 2-parameter model of nucleotide substitution (Kimura 1980) using a transition–transversion ratio (R) estimated from the data. R was estimated at 15 using the maximum-likelihood method in MEGA. The mtDNA genealogy was estimated using the maximum-likelihood method from the genetic distances estimated as described above. The consensus genealogy and support for each node was inferred from 10,000 bootstrap (over nucleotide positions) replicates (Felsenstein 1985). The genealogy was rooted with the homologous mtDNA control region sequences from a North Atlantic fin whale, Balaenoptera physalus, (GenBank™ accession number NC_001321.1; Árnason et al. 1991) and a North Pacific Bryde’s whale, B. brydei (GenBank™ accession number NC_006928.1; Sasaki et al. 2005). Furthermore, a neighbor-joining genealogy (Saitou and Nei 1987; Tamura et al. 2004) was estimated in MEGA using the same settings as for the maximum-likelihood genealogy and default settings for tree inference. Haplotype networks of both genealogies (without the Antarctic haplotype and the two outgroups) were estimated using the software HAPLOTYPE VIEWER (Ewing 2010).

Homogeneity tests and genetic divergence

The degree of differentiation was estimated as ΦST (Excoffier et al. 1992) using ARLEQUIN (ver. 3.5.2.2, Excoffier and Lischer 2010) applying Kimura’s 2-parameter model (Kimura 1980). The probability of ΦST being equal to or larger than the observed value of ΦST under the null hypothesis of a panmictic population was estimated from 10,000 permutations (without replacement) as implemented in ARLEQUIN. The 95% confidence intervals of the observed estimates were obtained from 10,000 bootstrap replicates as implemented in the package DIVERSITY (Keenan et al. 2013) in R (ver. 3.2.5, R Development Core Team 2016).

Estimation of effective population size, divergence time and migration rates

Effective population size, population divergence time and migration rates were estimated employing the coalescent approach implemented in the software IMA2P (ver. 1.0, Sethuraman and Hey 2016) which applies the Isolation with Migration model to genetic data. Compared to other demographic inference methods such as the methods implemented in the software BEAST (Drummond and Rambaut 2007) or MIGRATE-N (Beerli and Felsenstein 2001), which allow the estimation of either effective population size and divergence time or effective population size and migration rate, IMA2P allows the estimation of all three parameters (i.e., effective population size, divergence time and migration rate).

We applied the HKY model of sequence evolution (Hasegawa et al. 1985) and an annual, per-locus mutation rate at 2.58 × 10−5 (based upon a per-site mutation rate at 5.30 × 10−8 from Alter and Palumbi 2009) and a prior range from 4.87 × 10−6 to 4.87 × 10−5. The generation time was 26.19 years; i.e. the average of 29.08 years (Pacifici et al. 2013) and 23.30 years (Taylor et al. 2007). The inheritance scalar was set at 0.25.

The priors were defined from the posterior distribution from preliminary estimations (see Table S1 and Fig. S1) varying priors of θ (4Neµ, where Ne denotes the effective population size and µ the generational mutation rate), m (Nm/µ, where Nm denotes the number of migrants per generation) and divergence time (t = Tdivµ, where Tdiv denotes the time since splitting in generations) parameters. The final prior parameter values were set at θ = 250, m = 1.5 and t = 10 for the upper bound and zero for the lower bound for all parameters. The final Markov Chain Monte Carlo (MCMC) sampling comprised 1.0 × 108 steps, with samples drawn from the posterior every 100 steps and a preliminary burn-in at 1.0 × 106 steps. The Metropolis-Coupled Markov Chain Monte Carlo (MC3) was applied to improve the mixing. Stationarity was considered achieved when no perceivable trends were observed in the plot trend and an effective sample size (ESS) > 500 was obtained for all values. In addition, six independent runs, i.e. with different random number seeds, were examined for consistency in the final parameter estimates. The final parameter estimates of Ne, t and 2mNe were the average value of the six replicates (Table S2).

