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Variable outcomes of hybridization between declining Alosa alosa and Alosa fallax

Abstract Hybridization dynamics between co‐occurring species in environments where human‐mediated changes take place are important to quantify for furthering our understanding of human impacts on species evolution and for informing management. The allis shad Alosa alosa (Linnaeus, 1758) and twaite shad Alosa fallax (Lacépède, 1803), two clupeids sister species, have been severely impacted by human activities across Europe. The shrinkage of A. alosa distribution range along with the decline of the remaining populations' abundance threatens its persistence. The main objective was to evaluate the extent of hybridization and introgression between those interacting species. We developed a set of 77 species‐specific SNP loci that allowed a better resolution than morphological traits as they enabled the detection of hybrids up to the third generation. Variable rates of contemporary hybridization and introgression patterns were detected in 12 studied sites across the French Atlantic coast. Mitochondrial markers revealed a cyto‐nuclear discordance almost invariably involving A. alosa individuals with an A. fallax mitochondrial DNA and provided evidence of historical asymmetric introgression. Overall, contemporary and historical introgression revealed by nuclear and mitochondrial markers strongly suggests that a transfer of genes occurs from A. fallax toward A. alosa genome since at least four generations. Moreover, the outcomes of introgression greatly depend on the catchments where local processes are thought to occur. Undoubtedly, interspecific interaction and gene flow should not be overlooked when considering the management of those species.

