Onychoprion fuscatus. Earliest breeders had significantly different genetic structure from the latest breeders. Limited evidence was found for localised spatial structure, with a small number of individuals being more related to their nearest neighbours than the rest of the population. Therefore, population genetic structure is shaped by heterogeneities in collective movement in time and to a lesser extent space, that result in low levels of spatio-temporal genetic structure and the maintenance of genetic diversity.When and where animals breed can shape the genetic structure and diversity of animal populations. The importance of drivers of genetic diversity is amplified in island populations that tend to have more delineated gene pools compared to continental populations. Studies of relatedness as a function of the spatial distribution of individuals have demonstrated the importance of spatial organisation for individual fitness with outcomes that are conditional on the overall genetic diversity of the population. However, few studies have investigated the impact of breeding timing on genetic structure. We characterise the fine-scale genetic structure of a geographically-isolated population of seabirds. Microsatellite markers provide evidence for largely transient within-breeding season temporal processes and limited spatial processes, affecting genetic structure in an otherwise panmictic population of sooty terns  Although complete panmixia is rare, genetic differentiation is low in species with high dispersal capabilities, such as birds2, flying insects3 and fish4. In contrast, philopatric behaviour, and thus reduced dispersal, promotes inbreeding, which may result in genetic differentiation5. For geographically isolated populations, genetic diversity is often lower than in mainland populations6. For example, mainland populations of Kentish plovers Charadrius alexandrinus have high levels of gene flow and genetic panmixia, whereas those breeding on islands have lower genetic diversity, and genetic differentiation from the mainland populations increases with increasing distance from the mainland7.Population genetic structure is driven by a number of factors, including divergent selection, genetic drift and mutation8. Natal site fidelity over many generations may lead to kin groups9, with benefits of nesting near genetic relatives including reduced aggression and increased predator vigilance10. Fine-scale spatial genetic structure or isolation by distance (i.e. where similarities among genotypes decay with increasing distance11) has been recorded in a number of taxa, including mammals12, birds13, and fish14.In addition to philopatry, genetic diversity may be affected by other factors such as land barriers and separation during the non-breeding season15, whereby temporal barriers impede gene flow16, giving rise to differentiation within a species occupying the same spatial location. For such a scenario to influence a population’s genetic structure, variation in individual breeding timing must have some heritable basis17. In such populations early breeders are more likely to breed with other early breeders as are later breeders with other later breeders. Research has shown that breeding timing often has an additive genetic component (see review by Hendry and Day15), and thus the resulting offspring of such early and late pairings are more likely to breed at the same time as their parents. Therefore, ‘dispersal’ between reproductive times will likely decrease with increasing time , giving rise to higher genetic similarity between individuals breeding at similar times and greater differentiation between those breeding at different times. For example, spawning time in rainbow smelt Osmerus mordax led to genetic differentiation between early and late spawners to the same stream in eastern Canada18. Isolation by time can also operate regardless of spatial location such as in sockeye salmon Oncorhynchus nerka that bred at the same time but at different locations (in streams 20 km apart) that were more genetically similar than those individuals breeding in the same stream but at different times (i.e. 13–15 days apart)19. Temporal genetic impacts on reproduction have been reported in flowering plants17, invertebrates20, fish16 and birds21, including seabirds22. However, temporal effects on genetic structure are not common in the literature, especially in respect to within-breeding season effects23.Isolation over time may also result in heterogeneity in genetic structure24, despite their high dispersal capabilities. In fact, seabird populations show highly variable levels of genetic structure. In a meta-analysis, Friesen, et al.8 found evidence of genetic structure, at various geographical scales, in 40% of 53 species of seabirds. Previous research on seabirds has considered broad-scale variation in population genetics among geographically-isolated nesting colonies25. The usefulness of exploring within-population genetic mixing mechanisms for understanding species’ global gene flow and population dynamics was highlighted by Cristofari, et al.26, who suggested that basic genetic features and processes may be obscured when analyses focus on larger geographic scales. Although their study did not consider temporal effects, they provided evidence for the importance of fine-scale spatial genetic heterogeneity, driven by variations in habitat quality in a panmictic king penguin Aptenodytes patagonicus colony in the Crozet Archipelago in the Southern Indian Ocean.Seabirds are an ideal study system to investigate fine-scale genetic structure given that many species exhibit philopatryOnychoprion fuscatus