Tests of mutation-drift equilibrium and mismatch distributions

Estimates of Tajima’s D (Tajima 1989) and Fu and Li’s F* (Fu and Li 1993) and their statistical significance were computed using DNASP (ver. 5.10, Librado and Rozas 2009) to assess possible deviations from neutral evolution. Coalescent simulations (Hudson 1990; implemented in DNASP) were employed to estimate 95% confidence intervals for D and F* from 10,000 replicates. Furthermore, frequency distributions of the observed pairwise nucleotide site differences (‘mismatch distributions’) per sampling location were computed using DNASP and compared to the expected distribution for a population of exponentially expanding size (Watterson 1975; Slatkin and Hudson 1991; Rogers and Harpending 1992). The degree of statistical deviation from the expected distribution was evaluated with the χ2 test (Lindgren 1975).

Data access

All North Atlantic mtDNA haplotypes have been deposited in GenBank™ under accession numbers MH035689–MH035695. Interested readers are encouraged to contact the corresponding author(s) for microsatellite genotypes and access to raw data.

Results

Data analysis of microsatellite genotypes

Duplicate samples and missing data

The probability of identity (I) was estimated at 5.0 × 10−9 for the North Atlantic samples (a total of 11 loci, Tables 1 and 2) and at 1.1 × 10−5 for the North Pacific samples (a total of 7 loci, Table 1). Consequently, the expected number of pairs of unrelated individuals matching at all loci was at 4.2 × 10−7 in the North Atlantic and at 5.3 × 10−3 in the North Pacific. No matching pairs of multi-locus genotypes were observed among the North Pacific samples. A total of three pairs of matching multi-locus genotypes were detected among the North Atlantic samples; two sample pairs from the Gulf of Maine and one pair from the Azores. Also considering the samples’ corresponding sex and mtDNA haplotype, these were inferred as duplicate samples from the same individuals. Accordingly, only data from one sample of each identical pair were retained in the final dataset.

Table 1 Microsatellite diversity indices in North Pacific and North Atlantic samples
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The calibration with the North Atlantic dataset (i.e. size-calling of North Pacific alleles after amplification with North Atlantic primers) failed for four North Pacific samples, which were thus discarded. No ambiguous genotypes were found after re-typing the North Atlantic samples for all loci, yielding an inconsistency rate of < 5.2 × 10−4 per genotype.

The final microsatellite dataset was comprised of 569 unique multi-locus genotypes; n = 485 for the North Pacific, n = 43 for Iceland, n = 16 for the Gulf of Maine and n = 25 for the Azores. In total, eight genotypes were missing from the final dataset (i.e. 0.2%). Of the four additional microsatellite loci genotyped only in the North Atlantic samples, 6% of genotypes were missing.

Diversity estimates

Tables 1 and 2 list the diversity estimates observed for the microsatellite loci. The number of alleles ranged from 3 (GATA053) to 13 (GT023). The mean number of alleles was 8.4. Private alleles were detected in both ocean basins, as well as in each of the three North Atlantic sampling locations. When considering all 59 different alleles observed in the total dataset for 7 loci, 21 (35.6%) of these were private to the North Pacific and 10 (16.9%) were private to the North Atlantic. When considering all 68 different alleles observed in the North Atlantic dataset for 11 loci, 1 allele (1.5%) was private to the Gulf of Maine, 5 alleles (7.4%) were private to Iceland and 2 (2.9%) were private to the Azores.

Table 2 Measures of diversity for 4 microsatellite loci analysed only in the North Atlantic samples
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Mean HE for all seven microsatellite loci was similar in each ocean basin (Table 1); HE was estimated at 0.60 in the North Atlantic (ranging from 0.19 to 0.76) and at 0.58 in the North Pacific (ranging from 0.31 to 0.81). The mean HO was also estimated at 0.60 in the North Atlantic (ranging from 0.17 to 0.88) and at 0.56 in the North Pacific (ranging from 0.30 to 0.77). The estimates of the mean HE and HO at each North Atlantic sampling location were in the same range as the estimates obtained from the pooled samples in both ocean basins. The mean HE and HO for all 11 loci estimated from the North Atlantic samples were also similar (Table 2).

Deviations from Hardy–Weinberg expectations and linkage disequilibrium

In the total sample (i.e. the combined North Atlantic and North Pacific dataset), significant deviations from the Hardy–Weinberg genotype frequencies were detected at five (EV094, GATA053, GATA098, GT011 and GT023) of the seven loci after FDR correction (p-values < 0.0036). No significant deviations from the expected Hardy–Weinberg genotype frequencies were detected in either the North Atlantic or the North Pacific datasets after applying the FDR correction. Several instances of statistically significant linkage disequilibrium were detected among the seven loci after applying the FDR procedure (p-values < 0.0047) in the combined North Atlantic and North Pacific dataset. In contrast, no statistically significant degree of linkage disequilibrium was detected among samples from each ocean basin after applying FDR correction.

