Yacine Ben Chehida

Tjibbe Stelwagen

Jeroen P A Hoekendijk

Marisa Ferreira

Catarina Eira

Andreia T Pereira

Lidia Nicolau

Julie Thumloup

Michael C Fontaine
email: michael.fontaine@ird.fr

Andreia Torres-Pereira

Andreia Torres

Harbor porpoise losing its edges: genetic time series suggests a rapid population decline in Iberian waters over the last 30 years

Keywords: cetacean conservation genetics, genetic time series, genetic diversity, bycatch, approximate Bayesian computation, population genetic modelling

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The impact of climate change on species is expected to be especially noticeable at their distribution edges, where species meet the limits of their ecological tolerance. Marginal populations of temperate species at the rear (warm) distribution edge are often considered more susceptible to warming than central populations because of the warmer ambient temperatures they experience, but this overlooks the potential for local adaptation [START_REF] Bennett | Central and rear-edge populations can be equally vulnerable to warming[END_REF][START_REF] Vilà-Cabrera | Refining predictions of population decline at species' rear edges[END_REF].

Cetaceans are particularly vulnerable to climate change [START_REF] Macleod | Global climate change, range changes and potential implications for the conservation of marine cetaceans: a review and synthesis[END_REF] due to their crucial function as apex predators in marine ecosystems [START_REF] Bowen | Role of marine mammals in aquatic ecosystems[END_REF]. Climate change influences the availability of their prey and modifies the habitat, thereby impacting several (if not all) cetacean species and may even put some of them at a high risk of extinction [START_REF] Learmonth | Potential effects of climate change on marine mammals[END_REF]. As a response, several cetacean species are shifting their distribution ranges to track suitable habitats or adapt locally by switching to different food resources [START_REF] Lambert | Quantifying likely cetacean range shifts in response to global climatic change: implications for conservation strategies in a changing world[END_REF][START_REF] Learmonth | Potential effects of climate change on marine mammals[END_REF][START_REF] Macleod | Global climate change, range changes and potential implications for the conservation of marine cetaceans: a review and synthesis[END_REF][START_REF] Williamson | Cetaceans as sentinels for informing climate change policy in UK waters[END_REF]. For example, temperate and subpolar small cetaceans like the harbor porpoises (Phocoena phocoena) are likely to show a poleward shift [START_REF] Heide-Jørgensen | Harbour porpoises respond to climate change[END_REF][START_REF] Macleod | Global climate change, range changes and potential implications for the conservation of marine cetaceans: a review and synthesis[END_REF]. Due to an elevated metabolic rate and relatively short generation time, this small cetacean is heavily dependent on a continuous food supply [START_REF] Hoekendijk | Resilience of harbor porpoises to anthropogenic disturbance: Must they really feed continuously?[END_REF][START_REF] Wisniewska | Ultra-high foraging rates of harbor porpoises make them vulnerable to anthropogenic disturbance[END_REF].

This species is thus expected to be particularly sensitive to climate changes [START_REF] Heide-Jørgensen | Harbour porpoises respond to climate change[END_REF]. In addition, due to their coastal distribution, thousands of harbor porpoises are also killed accidentally each year by commercial fisheries. This by-catch mortality is threatening local populations to a level that is still hard to quantify, but it is likely unsustainable, especially with a highly fluctuating environment [START_REF] Carlén | Out of sight, out of mind: how conservation Is failing european porpoises[END_REF]IMR-NAMMCO, 2019;[START_REF] Vingada | Conservação de Cetáceos e Aves Marinhas em Portugal Continental. O projeto LIFE+ MarPro[END_REF].

Harbor porpoises are officially subdivided into three distinct subspecies: P. p. vomerina in the Pacific, P. p. phocoena in the North Atlantic, and P. p. relicta in the Black Sea [START_REF] Fontaine | Harbour porpoises, Phocoena phocoena, in the Mediterranean sea and adjacent regions: biogeographic relicts of the last glacial period[END_REF][START_REF] Read | Harbour porpoise Phocoena phocoena[END_REF][START_REF] Rosel | Variability of the mitochondrial control region in populations of the harbour porpoise, Phocoena phocoena, on interoceanic and regional scales[END_REF]. A fourth lineage or Evolutionary Significant Unit (ESU) has been suggested for the Afro-Iberian harbor porpoises (IBMA; currently unnamed subspecies, but possibly P. p. meridionalis following [START_REF] Fontaine | Postglacial climate changes and rise of three ecotypes of harbour porpoises, Phocoena phocoena, in western Palearctic waters[END_REF] owing to their distinctive ecology, morphology [START_REF] Donovan | Harbour porpoises in the North Atlantic: edited extract from the report of the IWC Scientific Committee[END_REF][START_REF] Smeenk | Note on the harbour porpoise Phocoena phocoena in Mauritania[END_REF], and genetic divergence [START_REF] Fontaine | Postglacial climate changes and rise of three ecotypes of harbour porpoises, Phocoena phocoena, in western Palearctic waters[END_REF] (Figure 1). However, no formal description or subspecies taxonomic assignment has been made for this distinct lineage so far [START_REF] Pierce | Stable isotope analysis reveals habitat partitioning among marine mammals off the NW African coast and unique trophic niches for two globally threatened species[END_REF]. IBMA porpoises are found around the relatively cold and productive upwelling waters off the coasts of Iberia and on the NW Atlantic coasts of Africa from southern Morocco, to Mauritania, and Senegal [START_REF] Fontaine | Harbour porpoises, Phocoena phocoena, in the Mediterranean sea and adjacent regions: biogeographic relicts of the last glacial period[END_REF][START_REF] Gaskin | The harbour porpoise Phocoena phocoena (L.): regional populations, status, and information on direct and indirect catches[END_REF]IMR-NAMMCO, 2019;[START_REF] Read | Harbour porpoise Phocoena phocoena[END_REF]. They are significantly larger in term of skull morphology, growth curve, and adult body size (typically ≥2m long) compared to the two other ecotypes (or subspecies) (~1.5m long) [START_REF] Galatius | Geographic variation of skeletal ontogeny and skull shape in the harbour porpoise (Phocoena phocoena)[END_REF][START_REF] Read | Harbour porpoise Phocoena phocoena[END_REF][START_REF] Sequeira | Harbour porpoise, Phocoena phocoena, in Portuguese waters[END_REF][START_REF] Smeenk | Note on the harbour porpoise Phocoena phocoena in Mauritania[END_REF]. Beside their distinctive morphological features, the IBMA porpoises belong to the Eastern Canary Coastal province with equatorward surface circulation in the form of the Canary current, rich in eddies and large upwellings that are permanent along the NW African coast and seasonal along the Iberian coast [START_REF] Álvarez-Salgado | The Portugal coastal counter current off NW Spain: new insights on its biogeochemical variability[END_REF][START_REF] Fraga | Upwelling off the Galacian Coast, northwest Spain[END_REF][START_REF] Moustahfid | Ecological-Fishery Forecasting of Squid Stock Dynamics under Climate Variability and Change: Review, Challenges, and Recommendations[END_REF]Pierce et al., in revision).