waste reproductive effort when hybrids are unviable or unfit ultimately leading to demographic swamping (Wolf, Takebayashi, & Rieseberg, 2001), or parental species displacement by hybrids when hybrid genotypes are viable and fertile leading to genetic swamping (Rhymer & Simberloff, 1996;Seehausen, 2006;Taylor et al., 2006;Todesco et al., 2016). Since the consequences of hybridization can vary considerably in time and space even within species, they need to be carefully studied to understand their ultimate effects.
Climate change, alterations of the physical landscape, or worldwide translocation of organisms by humans is dramatically increasing rates of hybridization and gene flow between species (Allendorf, Leary, Spruell, & Wenburg, 2001;Brennan et al., 2015;Grabenstein & Taylor, 2018). Anthropogenically driven changes such as habitat loss or degradation and shifts in species' distributions influence the degree of contact between groups of individuals and the integrity of reproductive barriers (Crispo, Moore, Lee-Yaw, Gray, & Haller, 2011). Two inherent aspects of evolutionary biology challenge the traditional view of species conservation.
Firstly, the species concept has long been and is still a discussed topic within fields that define species such as biology, taxonomy, evolutionary biology, or ecology and there is still no universally accepted definition of species. Secondly, and although "species" may be described across their distribution range, their constitutive populations may be locally adapted through ecological or geographic divergence and interbreeding (Fitzpatrick, Ryan, Johnson, Corush, & Carter, 2015). Despite a species-based conservation viewpoint seeing species as discrete fundamental units, academics and managers have begun to recognize the importance of intraspecific variation (Palkovacs, Dion, Post, & Caccone, 2007) and the value of hybridization in promoting adaptation (Becker et al., 2013;Jackiw, Mandil, & Hager, 2015;vonHoldt, Brzeski, Wilcove, & Rutledge, 2018). However, the high variability in the ultimate consequences of hybridization for species makes prediction and general recommendations difficult (Gompert & Buerkle, 2016;Mandeville et al., 2019). Crucial to understanding the impact of hybridization is characterizing the geographic patterns of introgression between sympatric species to evaluate hybridization in conservation planning.
The allis shad Alosa alosa (Linnaeus, 1758) and twaite shad Alosa fallax (Lacépède, 1803) are anadromous clupeids and sister species that have been severely impacted by human activities since the beginning of the 19th century and increasingly throughout the course of the 20th century, leading to the extirpation (Sabatié, 1993) of several populations across their distribution range. These two species currently have overlapping ranges in the Atlantic where they can be found in sympatry in many rivers along the European Atlantic coast from the British Isles through to Portugal.
Because A. alosa uses the higher parts of the rivers as spawning grounds, the construction of dams has particularly affected the abundance and persistence of its populations (Baglinière & Elie, 2000;Baglinière, Sabatié, Rochard, Alexandrino, & Aprahamian, 2003;Castelnaud, Rochard, & Le Gat, 2001). Only few of the 29 rivers colonized by A. alosa at the beginning of the 20th century remain fully functional for the reproduction of the species (Baglinière et al., 2003). In particular, the Garonne-Dordogne population that is geographically central to A. alosa current distribution range and that was once thriving has seen its abundance reduced by a factor 100 since 1994 (Rougier et al., 2012). In response, a moratorium on professional and recreational fishing was imposed in 2008. The shrinkage of the distribution range along with the decline of the remaining populations' abundance threatens the persistence of A. alosa.
Despite both having an anadromous life cycle, the two species have contrasting dispersal and migration behavior. Alosa alosa has a life cycle that widely exploits the marine and freshwater environments with good dispersal abilities at sea, and its reproductive grounds are located further upstream in the rivers Baglinière et al., 2003). On the other hand, A. fallax seems to have a stronger homing behavior, and fish stay closer to the coast at sea and reproduce closer to the estuary in the rivers. Those differences in the geographical extent of their life cycle have major impacts on their population dynamics such that A. alosa populations are more connected to each other compared to A. fallax . Moreover, A. fallax is generally less impacted by the presence of obstacles in the rivers that are usually upstream from their spawning ground even though some populations have also declined or were extirpated . Due to the poor ability of shads to cross fall height above a meter, the presence of dams even of small size may prevent A. alosa from reaching its natural spawning grounds and force the presence of both species within the same spawning area below the obstacles. As a consequence, dams and obstacles along the river alter species spatial segregation of reproductive sites that normally represent a significant species premating barrier, potentially resulting in increasing hybrid production .
Several studies have explored the extent of hybridization between A. alosa and A. fallax in Morocco (Sabatié, 1993), France and Portugal Boisneau, Mennesson-Boisneau, & Guyomard, 1992), Ireland (Coscia, Rountree, King, Roche, & Mariani, 2010), and England (Jolly, Maitland, & Genner, 2011), using molecular markers (allozyme, microsatellite, and mitochondrial markers) and the count of gill rakers. These studies revealed the presence of hybrids across the distribution range and the exchange of genetic material between both species, but gaps of knowledge are remaining. Firstly, those studies are either very localized or geographically sparse, meaning that information between geographically distant populations is missing. Those sampling scales miss out on important information to understand population dynamics given that those species are thought to migrate over small distances following a stepping stone model. Secondly, the Garonne-Dordogne system that harbors shad populations that are currently under concern and localized at the center of species distributions needs to be studied in detail to better compare its hybridization pattern to other neighboring rivers. Finally, the qualitative and quantitative importance of hybridization requires a finer characterization of hybrid individuals using appropriately designed genetic markers to improve resolution and increase the discrimination of different hybrid classes (McFarlane & Pemberton, 2019). This is a necessary step to improve our understanding of the dynamics and consequences of hybridization between A. alosa and A. fallax.
Our main objective was to study the geographic patterns of hybridization and the consequences of introgression between two declining shad species. We first refined species delimitation and hybrid identification method using a combination of gill raker count, a traditionally used morphological character for species identification (Sabatié, Boisneau, & Alexandrino, 2000), and genetic assignment based on species-specific single nucleotide polymorphism (SNP). We then quantified the level of contemporary hybridization by assigning individuals as purebred and hybrids up to the third generation in 12 studied sites across the French Atlantic coast.
Additionally, we studied mitochondrial DNA variation on the same individuals to test for potential sex-biased introgression, evaluate more long-term history of hybridization, and compare historical and contemporary hybridization patterns. Variation in hybridization outcomes across time and geographical scales helped refining our understanding of species reproductive barriers, the consequences of introgression and their implication in a conservation and management context.