are one of the most numerous and globally distributed seabirds, occurring throughout the tropical oceans. However, little is known about the genetic structure of their breeding populations . Like many pelagic seabird species28, sooty terns are in decline and a recent urgent call for the reassessment of their conservation status has been made29. Factors such as declining fish stocks, climate change, pollution and introduced predators at breeding grounds30 are thought to have contributed to such declines. We studied the population that breeds on Ascension Island in the South Atlantic which represents 40% of Atlantic sooty terns31; the Ascension population has declined by 84% from > 2 million to ~ 500,000 individuals between 1942 and 2005, respectively29. To investigate genetic structure within the Ascension Island sooty tern population we use microsatellite markers to address three questions: (1) What is the current level of genetic diversity and structure exhibited by the population? (2) Does the population show spatial heterogeneity in genetic structure at different spatial scales? (3) Is there evidence to support within-breeding season temporal genetic differentiation?Sooty terns Given the recent declines in the Ascension Island population of sooty terns, a within-population approach will aid our understanding of local population dynamics, as well as genetic diversity and potential plasticity to environmental change. To the best of our knowledge, this study is the first to combine within-population genetic, spatial and temporal data to understand population genetic structure of a colonial seabird.2 island in the South Atlantic . Sooty terns are small, long-lived colonially nesting seabirds that typically lay one egg per breeding attempt and have long maturation times of approximately 5 years31. During incubation both parents incubate the egg and once hatched the chick is guarded for a few days before being left alone to allow both parents to forage at sea31. Sooty terns are surface feeders having poor waterproofing and rely on larger marine predators, such as tuna Thunnus spp. to drive small fish to the surface where terns feed on them in so-called ‘facilitated foraging’32.The study took place between 25th October 2015 and 26th January 2016 on Ascension, a 97 km33 noted that individuals frequented nesting grounds at night before egg laying commenced, and would largely nest in the same location as these preliminary gatherings. Sooty terns on Ascension Island nest in spatially distinct colony clusters at two main breeding grounds: Mars Bay and Waterside , to give an estimate of population size at each site of: 127,764 ± 16,638 pairs (Mars Bay) and 66,315 ± 4929 pairs (Waterside) .Ashmoleide Fig. . We esti31 which we marked with a number corresponding to that of the nest using a non-toxic permanent marker as a back-up should the nest label have been lost. We captured adult birds using a telescopically handled fish-landing net and ringed them with uniquely numbered British Trust for Ornithology (BTO) metal rings. We also marked birds with a non-toxic permanent marker on one side of the breast (which remained visible at a distance of 5 m for up to four weeks), preventing recapture of the same individuals. We recorded the location of each sampling point using a hand-held GPS unit  accurate to ± 5 m. To increase nest location precision, we manually recorded each nest on a map and measured the triangular distances between each nest in relation to the sampling point. These were later uploaded to ArcGIS 10.235 to obtain each nest’s spatial coordinates. To estimate hatching dates, we visited nests every three to six days. We collected blood samples from 287 birds (including 77 pairs) at 210 nests. Approximately 100 µL of blood was taken by brachial venepuncture of each bird using a 27G needle and syringe, and stored in 1 mL of 70% ethanol. We made ad-hoc recoveries of recently deceased chicks and stored them at − 20 °C (n = 7). We extracted DNA samples from chick brain tissue.To assess spatial heterogeneity in genotypes, we monitored breeding individuals along five 90 m transects placed at random within colony clusters, across both breeding grounds Fig. . TemporaAll experiments were performed in accordance with relevant guidelines and regulations. Blood sampling took place following approval for overseas fieldwork from the local review process of the Animal Welfare Ethical Review Board (AWERB) of the University of Birmingham, UK, and under an Environmental Research Permit issued by the Ascension Island Government (AIG) (ERP-2015-13). The ringing and marking of birds was carried out under a UK BTO ringing licence held by LJHG (permit no. 6316). None of the sampled birds abandoned breeding attempts during the study.36 together with three sex-typing markers38. We used the same protocols as those described by Garrett et al.36 to extract DNA from blood and tissue samples, conduct PCRs, and amplify and genotype samples.Genotyping used 26 highly polymorphic autosomal microsatellite markers39 using unrelated individuals40. We selected non-relatives using the program Friends and Family v2141 with relatedness set to < 0.24 (n = 219), assuming a half-sibling relatedness of 0.25. Relatedness values range from − 1 to + 1, with negative values between two individuals suggesting that they are less related on average than two randomly selected birds from the population; positive values represent pairs that are more related than random pairs41. To correct for multiple tests, we applied a false discovery rate control42 to LD and HWE P-values. We assessed observed and expected heterozygosities using CERVUS