Homogeneity tests and genetic divergence

Pairwise estimates of ϴ ranged from 0.003 (Iceland-Azores comparison) to 0.20 (North Atlantic–North Pacific comparison, Table 3). Homogeneity was rejected for all loci separately and combined (p-values < 0.0001) between the North Atlantic and North Pacific Ocean basins. In contrast, no significant deviations from homogeneity were detected within the North Atlantic Ocean.

Table 3 Pairwise estimates of genetic divergence between sampling locations
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Bayesian clustering

The most probable value of K in the combined dataset (i.e. both North Pacific and North Atlantic) was estimated at two from the posterior mean likelihood values (P (K = 2|D) = ~ 1.0, Table S3). All samples from the same ocean basin were allocated to the same cluster (Fig. 2) at admixture probabilities of 100%. K = 1 was the most probable estimate for the combined North Atlantic dataset (P (K = 1|D) = ~ 1.0, Table S3).

Fig. 2
figure 2

Structure plots for the total dataset (top) and for the North Atlantic dataset (bottom), showing estimated probabilities of assignment to each of two populations (K) for all individuals’ microsatellite genotypes. Each column represents one individual’s genotype

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Data analysis of the mtDNA control region nucleotide sequences

Levels of polymorphism

The final dataset of mtDNA control region DNA sequences was comprised of the first 487 nucleotides at the 3′ end of the mtDNA control region in 572 samples (n = 488 for the North Pacific due to one failed mtDNA sequence and n = 84 for the North Atlantic; each sample representing a unique multi-locus microsatellite genotype). In total, 41 segregating sites which defined 65 different mtDNA sequence haplotypes were identified (Fig. 3), with none shared between ocean basins. Among the 41 segregating sites, three were segregating for three nucleotides, resulting in a total of 44 observed substitutions; one inferred insertion-deletion event, 38 transitions and five transversions. There were seven mtDNA haplotypes detected among the North Atlantic samples and 58 among the North Pacific samples. The mean haplotype and nucleotide diversity for each sampling location separately and for all samples together are listed in Table 4.

Fig. 3
figure 3

Frequency of mtDNA control region haplotypes per sampling location

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Table 4 Measures of diversity and neutrality estimated from the mtDNA control region sequences
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MtDNA genealogy

The final alignment of sei, Bryde’s and fin whale mtDNA control region sequences yielded a consensus sequence of 491 nucleotides (including alignment gaps). The maximum-likelihood genealogy (Fig. 4) estimated from the aligned sequences was comprised of two clades with sei whale mtDNA sequences supported by a bootstrap value at 90%. One clade contained six mtDNA haplotypes detected among the North Atlantic samples. The other clade contained all the mtDNA haplotypes detected in the North Pacific, the only Antarctic mtDNA haplotype as well as one North Atlantic mtDNA haplotype. The neighbour-joining genealogy showed a similar topology and similar bootstrap values (Fig. S2). The haplotype networks (see Fig. 4 and Fig. S2) were similar with a sister position of North Atlantic haplotype Hap_6.

Fig. 4
figure 4

Maximum-likelihood haplotype network (left) and genealogy (right) of mtDNA control region haplotypes. Each node in the haplotype network represents a haplotype and node sizes are proportional to haplotype frequencies. Each line segment between nodes represents one nucleotide difference. For convenience, only the North Atlantic haplotype designations and the designations of the four most frequently occurring North Pacific haplotypes are shown. The haplotype tree is drawn to scale, with branch lengths in the evolutionary distance unit of number of base substitutions per site. Only bootstrap values above 60% are shown

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Homogeneity tests and estimates of genetic divergence

Homogeneity was rejected (ΦST = 0.72, p < 0.001) between the North Atlantic and North Pacific Oceans (Table 3). However, no significant deviations from homogeneity were detected among the three North Atlantic sampling locations.