| Samples and DNA extraction
The primary source of genetic material for this study was collected as part of monitoring programs on Alosa alosa and Alosa fallax populations in French rivers of the Atlantic coast by fisheries departments, local migrating fish associations, and planning councils. Some genetic material was also retrieved from previous research project (Martin et al., 2015). A total of 634 sexually mature Alosa spp. individuals were sampled from 12 river localities across the French Atlantic coast, from the Vire river on the English Channel (Normandy region) through to the Nivelle river on the south Bay of Biscay and from one locality in the marine environment in the middle of the Bay of Biscay facing the Charente's river mouth and named hereafter Ocean (Table 1, Figure 1). Sample conditions were diverse, ranging from tissue samples collected on alive fish that were released after fin clipping to tissue samples collected on fish carcasses. All tissue samples were placed into vials containing molecular grade 95% ethanol. Total genomic DNA was extracted using Invitrogen™ PureLink™ Genomic DNA Mini Kit following the manufacturer's instructions and visualized on 1.5% agarose gels.
The number of gill rakers on the first left branchial arch, a morphological character commonly used to discriminate between A. alosa (>90 gill rakers) and A. fallax (<60 gill rakers), was counted under a binocular, following the recommendations of Sabatié (1993) and King and Roche (2008).

| Nuclear SNP genotyping
Species-specific nuclear SNP was developed from genomic and transcriptomic sequences originating from the complete sequencing of pooled individuals from the two species of interest (S. Sabatino, in prep.). Briefly, 14 pooled-DNA samples of anadromous and freshwater A. alosa and A. fallax were aligned to a de novo A. alosa genome generated via short-read and mate-pair sequencing data (S. Sabatino, in prep.). Variable loci were identified and evaluated using Popoolations2 (Kofler, Pandey, & Schlötterer, 2011) and FreeBayes (Garrison & Marth, 2012) and filtered for quality. Species-specific SNP loci for A. alosa and A. fallax were identified as those nearly fixed in the target species (greater than 0.9) and nearly invariant in the other. The Agena MassARRAY ® system (Gabriel, Ziaugra, & Tabbaa, 2009) was chosen as genotyping platform because it targets short sequences around the SNP of interest and is thus robust to degraded DNA (Fitak, Naidu,

