1. Introduction
The wellbeing of populations depends on an array of intrinsic and extrinsic factors, but effective population size and genetic diversity are among the most crucial. Reduction of genetic diversity in a population can lead to detrimental outcomes such as loss of adaptive potential and inbreeding depression, produced by the accumulation of slightly deleterious mutations due to reduced efficiency of purifying selection (Tanaka, 2000). Understanding the processes that influence current genetic variation is essential for the management and conservation of the diversity of a species (Kardos et al., 2021). Variation in genetic diversity is the result of historical and present demographic, geographic, ecological and behavioral mechanisms that influence gene flow, genetic drift and/or selection levels (Stange, Barret & Hendry, 2021). Overarching processes such as glacial contractions and post-glacial expansions have influenced current patterns of genetic structure and diversity in many European species, creating population subdivisions and hybrid zones by secondary contact (Hewitt, 1999; 2000; 2001). However, neutral evolutionary processes are not the only factor contributing to population subdivisions, as selective processes such as local adaptation could also produce different evolutionary trajectories (Barret & Schluter, 2008). Therefore, both neutral and adaptive processes must be considered when studying genetic diversity and population structure. Dispersal ability over vast geographical distances may facilitate gene flow among distant locations and hence hinder population differentiation (Slatkin, 1987). The marine habitat is a perfect example of an environment where a lack of physical barriers offers a continuous environment such that highly mobile species could present large homogenous populations and an absence of genetic structure. Yet, several cetacean species show fine-scale population structure, as for instance Northern bottlenose whale (de Greef et al., 2022), Finless porpoise (Zhou et al., 2018), Killer whale (Foote et al., 2016), and bottlenose dolphins (Louis et al., 2021).
The Harbour porpoise (Phocoena phocoena ) is a great example of complex genetic differentiation in a highly mobile cetacean species. It belongs to the Phocoenidae family, a group of seven species (Ben Chehida et al., 2020) containing some of the most threatened cetaceans (Carlén, Nunny & Simmonds, 2021): the Vaquita (Phocoena sinus ), one of the most endangered mammals on planet earth, with a population size of ~10 individuals (Jaramillo-Legorreta et al., 2019), and the Yangtze finless porpoise (Neophocaena asiaeorientalis  asiaeorientalis), with only ~1,000 individuals extant (Zhao et al., 2008). Porpoises are mainly affected by incidental bycatch (Brownell et al., 2019), pollutants such as PCBs (Berggren et al., 1999; Karlson et al., 2000), parasites (Dzido et al., 2021; Reckendorf et al., 2021; Ryeng et al., 2022), and noise pollution issued from offshore infrastructure developments, shipping routes and underwater explosions (Siebert et al., 2022). Harbour porpoises inhabit coastal and shelf waters across the Northern hemisphere and at least three subspecies have been described:P.p. vomeria in the North Pacific, P.p. phocoena in the North Atlantic, and P.p. relicta in the Black Sea. A fourth subspecies near the Iberian Peninsula and in Mauritanian waters (P.p. meridionalis ) has been proposed (Fontaine et al., 2007; 2014), although a formal description has not yet been made.