Estimation of effective population sizes, divergence time and migration rates

The parameter \(\theta\), which can be viewed as a proxy for long-term historic effective population sizes, was estimated at 6.2 (95% credible interval: 2.2–14) and 53 (95% credible interval: 39–73) for the North Atlantic and North Pacific samples, respectively; a difference of almost one order of magnitude (Table 5). The divergence time between the North Atlantic and North Pacific populations was estimated at ~ 163 thousand years ago (kya, 95% credible interval: 57–386 kya; Table 5). The number of effective migrants 2mNe from the North Pacific population into the North Atlantic population was estimated at 0.248 (95% credible interval: 0–1.97, Table 5) and at 0.007 from the North Atlantic population into the North Pacific population (95% credible interval: 0–1.47, Table 5).

Table 5 Estimates of relative effective population sizes, divergence times and migration rates
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Tests of neutrality and mismatch distributions

The observed estimates of Tajima’s D and Fu and Li’s F* for the separate and pooled sampling locations were all negative (Table 4), suggestive of population expansion. However, F* was only statistically significant for the Azores’ sample (p < 0.05) and for the pooled North Atlantic sample (p < 0.02) and D was only significant for the Azores’ sample (p < 0.05). The observed mismatch distributions (Fig. 5) corresponded to the expected frequency distributions of pairwise nucleotide site differences in an exponentially growing population.

Fig. 5
figure 5

Frequency distributions of the observed pairwise nucleotide site differences, or mismatch distributions, for the North Pacific, the North Atlantic and the separate North Atlantic sampling locations, compared to expected frequency distributions for a population of exponentially expanding size (red dotted line). For each distribution comparison, the χ2 value and number of degrees of freedom (d.f.) are given. None of the observed mismatch distributions deviated significantly from the expected distribution. The observed frequency distribution for the combined North Atlantic and North Pacific dataset is also given, but no expected distribution can be shown because assumptions (i.e. panmictic population) for their estimation do not hold

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Discussion

At an initial glance our results were consistent with the notion of a single panmictic population of sei whales in at least the western and central North Atlantic (but see below) which appear to have undergone a historic population expansion. Our results also supported the inference drawn by Baker et al. (2004) that sei whales in the North Atlantic and North Pacific Ocean are genetically distinct. The previous results were based solely on mtDNA sequences, and this study augmented the conclusion with nuclear microsatellite genotypes.

Differentiation within the North Atlantic Ocean

Low levels of genetic differentiation

We failed to detect any significant genetic heterogeneity among the three distinct sampling locations in the North Atlantic (the Gulf of Maine, off Iceland and the Azores) at nuclear or mtDNA loci. These findings suggested an absence of genetic population structure within the western and central North Atlantic. Pairwise estimates of ΦST and ϴ at mtDNA and nuclear loci were low, most close to zero (Table 3). The program STRUCTURE also failed to identify significant levels of genetic structure within the North Atlantic (and North Pacific). In other words, our analyses did not yield any results supporting the current designation of two of the three sei whale management units in the North Atlantic by the IWC (Fig. 1). Samples from the third, Eastern North Atlantic stock would provide for a more complete assessment, but according to the IUCN, sei whales seem to have been depleted in that area with no signs of recoveryFootnote 3.

However, low levels of genetic differentiation do not necessarily imply a single stock of sei whales in the North Atlantic but could have other causes (Palsboll et al. 2010). Firstly, our samples originated from two summer feeding grounds; namely, the Gulf of Maine and Iceland, and from a migratory corridor; the Azores. The sei whales utilizing these areas may have formed mixed assemblages of sei whales from different breeding populations and therefore do not show population structure. North Atlantic minke whales (Balaenoptera acutorostrata) present a similar problem of cryptic population structure. For instance, where Daníelsdottír et al. (1992) and Andersen et al. (2003) were able to detect some differentiation between minke whales from West Greenland, the Central and Northeast Atlantic, Anderwald et al. (2011) were not. The optimal sampling scheme would include the identification and sampling of sei whale breeding grounds as well as additional migratory corridors and feeding areas.