| Power of species-specific nuclear SNP for species and hybrid assignment
A first assessment of the genetic structure of natural populations was performed using Structure v 2.3.4 (Falush, Stephens, & Pritchard, 2003;Pritchard, Stephens, & Donnelly, 2000) using the admixture and correlated allele frequencies' models assuming 2 genetic clusters (K = 2). The analysis was independently run three times with a burnin of 100,000 MCMC iterations followed by 200,000 MCMC iterations that recorded the admixture coefficient (i.e., the proportion of the genome of each individual that belong to each of the two assumed genetic clusters). We checked that the two assumed genetic clusters delineated A. alosa and A. fallax without ambiguity across the studied area. Individuals from the Garonne/Dordogne population mostly consisted of purebred from the two species ( Figure 2) and were thus selected as representative of species allele frequencies for hybrids' genotype simulation. We used HybridLab (Nielsen, Bach, & Kotlicki, 2006) to simulate first- NewHybrids v 1.1 beta3 (Anderson & Thompson, 2002) was used to assign the simulated genotypes into the different hybrid classes. Among the 21 possible purebred or hybrid classes up to 3 generations, only 15 have different genotype frequency classes (defined as the expected proportion of the loci within an individual harboring zero, one or two alleles from one of the species) and can thus in principle be differentiated using NewHybrids (Anderson & Thompson, 2002;Pritchard et al., 2016). Theoretical genotypic frequencies of all hybrid classes are detailed in Table S2. We tested the performance of NewHybrids to assign simulated individuals from 15 identifiable purebred and hybrid classes to their correct class. A total of 675 simulated genotypes were submitted to NewHybrids with the number of simulated genotypes ranging from 100 for purebred to 50 for hybrids classes involving crosses between hybrids and purebreds (such as backcrosses) and 25 for hybrid classes expected to be rare in natural populations (F1 and crosses involving rare hybrid classes). Because missing genotypes can significantly affect the assignment power of multilocus genotypic data, we mimicked missing data observed in the real dataset by adding the same percentage of missing data to the simulated dataset. NewHybrids was run using uniform priors, a burnin of 5,000 iterations followed by 10,000 iterations that recorded the parameter estimates, the probability that the multilocus genotype originated from each of the 15 purebred or hybrid classes. Several runs were performed to assess the convergence and stability of the results. Each simulated multilocus genotype was assigned to the class that showed more than 50% of membership probability or to the most likely hybrid class. We then computed the efficiency (the proportion of correctly assigned individual), the accuracy (the proportion of true purebreds or hybrids assigned in each class), and the overall performance (the product of efficiency and accuracy) of the assignment procedure (Vähä & Primmer, 2006).
F I G U R E 2 Genetic clustering using Structure assuming two genetic clusters (top) and genetic assignment to three-generation hybrid classes using NewHybrids (bottom) for 675 simulated 77 nuclear SNP multilocus genotypes belonging to the 15 distinguishable purebred and three-generation hybrid classes (indicated in along the x-axis). Each individual is represented by a vertical bar representing the admixture coefficient (top) or the assignment probability to each hybrid class (bottom

| Species delineation and hybrid assignment in natural populations
Finally, NewHybrids was used to assign nuclear SNP genotyped in natural populations using the same procedure as for the simulated multilocus genotypes (uniform priors, burnin of 5,000 iteration followed by 10,000 iterations). Each simulated multilocus genotype was assigned to the class that showed more than 50% of probability, or to the most likely hybrid class.

| Link between genotypic classes and gill raker counts
The correlation between gill raker counts and Structure posterior probability of membership to A. alosa was tested with Spearman's rank correlation rho. Statistical differences in gill raker counts among and between hybrid classes were tested using a Kruskal-Wallis test and pairwise Wilcoxon's tests with Holm's correction for multiple testing (Holm, 1979).

| Mitochondrial sequencing
To further study hybridization between A. alosa and A. fallax, the distribution of their mitochondrial polymorphism among the different genotypic classes was explored. Because of the poor quality of the DNA extractions (degraded in most samples, especially from those extracted from fish carcasses), a short-read sequencing strategy was adopted.
Based on amplification success, potential negative interaction and overlap, a final set of 11 primer pairs (Table S1) was validated for use within a single multiplexed PCR. A three-round multiplex PCR approach was performed (Chen et al., 2016) to increase homogeneity of amplification and thus coverage of sequence between loci. In the first round, the amplification was carried out in a total volume of 5 µl, including 3 mM MgCl 2 , 0.05 µM of each primer, 200 mM of each dNTP, one unit of HotStartTaq Plus DNA polymerase (Qiagen), and 1 µl of suspended DNA. Fragments were amplified using an initial denaturation step of 94°C for 5 min, followed by 20 cycles consisting in a denaturation at 94°C for 30 s, an annealing at 58°C for 3 min, and an extension at 72°C for 30 s, and an additional final extension at 72°C for 10 min. In order to maximize amplification homogeneity across loci, a second PCR round was performed to consume all remaining primers and was carried out in a total volume of 10 µl called if the sequence with the highest number of reads had more than 30 reads and the second highest allele had less than 50% coverage compared to the main allele. If no unique sequence had more than 30 reads or 50% coverage compared to the main allele, a missing data was called.