While P. p. phocoena in the northern Bay of Biscay and further north in the North Atlantic are generally considered opportunistic feeder relying primarily on bottom dwelling on the shallow continental shelf (i.e. in waters of <200 m depth), the IBMA porpoise feeding ecology is still poorly known. It is hypothesized that they exhibit similar foraging behaviors as their counterparts on the continental shelf to the north, but with a greater emphasis on pelagic fish (Pierce et al., in revision;Pinela et al., 2010;[START_REF] Santos | The diet of harbour porpoise (Phocoena phocoena) in the Northeast Atlantic[END_REF]. From a genetic perspective, the Iberian and Mauritanian porpoises are closely related but they were found genetically differentiated from each other and form two distinct populations separated by at least 2500 km [START_REF] Ben Chehida | No leading-edge effect in North Atlantic harbor porpoises: Evolutionary and conservation implications[END_REF][START_REF] Fontaine | Harbour porpoises, Phocoena phocoena, in the Mediterranean sea and adjacent regions: biogeographic relicts of the last glacial period[END_REF][START_REF] Fontaine | Postglacial climate changes and rise of three ecotypes of harbour porpoises, Phocoena phocoena, in western Palearctic waters[END_REF].

The upwelling habitat of the Iberian porpoise (IB) population is at the border of a biogeographical transition zone between temperate and subtropical waters where species with different affinities co-occur and reach their respective ecological limits [START_REF] Bowen | Role of marine mammals in aquatic ecosystems[END_REF][START_REF] Macleod | Global climate change, range changes and potential implications for the conservation of marine cetaceans: a review and synthesis[END_REF]. Because species' habitat suitability at the margins between biogeographic zones are often suboptimal, populations like the Iberian harbor porpoises are expected to be among the first to show the impacts of environmental changes. This is particularly true with the small-scale Iberian upwelling system, which is known to fluctuate in response to ongoing climate change [START_REF] Casabella | Trends of the Galician upwelling in the context of climate change[END_REF][START_REF] Pires | Assessing future climate change in the Iberian Upwelling System[END_REF]. Such environmental fluctuation may impact the Iberian harbor porpoises in various ways, possibly leading to dramatic impacts on the population demography and genetic diversity. Porpoises may adapt locally, for example, by adapting to various prey species [START_REF] Heide-Jørgensen | Harbour porpoises respond to climate change[END_REF], by migrating to more suitable environments, or the population may also become extirpated if environmental changes outpace their ability to adapt. Previous population genetic studies [START_REF] Ben Chehida | No leading-edge effect in North Atlantic harbor porpoises: Evolutionary and conservation implications[END_REF][START_REF] Fontaine | Postglacial climate changes and rise of three ecotypes of harbour porpoises, Phocoena phocoena, in western Palearctic waters[END_REF][START_REF] Fontaine | Mixing of porpoise ecotypes in southwestern UK waters revealed by genetic profiling[END_REF][START_REF] Fontaine | Genetic and historic evidence for climate-driven population fragmentation in a top cetacean predator: the harbour porpoises in European water[END_REF], based on samples collected between 1990 and 2002, showed that the Iberian population was a source population with highly asymmetric geneflow, sending more migrants to the North into the P. p. phocoena populations on the northern side of the Bay of Biscay and to the South into the Mauritanian population than in the other direction (Figure 1). In fact, a tension zone was detected in the northern side of the Bay of Biscay with Iberian genetic ancestry decreasing sharply with the increasing latitude [START_REF] Fontaine | Rise of oceanographic barriers in continuous populations of a cetacean: the genetic structure of harbour porpoises in Old World waters[END_REF][START_REF] Fontaine | Postglacial climate changes and rise of three ecotypes of harbour porpoises, Phocoena phocoena, in western Palearctic waters[END_REF][START_REF] Fontaine | Mixing of porpoise ecotypes in southwestern UK waters revealed by genetic profiling[END_REF]. If recruitment does not keep up with the mortality and migration rate, the census size of the Iberian population may decrease, leading to a gradual reduction in number of reproducing individuals, in genetic diversity, and thus in effective population size over time.

Unlike previous studies, which inferred population structure between the Iberian population and other populations [START_REF] Ben Chehida | No leading-edge effect in North Atlantic harbor porpoises: Evolutionary and conservation implications[END_REF][START_REF] Fontaine | Postglacial climate changes and rise of three ecotypes of harbour porpoises, Phocoena phocoena, in western Palearctic waters[END_REF][START_REF] Fontaine | Mixing of porpoise ecotypes in southwestern UK waters revealed by genetic profiling[END_REF][START_REF] Fontaine | Genetic and historic evidence for climate-driven population fragmentation in a top cetacean predator: the harbour porpoises in European water[END_REF], the goal of the present study is to assess how mtDNA variation evolved over time in the Iberian population, comparing sampling cohorts spanning about 3 decades and how this information can guide conservation of the Iberian population. More precisely, using genetic diversity as a proxy for effective population size variation, we tested if mitochondrial genetic diversity decreased through time by comparing the genetic composition of two cohorts of porpoises, one sampled in the 1990 's (1990-2002), and the other one from the 2010's (2012-2015).

Material and methods

Sampling and molecular analyses. Tissue samples from 52 by-caught or stranded individuals collected along the Portuguese coast between 2012 and 2015 were obtained from the Portuguese Marine Animal Tissue Bank (MATB). These samples form the 2010's cohort of the Iberian population (also called the 'new' cohort or IB_N in this study) (Figure 1, Table 1 andS1). Tissues samples were stored in 70% ethanol and kept at -20ºC until molecular analyses. Genomic DNA was extracted with the DNeasy Blood and Tissue kit (QIAGEN Inc.) following the manufacturer's protocol. Following [START_REF] Fontaine | Postglacial climate changes and rise of three ecotypes of harbour porpoises, Phocoena phocoena, in western Palearctic waters[END_REF], we screened five mitochondrial coding regions (ATP-6, ATP-8, Cyt-b, ND5, and COI) for genetic polymorphisms using Sanger sequencing. Target regions were PCR amplified, quality controlled, and purified following the protocol described in [START_REF] Fontaine | Postglacial climate changes and rise of three ecotypes of harbour porpoises, Phocoena phocoena, in western Palearctic waters[END_REF]. Sanger sequencing was outsourced to GATC Biotech ltd. The resulting forward and reverse sequences were visually inspected, cleaned, and assembled with Geneious 10.0.9 [START_REF] Kearse | Geneious Basic: An integrated and extendable desktop software platform for the organization and analysis of sequence data[END_REF].

We augmented the dataset with the 82 previously published sequences from [START_REF] Fontaine | Postglacial climate changes and rise of three ecotypes of harbour porpoises, Phocoena phocoena, in western Palearctic waters[END_REF] encompassing the same five mitochondrial genes. These sequences were obtained for 7 82 harbor porpoises collected between 1990 and 2002 (Figure 1, Table 1 andS1). These included: 14 individuals collected along the French coast of the Bay of Biscay (BB), 23 further north in the North Atlantic (NAT) from the Channel to the Nordic waters of Norway and Iceland; 19 from the Iberian coast (a.k.a. the `old` Iberian cohort or IB_O), 14 from Mauritania (MA); and 12 from the Black Sea (BS). The 'old' Iberian cohort from [START_REF] Fontaine | Postglacial climate changes and rise of three ecotypes of harbour porpoises, Phocoena phocoena, in western Palearctic waters[END_REF] obtained between 1990 and 2002 included 19 stranded and bycaught porpoises from the Galician coasts of Spain and Portugal (Figure 1 and Table S1). Previous studies based on mtDNA data and nuclear microsatellite data demonstrated that the Spanish and Portuguese samples were part of a same panmictic genetic group [START_REF] Ben Chehida | No leading-edge effect in North Atlantic harbor porpoises: Evolutionary and conservation implications[END_REF][START_REF] Fontaine | Rise of oceanographic barriers in continuous populations of a cetacean: the genetic structure of harbour porpoises in Old World waters[END_REF][START_REF] Fontaine | Postglacial climate changes and rise of three ecotypes of harbour porpoises, Phocoena phocoena, in western Palearctic waters[END_REF][START_REF] Fontaine | Mixing of porpoise ecotypes in southwestern UK waters revealed by genetic profiling[END_REF]. The main focus of the present study thus consists in comparing the temporal evolution of the mtDNA variation between the two Iberian cohorts of porpoises: i.e. the 'new ' [2010's: 2012-2015, n=52] versus the 'old ' [1990's:1990-2002, n=19]). The temporal coverage of this serial sampling of the Iberian population is thus at most 25 years, which corresponds to 2.5 generations, assuming a generation time of 10 years [START_REF] Read | Harbour porpoise Phocoena phocoena[END_REF]. The mtDNA sequences from the other populations of [START_REF] Fontaine | Postglacial climate changes and rise of three ecotypes of harbour porpoises, Phocoena phocoena, in western Palearctic waters[END_REF] neighboring the Iberian population were included here to provide a broader perspective to interpret the phylogenetic analyses.