v3.0.743, which we used as an indication of population genetic diversity44. All markers tested were found to be in HWE except Ofu06, which displayed an excess of homozygotes. It also displayed a high estimated null allele frequency > 10%  and evidence of Linkage Disequilibrium (LD) in GENEPOP v4.247. We assigned clusters of individuals with similar variation to one of the n populations (K) identified using a burn-in of 100,000 iterations and 500,000 MCMC (Markov chain Monte Carlo) steps with values of K from 1 to 10. Ten independent runs per K were performed to check for consistency across runs. The likelihood of different values of K was then assessed using posterior probabilities. To estimate this variation in the posterior probability, each value of K was assessed through 10 iterations. We also used the Evanno, et al.48 method to calculate the most likely value of K (for K > 1) using Structure Harvester v 0.6.9449.We tested population-level variation in genetic structure using the program STRUCTURE v 2.3.450 using all individuals including chicks (n = 294). Wang’s estimator of relatedness51 had the best correlation coefficient and was used in subsequent analyses . All of the chicks genotyped (n = 7) were correctly identified as offspring of their expected parents except for one which was unrelated to its expected paternal parent, suggesting extra-pair paternity. We only analysed chick DNA to assess relatedness estimators and check the reliability of the markers; they were excluded from all other analyses. We estimated individual inbreeding coefficients using Ritland’s method-of-moments estimator (MME)52 in the R package ‘related’. We visualised observed inbreeding per individual by mapping a graduated colour ramp onto the colony using ArcGIS v10.235.We used pairwise relatedness and individual inbreeding coefficients as measures of fine-scale genetic structure in subsequent analyses. We assessed the performance of four relatedness estimators with the R package ‘related’53 to estimate the pairwise G″ST with the number of permutations and bootstraps set to 999. We used the G statistic, given the commonly used FST can have limitations such as a reduced value when heterozygosity is high54 when using highly variable loci. To look for evidence of genetic isolation by distance, we performed spatial autocorrelation analysis in GENALEX v 6.555 on all adult genotypes together with their spatial location. We used even distances up to 100 m and a separate test for all distances with even sample sizes . We analysed fine-scale genetic spatial structure using the two-dimensional local spatial autocorrelation (2D LSA) statistics from the program GENALEX v 6.559. Given the four nearest individuals are more likely to be an individual’s direct neighbours surrounding a focal nest (LJHG pers. obs.), we set the number of neighbours to four. We inferred local autocorrelation (lr) where P < 0.05 using permutation tests  to calculate significance. We mapped locations of significant lr results onto the colony using ArcGIS v10.2.We conducted heterogeneity tests in relatedness at transect- and sampling point-scales using permutations to detect within- and between-transect and sampling point differences. These permutation tests use the same number of individuals from within, compared to between, sampling points, the latter of which were selected at random using 10,000 simulations to obtain the 60, resulting in four such clusters . We compared genetic differentiation between breeding timing classes using G″ST statistics in GENALEX v6.5 with permutations and bootstraps set to 999. We mapped breeding timing classes onto the colony using ArcGIS v10.2. Inbreeding coefficients of individual birds were linearly regressed against hatching dates that had been Tukey transformed using the R package ‘rcompanion’61. We also compared pairwise relatedness with the absolute difference (in days) between pairwise hatching dates using a Mantel test in GENALEX v6.5.To assess within-breeding season temporal partitioning in genetic structure, we estimated breeding timing using hatching dates calculated as the number of days from the first reported hatching date (i.e. 24th September 2015) (AIGCFD pers. comm.), as laying dates were not always known. We considered evidence for a relationship between genetic structure and breeding timing by assigning timing classes and as a linear relationship. For the former, breeding pairs with known hatching dates (n = 203) were assigned a breeding timing class using a k means clustering algorithm in RK = 1 had the highest log-likelihood, Fig. K > 2 . Nor was there any distance-based structuring from autocorrelation analysis, which detects isolation by distance using an alpha of < 0.01  or between transects (r = − 0.007 ± 0.086) showed no significant difference (P = 0.55). However, individuals located within sampling points were significantly more related to one another (r = − 0.004 ± 0.090) than to individuals at other sampling points . There was some evidence of very fine-scale genetic structuring between a small number of neighbouring individuals, with the 2D LSA analysis yielding 12 individuals that were more related to their four nearest neighbours than those selected at random from the population . These individuals were located within three sampling transects .There was within-breeding season temporal genetic partitioning at the population level. Pairwise comparisons of genetic similarity between breeding timing classes found significant differences only between the earliest and latest timing classes with 34 days between average hatching dates , philopatry usually occurs once breeders have undertaken