| Mitochondrial introgression
To test for historical introgression, we looked at the association between nuclear genotype and mitochondrial haplotype for each individual and explored the distribution of purebred and hybrid classes among a haplotype network. The sequences of the different mitochondrial fragments were concatenated, and a haplotype network was computed and drawn using PopART (Leigh & Bryant, 2015) to visually assess the distribution of mtDNA diversity into the different purebred and hybrid classes on the broad scale. Default median-joining network settings were used to generate the haplotype networks with pie charts representative of the proportion of the different purebred and hybrid classes as defined by NewHybrids.

| Mitochondrial diversity
Intraspecific genetic diversity of the mitochondrial concatenated fragments and demographic parameters were examined within A. alosa and A. fallax purebreds for each sampling locality when sample sizes and number of haplotypes allowed it. To adjust for small sample size in some localities and to allow for comparisons between species, sampling localities were also pooled into groups of localities and genetic diversity and demographic parameters were also computed on those groups. Firstly, genetic diversity indices were estimated by computing the number of haplotypes (H), haplotype diversity (h), number of polymorphic sites (S), and nucleotide diversity (π) using DnaSP 6. 0 (Librado & Rozas, 2009). Secondly, to test whether populations were affected by recent demographic expansions, we tested for departures from mutation-drift equilibrium by computing Tajima's D (Tajima, 1989). Either positive selection or demographic expansions would lead to negative values of this statistic.
Furthermore, we tested each group for signal of recent population expansion by calculating Fu's F s (Fu, 1997). Population growth is expected to generate an excess of rare alleles, which would lead to negative values of F s . We tested for the significance of the two statistics using 100,000 coalescent simulations. All these analyses were conducted in DnaSP 6.0 (Librado & Rozas, 2009).

| Nuclear SNP genotyping
From the initial set of 80 SNP, we removed one SNP that failed to produce interpretable signal and two additional SNP that showed more than 50% of missing data across individuals. From the 671 genotyped individuals, 37 replicates and 41 individuals genotyped for less than 60 SNP were discarded reducing the dataset to 593 individuals. Missing genotypes in the whole dataset amount to 2.1% (1,071 missing genotypes over a total of 51,051 possible genotypes) which is reasonably low given the poor quality of some extracted DNA.

| Power of species-specific nuclear SNP for species and hybrid assignment
The admixture coefficient estimated using Structure assuming two genetic clusters (K = 2) presented a linear gradient along the 13 sim-

Assignment probability
Estimated class Only crosses involving first-or second-generation hybrids (such as backcrosses and F1) presented intermediate assignment probabilities. As a result, the first step of the assignment procedure that used an assignment probability threshold of 50% allowed the assignment of all but 10 multilocus genotypes. The remaining 10 multilocus genotypes were assigned to the most likely hybrid class. Overall, this assignment strategy had high efficiency (89%), accuracy (90%), and performance (83% , Table S4). Importantly, common and informative genotypic classes with regard to the dynamics of hybridization (i.e., purebred, F1 hybrids, backcrosses, and purebred × backcrosses) could be assigned with high performance with efficiency and accuracy well above 80% (Table S3). Crosses involving first-and second-generation hybrids (such as backcross × F1, F2-F3, backcross × backcross) that were found to be very rare in natural populations (see below) showed lower efficiency (from 60% to 92%) and accuracy (from 59% to 91%) but with an assignment performance that is still manageable (from 63% to 83% if we exclude F2-F3 at 35%).

| Species delineation and hybrid assignment in natural populations
Genetic clustering with Structure assuming two genetic clusters showed that 72% of individuals had an admixture coefficient higher than 0.99 for one of the two genetic cluster (Figure 3    the Ocean locality, there was a significant proportion of hybrids (15.3% and 8.5%, respectively), including some hybrids involving A. fallax.

Fal × FalBC
Garonne and Dordogne localities presented the lower proportion of hybrids (only 2.5% in total). In the Nivelle river, the trend is similar to Brittany or Loire with only A. alosa × A. alosa backcross hybrids representing 11.5% of the studied individuals. Adour population presented both A. alosa and A. fallax purebreds and 6.9% of hybrids consisting of F1 hybrids and latter generation hybrids only toward A. fallax.