One additional sequence from the closest outgroup species, the Dall's porpoise (Phocoenoides dalli) also generated in [START_REF] Fontaine | Postglacial climate changes and rise of three ecotypes of harbour porpoises, Phocoena phocoena, in western Palearctic waters[END_REF], was included in the present dataset to root the mitochondrial phylogeny. All mtDNA sequences (n=135) were aligned with MUSCLE [START_REF] Edgar | MUSCLE: multiple sequence alignment with high accuracy and high throughput[END_REF].

Phylogenetic and population genetic analyses. In order to place the Iberian porpoises into a global phylogeographic context, we first reconstructed a maximum-likelihood (ML) phylogeny with all the 135 mtDNA sequences (including the outgroup). Unique mtDNA haplotypes were identified using DnaSP v.5.10.1 [START_REF] Librado | DnaSP v5: a software for comprehensive analysis of DNA polymorphism data[END_REF]. Then, we used PhyML [START_REF] Guindon | New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0[END_REF] implemented in Geneious to estimate the ML tree based on all available unique haplotypes identified in the dataset using a nearest neighbor interchange topology search. We used the [START_REF] Tamura | Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees[END_REF]'s (or TN93) model of nucleotide evolution as identified as the best-fitting model for our sequence alignment using the Akaike Information Criterion (AIC) in jModelTest 2.1.10 ( [START_REF] Darriba | jModelTest 2: more models, new heuristics and parallel computing[END_REF]. Each node's support was evaluated by conducting 5,000 iterations of bootstrap resampling. The phylogenetic tree was rooted with the sequence from a Dall's porpoise. In addition to the phylogenetic trees, we also reconstructed a median-joining haplotype network (Bandelt et al. 1999) using PopART (http://popart.otago.ac.nz).

Various statistics informative on the mtDNA genetic variation of the two cohorts of Iberian porpoises ("old" vs. "new") were estimated using the C library libdiversity (https://bioinfo.mnhn.fr/abi/people/achaz/cgi-bin/neutralitytst.c) and the R package pegas v.1.2 (Paradis, 2010) (Table 1). Genetic diversity was estimated using the number of haplotypes (H), haplotype diversity (Hd), nucleotide diversity (π), and Watterson's theta (θW).

Differences in sample size can significantly impact the values of some genetic diversity estimators [START_REF] Goodall-Copestake | On the comparison of population-level estimates of haplotype and nucleotide diversity: a case study using the gene cox1 in animals[END_REF]. Therefore, a rarefaction approach was applied to estimate measures of genetic diversity while correcting for difference in sample size [START_REF] Sanders | Marine benthic diversity: a comparative study[END_REF]) using a custom python script. More precisely, for the two cohorts of Iberian porpoises, for each statistic and each sample size increment k, we randomly subsampled 5,000 times with replacement a number of k sequences available in each group, with k ranging between 2 and the kmax available sequences (up to a maximum of 50). At each sample size increment k, we estimated the mean and standard error values per statistic and per group. Subsequently, genetic differentiation between the two cohorts was assessed using the Hudson's estimator of FST [START_REF] Hudson | Estimation of levels of gene flow from DNA sequence data[END_REF] and φST [START_REF] Excoffier | Analysis of molecular variance inferred from metric distances among DNA haplotypes: application to human mitochondrial DNA restriction data[END_REF] using respectively the libdiversity C-library and the R-package haplotypes v.1.1.3.1. The significance levels of FST and φST were assessed by randomization tests (1,000 permutations). Finally, we investigated evidence of changes in mtDNA effective population size (Ne) between the two cohorts (old vs. new) by calculating values of Tajima's D [START_REF] Tajima | Statistical method for testing the neutral mutation hypothesis by DNA polymorphism[END_REF]) and Achaz's Y [START_REF] Achaz | Testing for neutrality in samples with sequencing errors[END_REF] accounting for differences in sample sizes by implementing the same rarefaction approach as described for the diversity statistics. The Achaz's Y statistic is analogous to the Tajima's D, but accounting for potential sequencing errors (see details in [START_REF] Achaz | Testing for neutrality in samples with sequencing errors[END_REF].

Approximate Bayesian Computation (ABC).

In order to evaluate the support for different demographic scenarios and assess whether there is a signal of a recent population decline in the Iberian population based on the mtDNA data, we applied an ABC approach relying on coalescent simulations [START_REF] Lopes | ABC: A useful Bayesian tool for the analysis of population data[END_REF]. More precisely, we compared six demographic scenarios: (1) a population with a large constant effective population size, (2) a small constant effective population size, (3) an old expansion (≥150 generations, gen.), (4) an old decline (≥150 gen.), (5) a recent decline (≤3 gen.), and (6) an old expansion (>150 gen.) followed by a recent decline scenario (≤3 gen.) (see Figure 2 and Table S2 for details). In the coalescent simulations, we leverage the time series data information available and specified explicitly at which time the samplings of the two cohorts were performed.

For each of the six scenarios, we ran 10,000 coalescent simulations using fastsimcoal2 v2.6 [START_REF] Excoffier | fastsimcoal: a continuous-time coalescent simulator of genomic diversity under arbitrarily complex evolutionary scenarios[END_REF]. Details on the coalescent simulations and the mutation model used are provided in the supplementary Table S2. We used the program Arlsumstat v3.5.2 [START_REF] Excoffier | Arlequin suite ver 3.5: a new series of programs to perform population genetics analyses under Linux and Windows[END_REF] to compute a set of 55 summary statistics listed in Table S3, augmented with the principal axes obtained from a linear discriminant analysis (LDA) on the summary statistics [START_REF] Pudlo | Reliable ABC model choice via random forests[END_REF], to describe and summarize the genetic variation of each simulation.

We employed a random forest classification (RF) procedure for the model selection [START_REF] Pudlo | Reliable ABC model choice via random forests[END_REF]. RF is a machine-learning algorithm that uses bootstrapped decision trees to classify and select the best scenario using the summary statistics as a set of predictor variables [START_REF] Breiman | Random Forests[END_REF]. RF was therefore applied to compare the likelihood of the six competing scenarios (Figure 2). This procedure provides classification votes that accounts for the number of times a scenario is selected as the best one among the trees used to constructed the random forest. Based on a forest of a 1,000 decision trees, the scenario that gathered the highest number of votes was selected as the most likely.