their first breeding attempts63, with natal dispersal often considerably exceeding adult breeding dispersal64. Pre-breeder dispersal in Leach’s storm-petrels Oceanodroma leucorhoa demonstrates such a mechanism for genetic mixing between subpopulations and the persistence of geographically isolated populations despite increased rates of predation65. Evidence of gene flow into the Ascension Island sooty tern population includes: (i) a retrap of a bird ringed as a juvenile off the coast of Brazil that subsequently bred on Ascension Island66, (ii) a pre-breeding bird originally from Ascension Island recovered in Abidjan in Côte d’Ivoire, West Africa, and (iii) recoveries of juvenile birds from the Dry Tortugas, FL, USA that were made in the Gulf of Guinea off West Africa67. Therefore, it is likely that before breeding for the first time, birds fledged from Ascension Island and the Dry Tortugas may forage together in the Gulf of Guinea. Preliminary genetic analyses on the differentiation between populations also revealed similarities within but not between ocean basins, with differences observed between the Atlantic and Indo-Pacific populations27. The relatively recent global scale population expansion, within the last 100,000 years, is also likely to influence contemporary genetic diversity68. Thus, inter-population dispersal could be a key process that maintains genetic diversity within the Ascension Island population and indeed within the species as a whole.High population genetic diversity, coupled with a lack of genetic differentiation between breeding colonies (i.e. Mars Bay and Waterside), suggests that landscape-scale barriers to gene flow are not operational on the island. There was also no evidence of genetic isolation by distance Fig. a or sex-69. The benefits of nesting near genetic relatives include reduced aggression and increased vigilance against predators10, reduced nest site competition70, infanticide avoidance71 and familiarity with an area with respect to habitat features and risks from predators72.At finer scales there was some limited evidence of genetic structuring. Individuals within sampling points (within 5 m of each other) were more related to one another than those located at other sampling points. Localised spatial structure was also evident in 12 individuals that were more related to their nearest neighbours than to the rest of the population . For example, if group members differ in their previous breeding season success rates, their optimum return times for the following season will also differ, given higher investment in offspring by successfully breeding adults. An extreme example of this is black-browed albatrosses Thalassarche melanophris that breed at South Georgia in the Southern Atlantic Ocean. They delay breeding by up to four years following successful fledging of young, whereas around half of breeders failing during incubation return to breed the following year73. Competition for nest sites was also thought to be a driving factor in the genetic divergence of two breeding populations of band-rumped storm petrels Oceanodroma castro that breed on the same colony in the Galápagos Islands but at different times of the year (6 months apart)74. Timing-related genetic mechanisms show high heritability75 and may arise due to high competition for resources such as space, food and mates23. Indeed, Casagrande et al.76 suggested that the relatively high genetic differentiation between early and late breeding Eurasian kestrels Falco tinnunculus, breeding one month apart in close proximity, may be a result of variable food supply and weather conditions. Differences in population-level genetic structure according to timing of breeding have also been observed in a salmonid fish Oncorhynchus spp. with differences in arrival times of as little as 2 weeks apart15, although, few studies have investigated within-breeding season effects on genetic structure in birds .There was evidence of genetic structuring related to intragroup breeding synchrony with earliest breeders having significantly different genetic structure from the latest breeders  that negatively affected wintering feeding grounds, resulting in delays in breeding timing and selecting against early laying. However, although high genetic diversity and life-history traits such as longevity promote plasticity in the face of a changeable environment, with single-season breeding failures having little effect on population persistence, recurrent breeding failures and low adult survival due to low food availability, for example, will result in population declines83, even with high genetic diversity. Anthropogenic negative impacts on seabirds through overfishing, climate change, introduction of invasive species84, egg harvesting85 and other disturbance at nesting grounds86 increase pressure on such a population that could exceed its adaptive capacity  (Fig. High levels of genetic diversity are thought to enhance species’ recovery after experiencing extreme climatic conditions. For example, Reusch, et al.ors Fig. . In specned Fig. . For exaOur findings provide evidence for genetic temporal partitioning and less support for fine-scale spatial genetic structure in an otherwise panmictic seabird population. Local-scale structure could be dependent on factors such as breeding success, information exchange and competition for resources. Heterogeneity in any potential benefits to individuals from collective group structure in time and, to a lesser extent, space may have led to the observed temporal synchrony and low levels of genetic spatial structure, thereby resulting in the observed high genetic diversity within the population. These processes, together with the potential for juvenile dispersal between populations, may lead to gene flow at a scale that mitigates philopatry and avoids inbreeding, thereby increasing the likelihood of population persistence over time through adaptability to environmental change. Future studies investigating population structure would benefit from assessing within-population processes and the inclusion of spatio-temporal parameters to enhance our understanding of a population’s ecology and evolution, and the maintenance of population genetic structure.Supplementary Information 1.Supplementary Information 2.