This study broadened our understanding of hybridization between
Alosa alosa and Alosa fallax, two sympatric species in the rivers of the French Atlantic coast. We developed a set of 77 species-specific SNP loci that enabled to identify up to the third-generation hybrids and allowed a better resolution than morphological traits. We were able to detect introgression along the French Atlantic coast with varying hybridization rate and introgression direction depending on the catchments. Moreover, the addition of mitochondrial markers revealed a cyto-nuclear discordance almost invariably involving A. alosa individuals with an A. fallax mitochondrial DNA suggesting that mitochondrial introgression is highly asymmetric.

| A refined molecular method for species delimitation and hybrid identification
Previous studies on European shads' hybridization have used allozyme  and microsatellite (Coscia et al., 2010;Jolly et al., 2011) markers in combination with gill raker counts to identify hybrids. They all demonstrated that individual genotypic data coincided with morphologically based species and hybrid identification, and they concluded that morphology was a reliable marker for looking at broad pattern of species identity (Jolly et al., 2011). In this study, the assignments of simulated purebreds and hybrids reclassified all but 47 individuals (7%) to their originating classes which highlight the performance of the 77 species-specific SNP to discriminate first-, second-, and third-generation hybrids (Nussberger, Greminger, Grossen, Keller, & Wandeler, 2013;Pujolar et al., 2014).
F I G U R E 5 Haplotype network for 341 Alosa alosa, Alosa fallax, and hybrid individuals constructed using a media-joining algorithm and 1,130 bp of mitochondrial fragments. Bayesian cluster assignment to genotypic classes is denoted by colors. The area of each pie chart represents the number of haplotypes. Base pair discrepancies are given by the hash marks. Three haplogroups A, B, and I are defined by dashed circles The combination of powerful markers, simulations to assess their power, and the use of an appropriate molecular statistical approach as implemented in NewHybrids can be helpful in identifying precisely advanced hybrid classes (Boecklen & Howard, 1997;Pritchard et al., 2016;Pujolar et al., 2014). We emphasis that, with the increase availability of abundant genome-wide markers in closely related species, identification of species diagnostic markers and their genotyping across many individuals using low density genotyping assay is an accessible way to refine our understanding on hybridization dynamics in many biological systems (Benjamin et al., 2018;Nussberger et al., 2013;Pujolar et al., 2014).
We also found clear correspondence between species delineation based on morphological character and genetic assignment. The number of gill rakers on the first gill arch, a trait commonly used to distinguish species and hybrids in the field (Sabatié et al., 2000), and the SNP-based posterior probability of membership to A. alosa were highly positively correlated. All A. alosa had more than 90 gill rakers, and all A. fallax had less than 60 gill rakers. In addition, F1 hybrids all fall in between (range 71-87) these two species-specific thresholds.
As this morphological trait is continuous across the hybrid classes and second-and third-generation hybrids showed high morphological variability due to complex segregation of parental species alleles, numerous second-and third-generation hybrids were morphologically indistinguishable from purebred, resulting in cryptic introgression. In cases where the detection of advanced generation hybrids is required (e.g., to monitor the impact of anthropogenic disturbance such as dam and obstacles on hybridization), the use of SNP loci to monitor hybrids is a powerful and accessible tool.

| Patterns of contemporary and historical introgression
Single nucleotide polymorphism-based genetic assignments allowed us to have a previously unknown overview of the pattern of contemporary hybridization along the French Atlantic coast. The presence of later (2nd or higher) generation hybrids indicates that F1 and backcrosses hybrids are fertile and can produce viable offspring. In addition, among the 14.2% of hybrids there was a majority of third-generation hybrids (11.5%), compared to F1 (1.7%) and second generation (1%), most of them resulting from A. alosa successive backcrossing clearly indicating an overall pattern of A. fallax introgressive hybridization. These third-generation hybrids, which cannot be identified using traditional morphological methods, still harbor a significant proportion of genes from the other species (typically an average of 12.5%). This is of great evolutionary significance as it promotes genetic variation within interacting species at a higher rate than the effect of mutation (Anderson, 1953;Hedrick, 2013).   (Coscia et al., 2010) and of the Solway Firth (Jolly et al., 2011) and in the present study across the French Atlantic coast or toward A. fallax in Portugal . These results highlight the potential for substantial variability in the genetic consequences of hybridization in Eurasian shads.