The prior error rate and the type-II error rate were estimated using the out-of-bag simulations (i.e. simulations not used to build the decision trees). In addition, we computed the confusion matrix, which provides a more global assessment of the performance of our RF procedure [START_REF] Schrider | Supervised Machine Learning for Population Genetics: A New Paradigm[END_REF]. The convergence of the results was evaluated over 10 independent RF runs, as recommended by [START_REF] Fraimout | Deciphering the Routes of invasion of Drosophila suzukii by Means of ABC Random Forest[END_REF].

Finally, to check whether the formulated scenarios and associated priors were compatible with the observed dataset, we plotted both the simulated datasets from the training set and the observed dataset on the two first principal axes of a Linear Discriminant Analysis (LDA) and of a Principal Component Analysis (PCA), following the recommendation of [START_REF] Pudlo | Reliable ABC model choice via random forests[END_REF]. We visually checked whether the summary statistics from the observed data was located within the clouds formed by those from the simulated data for at least one demographic scenario. In addition, we assessed the goodness-of-fit between the simulated and the observed data following [START_REF] Gelman | Bayesian Data Analysis[END_REF]: the summary statistics for which the cumulative simulated distribution was larger or smaller than 95% CI of the distribution compared to the observed value were considered as significantly different from the observed data. All ABC-RF analyses were performed using the R package abcrf v.1.8.1 [START_REF] Pudlo | Reliable ABC model choice via random forests[END_REF] in R 4.1.2 (R Core Team, 2016).

Results and Discussion

In total, we successfully sequenced 52 Iberian harbor porpoises from the new cohort 1). This result provides the first evidence of possible dispersal (or migration) from the Mauritanian population into Iberian waters, suggesting that these two populations may not be as isolated as previously thought. Haplotype 10 could also belong to a distinct unknown population as it is relatively divergent from the other Mauritanian haplotypes (Figure 3, S1 and S2). Indeed, the ML tree (Figure 3 and S1) shows that this haplotype forms a strongly supported (bootstrap support >70%) distinct lineage from the other Mauritanian porpoises. The haplotype network also illustrates this divergence (Figure S2): while the Mauritanian haplotypes are separated from each other with at most two mutational steps, at least ten mutational steps separate this haplotype 10 from the other Mauritanian haplotypes (Figure S2). Notably, both porpoises carrying this haplotype 10 were found on the southern extremity of the Portuguese coasts (8.78W-37.07N; Praia do Burgau, Portugal), at least 400km south from all other Iberian sampling locations (Figure 1). Stranding records of harbor porpoises were previously reported in Morocco, on the east coasts of Gibraltar, and in Cadiz (Spain; [START_REF] Rojo-Nieto | Strandings of cetaceans and sea turtles in the Alboran Sea and Strait of Gibraltar: a longterm glimpse at the north coast (Spain) and the south coast (Morocco)[END_REF]. If unsampled populations occur between the Iberian Peninsula and the Mauritanian waters, they are most likely confined to a narrow and seasonal upwelling area. This relatively divergent haplotype 10 closely related to the Mauritanian rather than the Iberian porpoises could be evidence of such unsampled populations. This hypothesis of such unsampled population could be plausible since harbor porpoise sightings, stranding, and bycatches have been reported from the North of Morocco to the Gambia [START_REF] Boisseau | Encounter rates of cetaceans in the Mediterranean Sea and contiguous Atlantic area. Marine Biological Association of the United Kingdom[END_REF]IMR-NAMMCO, 2019).

Haplotype 10 was excluded from subsequent analyses of genetic diversity and coalescent simulations because it could be part of a distinct population and therefore would artificially inflate measures of genetic diversity (Table 1 andS1). Future studies with additional samples from these relatively unsampled regions (southern Iberian coast and Morocco) and data from the nuclear genome are needed to confirm whether these two porpoises are genetically distinct from the other Iberian porpoises, possibly forming a distinct population, and whether or not they are admixing with the other Iberian porpoises.

Aside from the two porpoises carrying the haplotype 10, our results indicate that both cohorts sampled at different times along the Iberian coasts belong to the same mtDNA gene pool. We detected no genetic differentiation between the two Iberian cohorts suggesting no significant differences in haplotype frequencies between them (FST and φST values = 0; p-value > 0.54, Table S4). This result reflects the high prevalence of haplotype 4 which dominates in both cohorts (Figure 3, S1 andS2). However, once we accounted for differences in sample size between the two cohorts using a rarefaction method, we observed a significant reduction in genetic diversity in the new cohort compared to the old one as shown by the lower number of haplotypes (H; Figure 4a), and in the two measures of nucleotide diversity (π and θW; Figure 4b and4c, respectively). Rarefaction curves provided striking evidence of this reduction in genetic diversity (Figure 4). The non-overlapping standard errors from a standardized sample size beyond eight sequences suggest that the decline of genetic variation is likely highly significant. For example, the average number of haplotypes (H) observed from 19 sampled sequences in each cohort decreases from 10 to 6 when comparing the old vs. new cohorts. This suggests that the proportion of rare haplotypes has dropped substantially over the 25-years sampling period of this study. Consistent with this observation is the rapid loss in nucleotide genetic diversity as measured by π and θW (Figure 4b and4c). Such a substantial loss of genetic variation over 2.5 generations is often attributed to an enhanced effect of genetic drift rather than other evolutionary forces like selection [START_REF] Hartl | A Primer of Population Genetics and Genomics[END_REF]. This could suggest a dramatic decline of the Iberian population in term of effective population size, possibly reflecting a substantial decline in the number of reproducing individuals.

Tajima's D [START_REF] Tajima | Statistical method for testing the neutral mutation hypothesis by DNA polymorphism[END_REF]) and Achaz's Y [START_REF] Achaz | Testing for neutrality in samples with sequencing errors[END_REF] statistics estimated for the two cohorts sampled at different times along the Iberian coast also displayed negative values (Figure 5). At first glance, such negative values would be indicative of a population that underwent a "recent" expansion at evolutionary time-scales [START_REF] Epps | Landscape genetics in a changing world: disentangling historical and contemporary influences and inferring change[END_REF][START_REF] Smith | The role of historical and contemporary processes on phylogeographic structure and genetic diversity in the Northern Cardinal, Cardinalis cardinalis[END_REF], most likely due to the foundation of the Iberian population during the period that followed the Last Glacial Maximum (19kyr ago; [START_REF] Fontaine | Postglacial climate changes and rise of three ecotypes of harbour porpoises, Phocoena phocoena, in western Palearctic waters[END_REF][START_REF] Fontaine | Harbour porpoises, Phocoena phocoena, in the Mediterranean sea and adjacent regions: biogeographic relicts of the last glacial period[END_REF]).

However, the values estimated for the new cohort for both statistics were significantly less negative (based on non-overlapped standard error estimates) than those from the old cohort.

