4.1 Population structure and evolutionary history of Harbour
porpoises in the North Atlantic and adjacent waters
By using whole-genome resequencing data, as previous authors suggested
(Fontaine et al., 2007; 2014; Ben Chehida et al., 2021), we found
evidence of three harbour porpoise subspecies and at least three
populations within the North Atlantic subspecies (Wiemann et al., 2010;
Lah et al., 2016). The three subspecies are P.p. relicta in the
Black Sea, P.p. meridionalis in Iberia and P.p. phocoenain the rest of the Atlantic. Black Sea porpoises seem to be a relict
population from past glaciations based on the suggested lack of genetic
exchange with the other subspecies studied (Figure 2A,B). Our sample set
included two samples from Iberian waters, but only one of them was
genetically distinct and hence suggestive of belonging to a putative
Iberian/Mauritanian subspecies (Fontaine et al., 2007; 2014). PCA
results (Figure 2A,C) show that Iberian sample No-2 does not cluster
with the Black Sea nor with the Atlantic subspecies. Admixture results
grouped this sample with the Atlantic cluster, possibly sinceNGSAdmix does not create a new cluster for only one sample.
Nevertheless, while no other sample had any Black Sea ancestry, this
sample had a 10% membership to the Black Sea cluster, further
suggesting its distinct evolutionary trajectory. More samples from the
Iberian/Mauritanian waters should be resequenced to categorically
identify these porpoises as an own separate subspecies.
Regarding the population structure within the North Atlantic subspecies,P.p. phocoena , we did not find evidence of separation between
West and East North Atlantic porpoises, but we confirmed the two
proposed distinct porpoise lineages in the peripheral waters of the
Baltic Sea (Lah et al., 2016; NAMMCO, 2019). On one hand, PCA, admixture
analysis (Figure 2) and Fst levels (Figure 6) show that CA, ICE, BAS and
NOS porpoises belong to the same population, the so-called Atlantic
population. On the other hand, BES and PBS porpoises clustered
separately. Between NOS and BES, there is some gene flow (as indicated
by partial assignments to the two respective clusters for some
specimens; blue and red in Figure 2). Three very distinct PBS
individuals stand out in the genetic structure analysis (Figure 2, 6,
S4), which we assigned to the PBS population. This third Baltic cluster
was interpreted as the Proper Baltic Sea population for a series of
reasons: first, this cluster only comprised porpoises of the PBS region
(Figure 2, 6, S14); second, the three porpoises assigned to the PBS
population were bycaught during the breeding season, when a separation
between putative BES and PBS porpoises occurs (Carlén et al., 2018);
third, these three porpoises were bycaught in the easternmost locations
(16-18.58W) and in areas known to be important for the PBS population
(Carlén & Evans, 2020), i.e., one in the Gdansk Bay and the other two
in the waters surrounding the Swedish island of Öland (Figure S4). These
waters present the greatest densities of harbour porpoises during the
breeding season in the PBS region (Amundin et al., 2022) and have been
reported as potentially important breeding grounds for the PBS
population.
The result indicating lack of genetic structure over long distances in
the open Atlantic is at odds with the fine-scale population structure we
observe in the Baltic region. In an area separated by less than 1,000
kilometers we identified three distinctive lineages (Figure S4C):
Atlantic population in the Skagerrak strait, Belt Sea population in the
Danish Belts, the Sound and Arkona basin, and the Proper Baltic
population in the Baltic proper. We did not find an IBD pattern (Figure
S14C), highlighting that geographical distance does not have a major
impact on the genomic variation and is unlikely to be a driver of the
genetic differences found in North Atlantic harbour porpoises. It should
be mentioned, though, that previous authors have found weak patterns of
IBD in North Atlantic harbour porpoises (Fontaine et al., 2007; Ben
Chehida et al., 2021) when including more sampling locations along the
harbour porpoise distribution range. Resequencing more specimens from
the open Atlantic may be needed to assess the potential role of
geographical distance on North Atlantic harbour porpoise population
differentiation.
The demographic history analysis shows that North Atlantic harbour
porpoises have been strongly influenced by Pleistocene glaciations,
especially since the Last Glacial Period (LGP), when around 25 rapid
climate fluctuations occurred until the end of the Last Glacial Maximum
(LGM), ~ 19,000yBP (Kindler et al., 2014). TheNe of the three harbour porpoise subspecies were
highly correlated until the onset of the LGP
(~110,000yBP) when the Iberian and Black Sea subspecies
curves split from the North Atlantic subspecies. Our divergence estimate
differs from that of previous authors using a portion of the mitogenome
(Fontaine et al., 2014) that dated the most common recent ancestor of
North Atlantic subspecies during the LGM. Similar differences in
divergence estimates have been reported before in the finless porpoise,
when authors using mitochondrial control region data (Wang et al., 2008)
inferred a much younger divergence than authors using whole-genome
resequencing data (Zhou et al., 2018). The Iberian and Black Sea
subspecies originated from the ancestral North Atlantic population when
a small group of individuals may have colonized the Mediterranean and
Black Sea after the LGP, which left an imprint in the genome as a
founder effect. Both Iberian and Black Sea subspecies had lowNe (Figure 3B), which increased sensitivity to
genetic drift which in turn lead to loss of genetic variation, high
inbreeding levels (Figure 4A, S5) and a positive Tajima’s D (Figure 4C).Ne curves for populations of the North Atlantic
subspecies started to diverge around ~75,000yBP, which
roughly coincides with two rapid climate fluctuations during that same
period (Kindler et al., 2014). This may suggest that BES and PBS
lineages have diverged from the Atlantic population during an
interglacial period before the formation of the Baltic Sea at the end of
the LGM. However, our demographic analysis also indicate that after the
LGM, the Ne of the Baltic populations increased.