| Geographical patterns of hybridization
One of the most striking results was the variety of contemporary hybridization patterns across the studied rivers (see Figure 1 for detailed patterns of hybridization). Different processes may explain the observed diversity.
Before any other, one major factor that comes to mind to explain variable hybrid rates may involve the presence and the relative abundance of both species (Hubbs, 1955;Lepais et al., 2009;Rhymer & Simberloff, 1996) in the considered river system. Intuitively, a lack of conspecific partners would increase the chance that an individual will mate with a heterospecific partner and favor the introgression of alleles from the most abundant species (Currat, Ruedi, Petit, & Excoffier, 2008). Even if we are not able to rigorously test this hypothesis here because of the lack of presence/absence or abundance data, a couple of observations may be done. In Brittany rivers where A. fallax was not sampled or reported on the field (but may occur further downstream in the estuary for example), first-and second-generation hybrids could not be found. However, third-generation hybrids were present and probably originated either from other rivers through fish dispersal and colonization or from a punctual hybridization event concomitant with A. alosa recolonization in these rivers. In Charente, Dordogne-Garonne, and Adour rivers where both species were sampled, rates of hybrid classes were very different depending on the considered river: from moderate hybrid frequencies in the Charente and Adour rivers to low hybrid frequencies in the Dordogne-Garonne system.
The spatial segregation of spawning grounds is the most likely determining factor explaining this variability of reproductive isolation between species among rivers. In contrast to Brittany populations where hybrids represented more than 50% of the analyzed individuals, Garonne and Dordogne rivers contained only 2.5% of hybrids. In these rivers, dams are high enough upstream and allow both species to reach their natural reproductive grounds separated from each other along the river gradient. These rivers represent clear cases where spatial reproductive isolation is efficient in limiting hybridization. The situation of Adour and Charente differs markedly with the presence of dams located downstream forcing A. alosa to reproduce in spatial proximity to (Adour) or together with (Charente) A. fallax, which resulted in average to high proportions of hybrids (6.9% and 15.3% respectively). These rivers contained recent hybrids showing that contemporary processes linked to humaninduced environmental perturbations translate into the erosion of reproductive isolation (Grabenstein & Taylor, 2018) between A. alosa and A. fallax. Clearly, a reduced distance between species spawning grounds induced by obstacles increases chances of interspecific mating (Hasselman et al., 2014).

Similar observation was made in
Ireland where the presence of weirs impeded the capacity of A. alosa to travel far upstream (Coscia et al., 2010) as well as in the Solway Firth where both species are forced to share spawning grounds due to river barriers (Maitland & Lyle, 2005).
The presence of hybrids in the rivers may also not necessarily imply that they have been produced where they were captured. This hypothesis is also consistent with the fact that straying adults most likely colonize neighboring river basins (J. Martin et al., 2015).
An alternative scenario would involve the recolonization of Brittany by shoals composed of a mixture of the two shad's species with unequal abundance similarly to the Ocean sampling where most of the individuals are from one species and one or a few individuals belong to the other species. Previous studies had also found sympatric occurrence of both species at sea (Taverny, 1991 Highly polymorphic (Jolly et al., 2012;Martin et al., 2015) or genomewide (Catchen et al., 2013) neutral markers and spatially explicit simulation-based approach (Currat, Arenas, Quilodran, Excoffier, & Ray, 2019) should be able to shed new light on the demographic aspect of hybridization during this recent northern shad recolonization event.
Other factors such as the migratory behavior of the fish in the rivers may be at play in favoring the interaction between both species at the time of reproduction. It seems to be the case in the Loire river where the natural spawning grounds of both species are located far upstream because the lower reaches of the river are unsuitable for spawning (Boisneau, Ruaux, & Boisneau, 2011). The high hybridization rates (41.2%) with first-, second-and third-generation hybrids must be the result of hybridization favored by A. fallax high colonization front (Boisneau et al., 2011). This uncommon reproductive behavior of A. fallax together with the effect of effective barriers to upstream migration brings both species reproductive grounds closer and promotes the regular production of F1 hybrids. These F1 will then reproduce with the more frequent species (A. alosa) in these "shared" upstream reeds and form recurrent backcrossing, explaining the directional introgression observed in Loire. through the transfer of locally adapted genes that will increase its adaptive potential.