For example, the Tajima's D values obtained from 19 randomly sampled sequences increased from -1.5 for the old cohort to -1.25 for the new (Figure 5a). The change in Achaz's Y values, which account for possible sequencing errors [START_REF] Achaz | Testing for neutrality in samples with sequencing errors[END_REF], show an even stronger increase from -1.75 to -0.95 between the old and new cohorts (Figure 5b). It is remarkable to see how both values have become dramatically less negative in the time lapse of only 2.5 generations between the two cohorts. This increase in D and Y values also provides a strong indication of a population losing rare haplotypes as it would be expected for a small and isolated population strongly subject to genetic drift that could be declining. Such a loss of genetic diversity may reflect different demographic scenarios, for example a small population that did not experience any population size changes, but still experiencing strong genetic drift. Alternatively, a scenario of ancient expansion and a very recent decline could be also possible. We used a simulation-based Approximate Bayesian computation analysis relying on the Random-Forest classifier (ABC-RF) to explored statistically six plausible demographic scenarios (Figure 2) describing the evolutionary history of the Iberian population since it diverged from the Mauritanian population within the last 150 generations [START_REF] Fontaine | Postglacial climate changes and rise of three ecotypes of harbour porpoises, Phocoena phocoena, in western Palearctic waters[END_REF]. Out of the six scenarios compared, the analysis strongly supported the SC6 scenario assuming an "old" expansion (>150 generations) followed by recent decline with the past 3 generations (SC6; Figure 2, S3 to S5). Ten replicates of the ABC-RF analysis supported strongly and unanimously this result (Figure S3). Scenario SC6 gathered in average (± SD) over the ten runs 56.6 ± 1.3% of the RF votes (Figure 2 andS3), with a posterior probability of 66.3 ± 2.5%, a prior error rate of 9.5 ± 0.0%, and type II errors ≤ 3.1% ± 0.0%. Furthermore, the confusion matrix (Figure S5) depicting the overall performance of the RF classification analysis indicated high power to distinguish between the competing scenarios (>90%). Such a high-resolution power to discriminate among the different demographic scenarios likely comes from the multiple sampling time points included in the ABC analysis.

The goodness-of-fit analysis provided additional support to the demographic model identified by showing that scenario SC6 was the only scenario that produced simulated values that closely matched the observed values for each summary statistics used in this analysis (Table S5, Figure S4 andS6). This scenario SC6 (Figure 2) suggests that the effective size (Ne) of the Iberian population expanded at least ~150 generations ago, thus ~1500 years ago, assuming a rough generation time of 10 years [START_REF] Fontaine | Postglacial climate changes and rise of three ecotypes of harbour porpoises, Phocoena phocoena, in western Palearctic waters[END_REF]. Then, under this scenario, Ne values of the Iberian population would have declined by a factor ≥ 10 within the last three generations (thus 30 years). Thus, this ABC analysis, together with the rarefaction results, adds an additional line of evidence suggesting a rapid loss of genetic variation over the last three generations due to a very recent decline in effective population size. The reader should note however that the effective population size (Ne) is a population genetic concept that differs from and is usually smaller than the census population size. It does not represent the number of individuals actually alive at any given time point. Following [START_REF] Frankham | Conservation Genetics[END_REF], Ne is the number of individuals that would result in the same loss of genetic diversity, inbreeding, genetic drift, etc., if they behaved in the manner of an idealized population. Such idealized population would assume non-overlapping generations, random mating, and no migration, mutation or selection. Ne would thus roughly represent the number of breeding individuals in this idealized population, or more precisely the number of breeding females since we analyzed here maternally inherited fragments of the mitogenome that behave as a single nonrecombining haploid unit. Therefore, our results are not intended to estimate exactly how many Iberian porpoises there were at different points in history, but rather they suggest that the number of reproducing females may have been substantially declining in the Iberian population over the last 2.5 generations. However, such interpretation must be taken with caution considering all the assumptions related to the Ne concept.

In this study, we also confirmed previous evidence of gene flow from the Iberian population to more northern populations with Iberian haplotypes present in the Bay of Biscay and in the English Channel where admixture between the two lineages, IBMA (or P. p. meridionalis) and P. p. phocoena, has been reported (see Figure 3, S1 and S2;Table S4, and also Ben Chehida et al., 2021;[START_REF] Fontaine | Postglacial climate changes and rise of three ecotypes of harbour porpoises, Phocoena phocoena, in western Palearctic waters[END_REF][START_REF] Fontaine | Mixing of porpoise ecotypes in southwestern UK waters revealed by genetic profiling[END_REF]. Additionally, we did not find any evidence of gene flow from the populations located further North into the Iberian population, as previously reported [START_REF] Ben Chehida | No leading-edge effect in North Atlantic harbor porpoises: Evolutionary and conservation implications[END_REF][START_REF] Fontaine | Rise of oceanographic barriers in continuous populations of a cetacean: the genetic structure of harbour porpoises in Old World waters[END_REF][START_REF] Fontaine | Postglacial climate changes and rise of three ecotypes of harbour porpoises, Phocoena phocoena, in western Palearctic waters[END_REF][START_REF] Fontaine | Mixing of porpoise ecotypes in southwestern UK waters revealed by genetic profiling[END_REF][START_REF] Fontaine | Genetic and historic evidence for climate-driven population fragmentation in a top cetacean predator: the harbour porpoises in European water[END_REF]. Indeed, [START_REF] Fontaine | Postglacial climate changes and rise of three ecotypes of harbour porpoises, Phocoena phocoena, in western Palearctic waters[END_REF] estimated very asymmetric values of gene flow using the coalescent approach of MIGRATE [START_REF] Beerli | Comparison of Bayesian and maximum-likelihood inference of population genetic parameters[END_REF][START_REF] Beerli | How to use MIGRATE or why are Markov chain Monte Carlo programs difficult to use?[END_REF] based on microsatellite data. While no evidence of immigration was detected toward the Iberian porpoises from the neighboring populations, ca. 3 individuals per generation were estimated migrating from the Iberian to the Mauritanian populations, and 70 per generation from the Iberian to the northern Bay of Biscay populations (see table 2 in [START_REF] Fontaine | Postglacial climate changes and rise of three ecotypes of harbour porpoises, Phocoena phocoena, in western Palearctic waters[END_REF]. In the northern Bay of Biscay and adjacent waters, a geographically restricted admixture zone was reported forming a well-delimited tension zone [START_REF] Fontaine | Mixing of porpoise ecotypes in southwestern UK waters revealed by genetic profiling[END_REF]. Because the population size of P. p. phocoena in the North Sea and adjacent waters is much larger than that of the Iberian population (census N estimates from the SCANS-III survey [START_REF] Hammond | Estimates of cetacean abundance in European Atlantic waters in summer 2016 from the SCANS-III aerial and shipboard surveys[END_REF]: ~400,000 vs. ≤ 3,000 individuals respectively), the observed northward migration may possibly lead to the assimilation of the Iberian gene pool by P. p. phocoena. In the long term, this process could be an additional cause of concern as it could result in the disappearance of the unique Iberian genetic pool. This scenario is advanced by [START_REF] Macleod | Global climate change, range changes and potential implications for the conservation of marine cetaceans: a review and synthesis[END_REF], which used habitat modeling to show that species with a similar range as the Iberian harbor porpoises (Figure 1C in MacLeod, 2009) may shift their range northward in response to a 5°C increase in water temperature due to global warming (Figure 1D in MacLeod, 2009). This model suggests that the future range of IBMA could completely overlap with the current range of P. p. phocoena (Figure 1D in MacLeod, 2009) to be taken with some caution since they do not account for local population adaptation. In the case of IBMA porpoises, their morphological and genetic distinctiveness together with the peculiar upwelling habitat they inhabit suggest that they are adapted to the local conditions.

Future genomic studies could assess whether selective processes have impacted the genome of IBMA porpoises and whether there is genetic evidence of such local adaptation process compared to other populations.