The North Atlantic subspecies (P.p. phocoena ) has a continuous distribution in the North Atlantic extending from the French Biscayan waters to the Baltic and Barents Sea and from the Norwegian Sea to the western North Atlantic coast of Canada and the United States, crossing Faroese, Icelandic and Greenlandic waters (Gaskin, 1992; Read 1999). The genetic structure of North Atlantic harbour porpoises has been widely studied (Alfonsi et al., 2012; Luna et al., 2012; Quintela et al., 2020 among others) and microsatellite data suggests that both sides of the Atlantic belong to the same lineage (Chehida et al., 2021). However, genetic and distribution densities suggest the presence of different ecotypes or populations on the peripheral waters of the Baltic Sea (Tiedemann et al., 1996; Wiemann et al., 2010; Lah et al., 2016) and West Greenland (Olsen et al., 2022). The Baltic Sea is a remarkable sub-basin of the Atlantic Ocean formed less than 10,000 years before present (BP) as a postglacial marine environment. Baltic populations of several marine organisms are genetically distinct from conspecifics from the North Sea and the Atlantic, possibly due to isolation, bottlenecks, and local adaptation (Wennerstrom et al., 2013). A series of small basins are separated by shallow underwater ridges ranging from the North Sea through Skagerrak, Kattegat, Belt Seas to the entrance of the proper Baltic Sea, making dispersal and gene flow limited (Johannesson & André, 2006). Moreover, the Baltic Sea is an extreme marine environment with low winter temperatures and one of the strongest salinity gradients in the world, ranging from ~34 practical salinity units (psu) in the Skagerrak to ~2 psu in the innermost parts of the Baltic (Feistel et al., 2010). These conditions make Baltic species a prime system to study local adaptation (DeFaveri et al., 2014; Wrange et al., 2014; Sjöqvist et al., 2015) and speciation in the marine environment (Riginos & Cunningham, 2005, Stuckas et al., 2009; Pereyra et al., 2009). Passive acoustic (Carlén et al., 2018), telemetry (Svegaard et al., 2015), morphological (Huggenberger et al., 2002; Galatius et al., 2012), and genetic data (Tiedemann et al., 1996; Wiemann et al., 2010; Lah et al., 2016) suggest the presence of three harbour porpoise populations in the Baltic region: one in the North Sea, Skagerrak and northern parts of Kattegat (North Sea population, NOS), another in southern parts of Kattegat and Belt Seas (Belt Sea population, BES) and a third one in the Baltic Proper (Proper Baltic Sea population, PBS). Although overlap between the three populations has been reported based on both genetic (Wiemann et al., 2010; Lah et al., 2016) and satellite tracking data (Svegaard et al., 2015), borders among them have been postulated based on geographical separation during the reproductive season (Svegaard et al., 2015; Carlén et al., 2018, Amundin et al., 2022).
Harbour porpoise abundance estimates vary greatly among regions: Black Sea porpoise population size is unknown, but declined by ~90% between the 1930s and the 1980s (Birkun, 2002); the European Atlantic Shelf is estimated to be inhabited by ~375,000 individuals (Hammond et al., 2013), ~20,000 animals are estimated in the Belt Sea and only ~500 in the Proper Baltic Sea (Amundin et al., 2022). The Black Sea subspecies and the Proper Baltic Sea population are considered endangered and critically endangered, respectively. To date, no assessment of genetic diversity, Ne or population structure has been conducted on North Atlantic porpoises at the whole-genome level. Historically, conservation and evolutionary geneticists have leaned on a handful of molecular markers, such as mitochondrial DNA and microsatellites, for the study of genetic variation among populations (Schweizer et al., 2021). However, with the development of high-throughput sequencing there has been a transition from genetics to genomics (Formenti et al., 2022). The ever-decreasing cost of reduced-representation and whole genome sequencing methods has positioned conservation genomics as a prominent tool for the characterization of biodiversity and preservation of species (Fuentes-Pardo & Ruzzante, 2017). Nowadays, the democratization of sequencing costs allows to resequence the genome of a set of individuals to assess genetic variation across thousands or millions of markers and address long-standing questions in evolutionary biology not fully resolved with traditional markers or reduced-representation methods (Foote et al., 2021; Robinson et al., 2022; Wolf et al., 2022). This increase of statistical power to unravel subtle patterns not fully captured by less dense datasets has had and will continue to have a remarkable impact in the field of conservation genomics (Lou et al., 2021; Szarmach et al., 2021).
Here, we used genomics approaches to study the population structure, genetic diversity, evolutionary history and local adaptation of the Harbour porpoise (Phocoena phocoena ). We generated the most comprehensive data set of North Atlantic harbour porpoises so far, by resequencing the whole-genome of 74 harbour porpoises from eight regions across the North Atlantic and adjacent waters. Our results shed light on the expansion of harbour porpoise populations across the North Atlantic, demonstrate that genome-wide data can unravel subtle population structure and contribute to understand how marine species adapt to their local environment. The results have great conservation implications as we found major levels of inbreeding and low genetic diversity in the endangered Black Sea subspecies and identified the critically endangered Proper Baltic Sea porpoises as a separate population.
2. Material and Methods