| Evolutionary consequences of directed introgression
While demography, sex-biased asymmetric hybridization and selective forces can all explain cyto-nuclear discordance (Toews & Brelsford, 2012), the observed cyto-nuclear discordance across our study area (i.e., introgression of A. fallax nuclear genes but not mitochondrial genomes in Brittany; more ancient introgression of A. fallax mitochondrial genomes without detectable introgression of A. fallax nuclear genome) may involve some sort of selection on the mitochondrial genome as shown by individual-based simulations (Bonnet, Leblois, Rousset, & Crochet, 2017). Another argument for the adaptive potential of hybridization in shads is the distribution of gill raker counts that are intermediate and highly variable among the different hybrid classes. This morphological trait is used for species discrimination, but is also an important functional trait that allows species to filter nutritive particles of different sizes depending on their trophic specificity (MacNeill & Brandt, 1990;Palkovacs et al., 2007;Palkovacs, Mandeville, & Post, 2014). In such context, hybrids may be able to explore different trophic niche and be more efficient than parental species in adapting to environmental changes. However, gill raker counts probably only represent the tip of the iceberg regarding the adaptive consequence of phenotypic variation generated by hybridization and introgression especially for ecologically relevant traits.
Because long-term directional introgression created significant gene flow across the species barrier, new gene combinations are exposed to natural selection and opportunity for adaptive introgression linked to local selection is high. This species complex, and in particular populations containing high frequency of same generation hybrids such as in Brittany, would be very suitable to test adaptive introgression using a genome-wide approach. As each of such hybrid contains 87.5% and 12.5% of gene from the two spe- should make European shads a model system to study adaptation by introgression in the near future. However, it will be especially important to obtain genome-wide estimates of shared ancestral polymorphism between A. alosa and A. fallax, to determine the extent to which ancient hybridization events contributed to what is observed and to build an appropriate null model of introgression that explicitly accounts for demographic effects to tear apart neutral from adaptive directional introgression (Currat et al., 2008).

| CON CLUS IONS
In this study, we provided evidence that gene flow between A. alosa and A. fallax is a contemporary and ancient phenomenon, mainly occurring from A. fallax genome toward A. alosa genome and that is part of the natural history of the species complex. Ongoing directional introgression has been found to be linked to recent demographic expansion and habitat perturbation. The significant level of directional introgression may have consequences on the evolutionary trajectory of Alosa species complex in increasing its adaptive potential through gene transfer and thus the resilience of introgressed populations. However, the presence of high rates of hybrids may also point out localized environmental perturbation, such as obstacles to migration, eroding prezygotic reproductive isolation due to reduced spatial segregation of spawning grounds. If such disturbance remain localized, it would not constitute a threat to the evolutionary and ecological integrity (vonHoldt et al., 2018) and the resilience of the species complex as a whole.

ACK N OWLED G EM ENTS
This research was supported by the Agence de l'Eau Adour-Garonne and the Région Nouvelle-Aquitaine. SNP genotyping and mitochondrial genes' sequencing were performed at the Genome