The results of this study support the hypothesis that the Iberian population is both small and isolated. Furthermore, both the sharp decline in mtDNA genetic diversity over the past 25 years and northward migration may represent reasons for concern for the long-term persistence of the Iberian porpoises. One plausible explanation is that the decay of mtDNA genetic variation we reported here could simply reflect the fact that Iberian porpoises constitute a stable population with a very low effective population size (Ne) (SC2 in Figure 2). This scenario SC2 was not the best model according to our simulation-based ABC-RF analysis, but the third best one, receiving only 16% of the total RF classification votes, but it remains plausible (Figure 2, S3 to S6, Table S5). In such a condition, genetic drift would have a disproportionate influence on the genetic make-up of this population and could lead to the observed loss of genetic variation over a few generations. Several previous studies showed that the Iberian population has indeed a very low Ne at nuclear microsatellite loci (Ne < 100 individuals; Ben [START_REF] Ben Chehida | No leading-edge effect in North Atlantic harbor porpoises: Evolutionary and conservation implications[END_REF][START_REF] Fontaine | Postglacial climate changes and rise of three ecotypes of harbour porpoises, Phocoena phocoena, in western Palearctic waters[END_REF][START_REF] Fontaine | Genetic and historic evidence for climate-driven population fragmentation in a top cetacean predator: the harbour porpoises in European water[END_REF]. The mtDNA employed in this study has an effective size four times smaller compared with nuclear autosomal loci. This means that the stochastic loss of genetic variation due to genetic drift would be drastically increased for mtDNA compared to nuclear loci. Thus, the Iberian population might be at a stable census size losing rare haplotypes through the enhanced effect of genetic drift at the mtDNA. Another more plausible explanation receiving the highest support in our ABC-RF analysis (scenario SC6, Figure 2, S2 to S6, Table S5) is that the decay of mtDNA genetic variation might be associated with a rapid and recent decline of the number of reproductive females in addition to the overall low Ne [START_REF] Lande | Genetics and demography in biological conservation[END_REF], as depicted by the scenarios SC6 in Figure 2. This scenario SC6 support a severe population decline in the last three generations, leaving at most 10% of the population pre-decline Ne. This hypothesis is even more plausible since there is accumulating evidence that the Iberian population is sending more migrants to adjacent populations than the reverse [START_REF] Ben Chehida | No leading-edge effect in North Atlantic harbor porpoises: Evolutionary and conservation implications[END_REF][START_REF] Fontaine | Postglacial climate changes and rise of three ecotypes of harbour porpoises, Phocoena phocoena, in western Palearctic waters[END_REF][START_REF] Fontaine | Genetic and historic evidence for climate-driven population fragmentation in a top cetacean predator: the harbour porpoises in European water[END_REF]. Thus, in addition to being small and isolated, the recent and strong population decline envisioned under SC6 of the ABC-RF analysis would imply that the Iberian porpoises may become increasingly subject to demographic stochasticity [START_REF] Allendorf | Conservation and the Genetics of Populations[END_REF].

Because this population is also known to be subject to strong genetic drift and environmental stochasticity [START_REF] Casabella | Trends of the Galician upwelling in the context of climate change[END_REF][START_REF] Pires | Assessing future climate change in the Iberian Upwelling System[END_REF], all these effects can accumulate and reinforce each other, potentially precipitating the decline of the Iberian porpoises. This socalled extinction vortex [START_REF] Allendorf | Conservation and the Genetics of Populations[END_REF] can be particularly severe for a long-lived K strategist species, inhabiting a marginal habitat highly subject to environmental variation like the Iberian harbor porpoises [START_REF] Casabella | Trends of the Galician upwelling in the context of climate change[END_REF][START_REF] Pires | Assessing future climate change in the Iberian Upwelling System[END_REF] and could put them at a high risk of extinction.

The present study relies on a single molecular marker (mtDNA). Therefore, future studies with additional samples and data from the nuclear genome are highly needed to confirm the trends highlighted here. Indeed, genome-wide data can provide further insights into the temporal dynamics of the Iberian population. For example, the reconstruction of historical demography based on genome-wide data [START_REF] Liu | Stairway Plot 2: demographic history inference with folded SNP frequency spectra[END_REF][START_REF] Schiffels | MSMC and MSMC2: The Multiple Sequentially Markovian Coalescent[END_REF][START_REF] Terhorst | Robust and scalable inference of population history from hundreds of unphased whole genomes[END_REF] can be used to test whether these harbor porpoises maintained a long-term low Ne [START_REF] Morin | Reference genome and demographic history of the most endangered marine mammal, the vaquita[END_REF][START_REF] Robinson | The critically endangered vaquita is not doomed to extinction by inbreeding depression[END_REF] since their post-glacial emergence [START_REF] Fontaine | Harbour porpoises, Phocoena phocoena, in the Mediterranean sea and adjacent regions: biogeographic relicts of the last glacial period[END_REF][START_REF] Fontaine | Postglacial climate changes and rise of three ecotypes of harbour porpoises, Phocoena phocoena, in western Palearctic waters[END_REF][START_REF] Fontaine | Genetic and historic evidence for climate-driven population fragmentation in a top cetacean predator: the harbour porpoises in European water[END_REF], or if on the contrary, the population declined recently [START_REF] Hu | Genomic consequences of population decline in critically endangered pangolins and their demographic histories[END_REF]. A simulation framework, similar to the one used by [START_REF] Robinson | The critically endangered vaquita is not doomed to extinction by inbreeding depression[END_REF], could be applied to assess whether these porpoises are already facing deleterious effects of the mutation load, despite their current low Ne, or if purging processes have been ongoing similar to those recently reported for the vaquita (Phocoena sinus) [START_REF] Morin | Reference genome and demographic history of the most endangered marine mammal, the vaquita[END_REF][START_REF] Robinson | The critically endangered vaquita is not doomed to extinction by inbreeding depression[END_REF].

Additional data sources also point to large fluctuations in population size in the Iberian porpoises and require careful considerations: the SCANS-III survey in summer 2016 estimated a census size as low as 2,898 animals (CV=0.32) in Iberian waters and Bay of Biscay [START_REF] Hammond | Estimates of cetacean abundance in European Atlantic waters in summer 2016 from the SCANS-III aerial and shipboard surveys[END_REF]. Annual aerial surveys in Portuguese continental waters between 2011 and 2015 estimated an overall average abundance of 2,254 porpoises and corresponding density of 0.09 ind/km 2 (CV=21.99%) with large interannual fluctuations. The highest annual abundance estimated for that period was 3,207 individuals (CV=38.14%) in 2013, followed by a sharp decrease in 2014, when only 1,653 individuals (CV=43.27%) were estimated [START_REF] Torres-Pereira | Harbour Porpoise Abundance in Portugal over a 5-Year Period and Estimates of Potential Distribution[END_REF]. [START_REF] López | Abundance estimates for harbour porpoise (Phocoena phocoena) in the Spanish area of the Iberian Peninsula Management Unit[END_REF] estimated an abundance of 683 (CV=0.63, 95%CI: 345-951) animals for the Spanish part of the IBMA range (Galicia and Bay of Biscay).