Secondly, it is important to consider the uncertainty of the divergence estimates as well as the assumptions underlying equating divergence estimates with contemporary connectivity. The upper bounds of 95% confidence intervals estimated for the point estimates of FST among the North Atlantic sampling locations ranged from 0.08 to 0.14 and from 0.03 to 0.05 for mtDNA and microsatellite data, respectively. Applying Wright’s drift-migration equilibrium, the relation between FST and Nm, i.e. FST = 1/(4 Nm + 1), implies that these upper bounds would correspond to between 3 and 8 migrants per generation (females for mtDNA). The failure of the program STRUCTURE to detect more than a single cluster among the North Atlantic samples should similarly be interpreted with caution. Several in silico assessments (e.g. Latch et al. 2006; Waples and Gaggiotti 2006) of the program have shown that STRUCTURE fails to detect more than one cluster when the degree of population genetic divergence is below 0.05–0.025, which corresponds to 5–10 migrants per generation assuming drift-migration equilibrium. In other words, the 95% confidence intervals of our divergence estimates included levels of divergence that both support a single stock (i.e. FST ~ 0) and multiple stocks (i.e. 3–8 migrants per generation). Along the same vein, the failure of STRUCTURE to detect more than one cluster in the North Atlantic does not negate the presence of multiple stocks given the relatively low migration rates possible given the observed outcome. From a conservation point of view, genetic differentiation alone might therefore not be a sufficient criterion to delineate useful management stocks.

Possible historic population expansion

The degree of population genetic divergence estimated as FST does not necessarily reflect contemporary gene flow (i.e. migration) but is heavily influenced by population history. The negative values of the observed estimates of Tajima’s D and Fu and Li’s F* were indicative of a historic population expansion, which makes sense given the geological history of the North Atlantic Ocean. The Gulf of Maine and the seas off Iceland were inaccessible to baleen whales during the last glacial maximum (LGM, 26.5–19 kya; Clark et al. 2009). The ice caps and summer sea ice extent have since retreated making the current summer foraging areas, such as the Gulf of Maine and the waters off Iceland, accessible to sei whales. Our results suggested an expansion of the North Atlantic sei whale population(s) after the LGM during the retreat of the summer sea ice as previously reported in case of the North Atlantic fin whale (Balaenoptera physalus; Bérubé et al. 1998) and minke whale (B. acutorostrata; Pastene et al. 2007; Anderwald et al. 2011). Albeit all values being negative, estimates of Fu and Li’s F* were only significant for the pooled North Atlantic sample and for the Azores, and Tajima’s D only for the Azores (Table 4). However, when the single individual from the Azores with haplotype Hap_6 (see below) was excluded, all estimates of D and F* became statistically insignificant. The observed frequency distributions of pairwise nucleotide site differences (Fig. 5) fitted the expected distribution for an exponentially growing population. Thus, we found a trend toward population expansion, but statistically insignificant, likely due to low statistical power from low sample sizes, and limited sequence variation in the sei whale mtDNA control region.

Among the seven mtDNA haplotypes detected in the North Atlantic, six differed from each other by a single substitution, suggesting a recent coalescence of these lineages consistent with the presumed recent population expansion. The seventh mtDNA haplotype (Hap_6) was detected in a single sample taken in the Azores. The haplotype differed from the remaining six North Atlantic mtDNA haplotypes by twelve substitutions and was placed as a sister group to the North Pacific haplotypes in the genealogy estimated in our study (Fig. 4). However, the bootstrap support for this haplotype’s branch was low leaving its position rather uncertain. Increased outgroup sampling and additional markers (i.e. the partial or complete mitogenome) may provide a more strongly supported topology. Although anecdotal at this point, the seventh divergent mtDNA haplotype might represent a recent immigrant maternal lineage, e.g. from the South Atlantic, or represent a rare North Atlantic mtDNA lineage. More data and samples are required to discern among these two possibilities.

Population expansion reduces the rate of genetic drift and hence the rate of population genetic divergence compared to constant-sized populations (e.g. Rogers and Harpending 1992; Kimmel et al. 1998; Waxman 2012). This effect would be even stronger if the ‘new’ populations were founded from the same historical population (e.g. Avise et al. 1988). In other words, a recent population history and expansion of sei whales in the North Atlantic may have contributed to the low levels of spatial population genetic divergence observed in our study. Basic population genetic theory relates the degree of genetic divergence among populations to the number of migrations, more precisely the product of the effective population size (Ne) and the probability that an individual is an immigrant (m; Wright 1951; Slatkin and Barton 1989). In other words, the number of immigrants per generation (i.e. 2mNe) determines the degree of genetic divergence among populations, meaning that populations with large Ne’s will diverge at a slower rate compared to populations with smaller Ne’s. Consequently, expanding populations will diverge at decreasing rates compared to similar-sized non-expanding populations, all other factors being equal. One example is depicted in Fig. S3, which illustrates the pronounced difference in estimates of FST in constant and expanding populations in the time following a population divergence.