This would be compatible with a scenario in which - as the ice sheets
retreated from northern regions at the end of LGM - a group of porpoises
colonized the newly formed Baltic Sea, originating the modern BES and
PBS populations. Nevertheless, other processes like gene flow and linked
selection could be cofounding factors in the SMC++ demographic
inferences (Mazet et al., 2016; Schrider et al.,2016), thus these
results must be interpreted with caution and
time/Ne estimates should not be taken literally.
The maximum likelihood tree inferred with Treemix is also
compatible with the existence of three subspecies in the North Atlantic
and adjacent waters. Previous phylogenies reconstructed with mitogenome
data (Ben Chehida et al. 2020) found that BLS porpoises are basal in the
North Atlantic, thus we were confident rooting our tree with the BLS
subspecies. The first split in our graphical representation of
historical relationships among North Atlantic harbour porpoises was the
IBE subspecies from the North Atlantic subspecies. Among the North
Atlantic subspecies, Treemix analysis placed present-day NOS
harbour porpoises as basal, compatible with a northward post-glacial
expansion from a southern refugia. As the ice sheets retreated, the
ancestral North Atlantic subspecies may have started to colonize novel
environments in the Baltic, Iceland, Barents Sea, and Canadian waters.
4.2 Local adaptation of
Baltic porpoises to low salinity levels
Species and populations inhabiting highly divergent environments are
expected to be under different selective pressures, which could cause
each local population to evolve traits that provide an advantage under
its local environmental conditions (Kawecki & Ebert, 2004). However,
the role of ecological specialization on population differentiation and
speciation remains poorly understood (Savolainen, Lascoux & Merilä,
2013). This is particularly true for cetacean species, with only a few
recent studies attempting to address the genetic basis of local
adaptation (Barceló et al., 2022; Pratt et al., 2022; de Greef et al.,
2022; Louis et al., 2021; Zhou et al., 2018). Ecologically and
geographical marginal environments often host populations at the edge of
the species distribution and under extreme selection regimes
(Johannesson & André, 2006). Examples of such populations are the Belt
Sea and Proper Baltic Sea harbour porpoise populations that occur in the
peripheral waters of the Baltic Sea, separated from the North Sea by a
pronounced salinity gradient.
The GEA results show that including BLS samples in the RDA had a major
impact on the outcome of the analysis, especially on the number of
putative SNPs inferred to be under selection. The genomic variation of
BLS porpoises was highly associated with high temperature (Figure S12),
with ~6,000 SNPs correlated with SST and only a few
associated with the other variables (Table S4). As water temperatures in
the Black Sea are significantly higher than in the rest of the locations
(Figure S6) and BLS porpoises are highly divergent, we could not discern
whether these ~6,600 SNPs were indeed associated with
temperature or were rather very distinct for different evolutionary
pressures or pronounced genetic drift in BLS porpoises. Thus, to
identify candidate genes associated with environmental variables we
focused on the dataset without BLS, where the population divergence was
not as strong.
Our seascape genomics analysis provides statistical support for an
influence of salinity on population differentiation in the Baltic Sea
(Figure 5, S12). SSS was highly significant in both RDA and pRDA, while
SST was significant only in the RDA. The salinity gradient could have
contributed to the origin of a soft barrier between the Atlantic and the
Baltic, leading to adaptive divergence in BES and PBS porpoises, as seen
in other cetaceans as the finless porpoise (Zhou et al., 2018). From 272
inferred candidate SNPs, 107 were annotated with genes potentially
associated with the salinity gradient in the Baltic (Table S5). We
identified three solute carrier group (SLC) genes, a group of membrane
transport proteins. Particularly interesting was the SLC10A1 gene, a
sodium ion transport with a critical role in the osmoregulation of bile
acids in the liver (Kubitz & Häusinger, 2007). We also obtained hits in
osmoregulatory genes (AQP9, DYNC2H1) previously inferred to be under
selection in cetaceans (Xu et al., 2013, São Pedro et al., 2015, Zhou et
al., 2018). Further steps to evaluate the candidate SNPs and genes
should include the annotation of Gene Ontology (GO) terms as well as
genes within 20kb of the candidate SNP (Pratt et al., 2022).