Globally, these low census sizes were associated with particularly high bycatches rate (always ≥8%) with the best estimates varying from 90-197 deaths per year [START_REF] Pierce | Stable isotope analysis reveals habitat partitioning among marine mammals off the NW African coast and unique trophic niches for two globally threatened species[END_REF] to 130-330 deaths per year [START_REF] Read | Harbour porpoise (Phocoena phocoena) and fisheries interactions in the north-west Iberian Peninsula[END_REF]. However, it is important to mention that these estimates are mainly based on "on-board observer" and aerial surveys data that deliver wide confidence limits. This is due to low observer coverage of fishing activities in the region, low density of porpoises, and estimates from various sources (observer data, strandings, aerial survey, and interviews). Furthermore, Iberian harbor porpoises are regularly found stranded, often with signs of bycatch mortality, potentially indicating unsustainable mortality rates (IMR-NAMMCO, 2019;[START_REF] López | Trends in strandings and by-catch of marine mammals in north-west Spain during the 1990s[END_REF][START_REF] Pierce | Stable isotope analysis reveals habitat partitioning among marine mammals off the NW African coast and unique trophic niches for two globally threatened species[END_REF][START_REF] Read | Harbour porpoise (Phocoena phocoena) and fisheries interactions in the north-west Iberian Peninsula[END_REF][START_REF] Torres-Pereira | Harbour Porpoise Abundance in Portugal over a 5-Year Period and Estimates of Potential Distribution[END_REF]Torres-Pereira et al., 2023a;[START_REF] Vingada | Conservação de Cetáceos e Aves Marinhas em Portugal Continental. O projeto LIFE+ MarPro[END_REF]. In fact, based on Portuguese stranding data, an average of 9.19% (CI=5.25-16.10%) of the porpoise population was estimated to be removed annually by fisheries between 2011 and 2015, corresponding to an average of 207 porpoises removed per year, which largely surpasses the Potential Biological Removal rate conservatively estimated for the same period (22 porpoises, CI: 12-43) (Torres-Pereira et al., 2023a). An additional cause for concern is related to potential overexploitation of the feeding resources [START_REF] Méndez-Fernandez | Ecological niche segregation among five toothed whale species off the NW Iberian Peninsula using ecological tracers as multi-approach[END_REF][START_REF] Santos | The diet of harbour porpoise (Phocoena phocoena) in the Northeast Atlantic[END_REF]. A scarcity in prey availability can be harmful because it enhances interspecific competition, as already reported between harbor porpoises and other marine mammals, for example with the bottlenose dolphin Tursiops truncatus whose ecological niche largely overlaps with the harbor porpoise in some portions of Iberian waters [START_REF] Méndez-Fernandez | Ecological niche segregation among five toothed whale species off the NW Iberian Peninsula using ecological tracers as multi-approach[END_REF][START_REF] Spitz | Diet overlap between harbour porpoise and bottlenose dolphin: An argument in favour of interference competition for food? Estuarine[END_REF]. Hostile interactions between the two species have been documented in Iberian waters and hypothesized to cause harbor porpoises to disperse to less productive narrow shelf areas [START_REF] Alonso | Evidence of violent interactions between bottlenose dolphin (Tursiops truncatus) and other cetacean species in NW Spain[END_REF][START_REF] Spitz | Diet overlap between harbour porpoise and bottlenose dolphin: An argument in favour of interference competition for food? Estuarine[END_REF].

Conclusions

The Afro-Iberian (IBMA) lineage of harbour porpoises was previously recognized as a unique Evolutionarily Significant Unit [START_REF] Crandall | Considering evolutionary processes in conservation biology[END_REF][START_REF] Moritz | Strategies to protect biological diversity and the evolutionary processes that sustain it[END_REF] distinct from the other sub-species devribed in the N. Atlantic and in the Black Sea. IBMA display unique features in terms of genetics, morphology, ecology and evolutionary history following [START_REF] Fontaine | Postglacial climate changes and rise of three ecotypes of harbour porpoises, Phocoena phocoena, in western Palearctic waters[END_REF] (see also the review of [START_REF] Fontaine | Harbour porpoises, Phocoena phocoena, in the Mediterranean sea and adjacent regions: biogeographic relicts of the last glacial period[END_REF]. Nevertheless, they are currently not recognized as a distinct subspecies [START_REF] Pierce | Iberian harbour porpoise -an update on fishery bycatch mortality[END_REF][START_REF] Pierce | Stable isotope analysis reveals habitat partitioning among marine mammals off the NW African coast and unique trophic niches for two globally threatened species[END_REF]. There is also currently no mention of these porpoises in the IUCN Red List of Threatened Species despite the multiple threats faced by this vulnerable lineage. Given the results of this study, the previous findings on their genetic distinctiveness from other populations at both mtDNA and nuclear markers [START_REF] Ben Chehida | No leading-edge effect in North Atlantic harbor porpoises: Evolutionary and conservation implications[END_REF][START_REF] Fontaine | Harbour porpoises, Phocoena phocoena, in the Mediterranean sea and adjacent regions: biogeographic relicts of the last glacial period[END_REF][START_REF] Fontaine | Rise of oceanographic barriers in continuous populations of a cetacean: the genetic structure of harbour porpoises in Old World waters[END_REF][START_REF] Fontaine | Postglacial climate changes and rise of three ecotypes of harbour porpoises, Phocoena phocoena, in western Palearctic waters[END_REF][START_REF] Fontaine | Mixing of porpoise ecotypes in southwestern UK waters revealed by genetic profiling[END_REF][START_REF] Fontaine | Genetic and historic evidence for climate-driven population fragmentation in a top cetacean predator: the harbour porpoises in European water[END_REF], and the previous concerns raised about their viability (IMR-NAMMCO, 2019; [START_REF] Pierce | Iberian harbour porpoise -an update on fishery bycatch mortality[END_REF][START_REF] Pierce | Stable isotope analysis reveals habitat partitioning among marine mammals off the NW African coast and unique trophic niches for two globally threatened species[END_REF], we recommend immediate actions and further research from the community in the following areas: (1) a formal taxonomic description is needed for these IBMA porpoises to be officially recognized as a distinct subspecies, following the guidelines of the International Commission on Zoological Nomenclature as recommended by the Taxonomic Committee of the Society for Marine Mammalogy; and (2) a better assessment for the Iberian porpoises in term of their life history traits, ecology, behavior, genetics and genomics, and the threats to the viability for this unique group. This knowledge will help designing better conservation measures and recommendations. It is indeed paramount to ensure that all measures are taken to gain additional knowledge and to apply effective conservation efforts to protect the Iberian population, as recently underline by the OSPAR Marine Mammal Expert Group (OMMEG) (ICES-WGMM, 2021; [START_REF] Pierce | Iberian harbour porpoise -an update on fishery bycatch mortality[END_REF]. Recently, both countries (Portugal and Spain) updated the conservation status of the harbor porpoises inhabiting Iberian waters: from "Vulnerable" to "Endangered" in Spain (Boletin oficial del estado, 2020) and to "Critically Endangered" in Portugal (Torres-Pereira et al., 2023b). In Portugal, a new Marine Protected Area (PTCON0063) in the northwestern coast was recently designated whereas another one was enlarged in the southwestern coast (PTCON00012) in order to incur higher protection for harbor porpoise hotspots in the country [START_REF] Vingada | Conservação de Cetáceos e Aves Marinhas em Portugal Continental. O projeto LIFE+ MarPro[END_REF]. Nonetheless, none of the conservation and mitigation measures within the areas' management plan (Portaria 201/2019) is currently in place and bycatch mortality remains effectively unaddressed. In the future, improving our understanding of the demographic trends and the threats these porpoises are facing is required to devise suitable and tailored larger scale conservation plans for the whole Iberian population. Such measures are timely needed to mitigate the decline of this unique lineage potentially on the brink of extinction. S5). The colored scenario, SC6, was the scenario gathering the highest number of Random Forest votes. The time scale is indicated by the arrow on the left, in generations before present (gen.).