Effects of whaling on population structure

It is possible that whaling of sei whales may have influenced the contemporary population genetic structure among North Atlantic sei whales. However, the possible effects could either increase or decrease post-whaling population genetic structure (Baker and Clapham 2004). For instance, differential rates of post-whaling recovery among populations could lead to source-sink dynamics and hence reduce pre-whaling population genetic divergence. In contrast, severe reductions of abundance in some populations might result in reduced levels of gene flow among populations and elevated rates of genetic drift which increase pre-whaling divergence. Among the baleen whales there are examples of both rapid post-whaling recolonization, i.e. source-sink dynamics (e.g. Best 1993; Clapham et al. 1999; George et al. 2004; Rugh et al. 2005), as well as slow or absent post-whaling recovery, i.e. increased isolation (e.g. Clapham et al. 1999; Wade et al. 2011; Pomilla et al. 2014), although none of these studies have demonstrated any discernible effects on change in pre-whaling population genetic divergence.

Timing and level of gene flow between the North Atlantic and North Pacific Oceans

We detected high and significant degrees of genetic divergence between the samples from the North Atlantic and North Pacific oceans (Table 3). Haplotype diversity was high for the North Pacific and intermediate for the North Atlantic (Table 4). The estimates of nucleotide diversity were low but within the range reported for other rorquals (e.g. Bérubé et al. 1998; Anderwald et al. 2011). The global haplotype genealogy revealed a clear separation of the North Atlantic and North Pacific haplotypes (Fig. 4). The single Antarctic mtDNA haplotype included in our analysis clustered together with the North Pacific mtDNA haplotypes, which was consistent with previous findings by Baker et al. (2004).

The inter-oceanic migration rate estimates pointed to migration rates of only ~ 1 migrant per four generations or less (Table 5). Divergence time estimates suggested that the North Atlantic and North Pacific sei whale populations separated ~ 163 kya during the penultimate Pleistocene glaciation; the Illinoian glaciation (140–350 kya; Lisiecki and Raymo 2005a, b). This is known to be one of the coldest glacial periods over the last million years (Colleoni et al. 2016). The extent of sea ice during colder conditions might have facilitated the population divergence between the North Atlantic and North Pacific sei whales, as has been suggested for other species and populations during the Pleistocene glaciations (e.g. Hewitt 2000, 2004).

The estimates of \(\theta\), a proxy for effective population size, indicated that the median effective population size of the North Pacific sei whale population was much larger (approximately nine times) compared to the North Atlantic population (Table 5). This was also reflected in the differences in haplotype and nucleotide diversities between the two oceans (Table 4). Looking at heterozygosity alone (Tables 1, 2) we saw no indication of a genetic bottleneck in the North Atlantic preceding the presumed population expansion after the LGM, which could have explained the differences in genetic diversity between the two oceans. However, providing detailed insight into the demographic history of both populations is reserved to future studies.

Although the estimates of \(\theta\) can be converted into estimates of effective female population sizes, we refrained from doing so given that the interpretation of such an estimate is far from straightforward (as reviewed by Palsbøll et al. 2013). Similarly, the inferred population divergence time should not be taken too literally. Direct gene flow between the North Atlantic and North Pacific after the rise of the Panama Isthmus (~ 3.5 million years ago; e.g. Coates et al. 1992) has only been possible through the Northwest Passage during a few brief periods with elevated temperatures. Our divergence time estimate was likely heavily influenced by past periods of gene flow between the hemispheres, as well as the mtDNA mutation rate and generation time employed in our estimation (Avise et al. 1988). The inclusion of samples from the Southern Hemisphere would likely result in very different estimates.

Concluding remarks

In conclusion, while our results did not seem to support the current division by the IWC of North Atlantic sei whales into three different stocks, the uncertainty in our estimates was sufficiently high that we could not rule out the presence of multiple stocks either. The available satellite tagging data suggests that sei whales travel across wide latitudinal and longitudinal ranges, which might explain the low levels of genetic divergence estimated in this study. In order to aid further efforts in the management and conservation of sei whales, we propose additional sampling across the species’ entire range, including breeding and feeding grounds and migratory corridors, as well as increased sample sizes. The low levels of variation in the North Atlantic sei whale suggest that increasing the number of loci may also enhance the precision of estimates of divergence and gene flow (e.g. single nucleotide polymorphism, or SNP, genotypes from genotyping-by-sequencing approaches).