Tables and Figures

  (2012-2015) for fragments of five mitochondrial genes including Cytochrome b (Cyt-b: 1,111 base-pairs, bp), and ATP-6 and -8 (842bp), NADH dehydrogenase 5 (ND5: 707bp), and the Cytochrome c oxidase subunit I (COI: 1,515 bp), When aligned with the 82 previous sequences of harbor porpoises from Fontaine et al. (2014) and an additional one of the Dall's porpoise used here as outgroup, the final alignment of 4,175 base-pairs (pb) and included 135 sequences and contained 384 segregating sites with 267 singletons and 117 parsimony informative sites defining 61 distinct haplotypes. Without the outgroup, the 134 mtDNA sequences of harbor porpoise contained 170 segregating sites with 65 singletons and 105 parsimony informative sites defining 60 distinct haplotypes (Table 1). Consistent with previous studies, the phylogenetic relationships among mtDNA haplotypes depicted by the ML tree (Figure 3 and S1) and by the haplotype network (Figure S2) revealed the three main monophyletic lineages corresponding to the three previously identified subspecies or ESU's (Fontaine et al., 2014): (i) P. p. relicta in the Black Sea; (ii) P. p. phocoena composed of the porpoises from the North Sea to the waters of Norway; and (iii) the Afro-Iberian (IBMA) porpoises (unnamed ssp. possibly P. p. meridionalis), which comprises two distinct monophyletic sub-lineages in the upwelling waters of Iberia and Mauritania, respectively.Evidence of migration from the Iberian to the Mauritanian populations has been previously documented[START_REF] Ben Chehida | No leading-edge effect in North Atlantic harbor porpoises: Evolutionary and conservation implications[END_REF][START_REF] Fontaine | Postglacial climate changes and rise of three ecotypes of harbour porpoises, Phocoena phocoena, in western Palearctic waters[END_REF] and is also observed here with two haplotypes (Hap_55 and Hap_59) found in the Mauritian population, but clustering with the Iberian lineage (Figure 3, S1 and S2). However, we also identified one haplotype (Hap_10 or Haplotype 10, see Figure 3, S1 and S2) present in two porpoises from the new Iberian cohort, but clustering with the Mauritanian lineage. This haplotype was carried by two individuals in the southernmost part of the Iberian sampling (red arrow in Figure

  . It is worth mentioning that Ben Chehida et al. (2021) recently modeled the future distribution of the harbor porpoises in the North Atlantic under the most aggressive scenario of the Intergovernmental Panel on Climate Change (IPCC) in 2050 (RCP8.5; Schwalm et al., 2020) and did not capture this strong northward range shift. The discrepancy between the two models may be related to differences in assumed temperature increase, 2°C and 5°C, in Ben Chehida et al. (2021) and MacLeod (2009), respectively. In addition, the environmental niche modeling in Ben Chehida et al. (2021) did not account for any biotic factors in the simulations. However, environmental niche modelling such as those conducted by MacLeod (2009) and Ben Chehida et al. (2021) needs

Figure 1 .

 1 Figure 1. Map showing the sampling locations and sample sizes. Geographic locations are

Figure 2 .

 2 Figure 2. Alternative scenarios tested in the ABC-RF procedure. See TableS2for further

Figure 3 .

 3 Figure 3. Cladogram derived from the maximum-likelihood mitochondrial phylogeny among unique mitochondrial haplotypes. A branch length transformation proportional to the number of tips under the node (as implemented in FigTree) was applied to facilitate the visualization of the branching patterns (see Figure S1 for the actual phylogenetic tree with branch lengths proportional to sequence divergence). The color-coded labels show the geographic origin of the haplotype. The numbers within the boxes refer to the number of individuals carrying the haplotype. No number in a box means that the haplotype was observed only once. Population code are as follow: BS=Black Sea (N=12)*; NAT=North Atlantic (N=23)*; BB=Bay of Biscay (N=14)*; MA=Mauritania (N=14)*; IB_O=Iberian Old (N=19)*; IB_N= Iberian new (N=52)**. [* Fontaine et al. 2014; ** This study].

Figure 4 .

 4 Figure 4. Rarefaction curves showing three statistics describing mitochondrial genetic

Figure 5 .

 5 Figure 5. Rarefaction curves showing two neutrality indexes indicative of demographic

Table 1 :

 1 Mitochondrial genetic diversity. The statistics display per groups and overall include the mtDNA sample sizes (N), the number of segregating sites (S), singleton mutations (Singl.); sites informative in parsimony (Parsim.), number of haplotypes (# Hap), haplotype diversity (Hd), the nucleotide diversity (π), Watterson's theta (θW),Tajima's D, Y, Achaz's Y. Population codes are as follow: BS=Black Sea; NAT=North Atlantic; BB=Bay of Biscay; MA=Mauritania; IB_O=Iberian Old 1990's cohort; IB_N= Iberian new 2010's cohort. Superscript next to each population code indicate the origin of the mtDNA sequence : 1. Fontaine et al. (2014); 2. Fontaine et al. (2014) + this study; 3. This study. See Table S1 for more details on the samples.

		N	S	Singl. Parsim. # Hap	Hd	π (%) θW (%)	D	Y
	BS 1	12	23	21	2	10	0.96	0.10	0.18	-2.04 -1.56
	NAT 1	23	99	57	42	18	0.97	0.41	0.64	-1.46 -0.83
	BB 1	14	66	19	47	10	0.95	0.52	0.50	0.22 0.05
	MA 1	14	11	1	10	7	0.90	0.07	0.08	-0.66 -1.93
	All IB 2	71	27	7	20	17	0.72	0.05	0.13	-1.85 -2.12
	IB_O 1	19	12	6	6	10	0.74	0.05	0.08	-1.53 -1.77
	IB_N 3	52	24	5	19	11	0.70	0.06	0.13	-1.77 -2.19
	IB_N* 3	50	14	7	7	10	0.73	0.04	0.08	-1.58 -1.26
	All	134 170	65	105	60	0.91	0.40	0.74	-	-

*Excluding haplotype 10

Acknowledgements

We thank all the people and networks involved in the sampling collection of harbor porpoises over the years across the distribution range of the harbour porpoises, including KA Tolley, T Jauniaux, J Haelters, AA Llavona, B and AA Ozturk, V Ridoux, F Caurant, W Dabin, E Rogan, M Sequeira, U Siebert, GA Vikingsson, A Borrell, AA Aguilar, and the national stranding networks: Réseau National d'Échouage and PELAGIS in France; Royal Belgian Institute of

Data Availability

The data sets and scripts supporting this study are available in the IRD Porpoises Genetics and Genomics Dataverse Repository (https://dataverse.ird.fr/dataverse/porpoise_genomics) at https://doi.org/10.23708/GOPRGV. Mitochondrial haplotypes were also deposited on the NCBI-GenBank under the Accession Numbers (Accession numbers are listed in Table S1).

We are also grateful to the Centre for Information Technology of the University of Groningen, and in particular Fokke Dijkstra and Bob Dröge, for their continuous support and for providing access to the Peregrine high-performance computing cluster. This study was funded by the University of Groningen (The Netherlands). YBC was supported by a PhD fellowship from the University of Groningen.

Conflict of interest

The authors declare that they have no conflict of interest.

Author Contributions