The arrival of Neolithic culture in North Africa, especially domestic animals has been essentially documented from archaeological records. As the data relative to sheep are scarce, we studied the genetic relationship between Moroccan sheep breeds and Mediterranean ones using the sequencing of 628 bp of the mitochondrial DNA control region in 193 Moroccan individuals, belonging to six breeds, and 652 sequences from other breeds in Europe and Middle East. Through Network analysis and an original phylogenetically derived method, the connection proportions of each Moroccan breed to foreign ones were estimated, highlighting the strong links between Moroccan and Iberian breeds. The first founders of Moroccan sheep population were issued at 79% from Iberia and 21% from a territory between Middle East and Africa. Their calculated expansion times were respectively 7,100 and 8,600 years B.P. This suggests that Neolithization was introduced by a double influence, from Iberia and from another route, maybe Oriental or Sub-Saharan. The consequence of the environmental changes encountered by founders from Iberia was tested using different neutrality tests. There are significant selection signatures at the level of Moroccan and European breeds settled in elevated altitudes, and an erosion of nucleotide diversity in Moroccan breeds living in arid areas. These changes were the result of cultural exchanges, followed by population movements well documented in Western Mediterranean2. Recent researches conducted by several archaeological teams proposed, with a high accuracy, the different steps of the diffusion of Neolithic, since about 120 sites have been studied and dated in the range of 12,000  to 4,000 years B.P. (late Neolithic)3. Indices of cereal and legume cultivation were attested at around 7,460 to 7,200 years B.P. and of sheep domestication at around 7,460 years B.P., at Kaf-That-el-Ghar in the Mediterranean side of the Tangier Peninsula4. It has been questioned whether the route of the migration was from south to north across the Gibraltar strait or the reverse5. Beside the slight delay relatively to south Spain Neolithic culture recorded at around 7,700 years B.P.1, the common pottery production in either sides of Gibraltar strait argue for the European source of this culture found in Northwest Africa7. The progressive development of agriculture was associated with a local warming deduced from the measurement of δ18Ο on shell margin of Phorcus turbinatus specimens collected and consumed by humans in Ifri Oudadane site8.The introduction of domesticated mammals in Northwest Africa was linked to a change in food production from hunting-gathering to farming9 are controversial, and many of them are not based on convincing evidence. The majority of these authors reported that except the Mountain population that has existed in the country for a very long time, the other local breeds were introduced into the country by Phoenicians, Romans, Arabs or brought from the sub-Saharan regions. The main breeds recognized by the public authorities with a well-defined standard are the Sardi, Timahdite, Boujaad, D’man, Beni-Guil, Blanche de Montagne and the Noire de Siroua, whereas the remaining breeds are composed by non-standardized populations. At the national level, characterization work on sheep breeds has been based on morphological characters10 and blood protein polymorphism11. Recently, a study based on SNPs markers highlighted the differentiation between most of Moroccan and Algerian breeds, with a special focus on inter-population exchanges12. However, the information about the link between Moroccan and European breeds is still lacking. The use of mitochondrial DNA (mtDNA) makes it possible to disentangle the origin, evolutionary history and phylogeny of many populations. So far, five maternal lineages, or haplogroups, have been identified in domestic sheep around the world: haplogroups A of Asian origin, B of European origin, C encountered in South Asia, and D and E, considered as rare, encountered in Eurasia17. According to Tapio et al.16 and Sanna et al.18, the haplogroup B expanded around 6,400 years ago and reached Western Europe before the haplogroup A. As a result, many breeds represent an admixture of two to three haplogroups.The origin of local sheep populations in Morocco is still obscure. The different scenarios reported in the literature19.The aim of this work was to assess the relationships between Moroccan and Mediterranean breeds by using the control region (CR) of the mtDNA in order to reveal the history of sheep settlement in western Mediterranean, including their periods of expansion. Moreover, it was interesting to compare the genetic variability of such characterized breeds to foreign ones, and to highlight the selective pressure that might have affected the Moroccan breeds relatively to Mediterranean ones. Among the different sources of selection, we focused on the climatic constraints, taking advantage of the highly contrasted areas of Morocco described in a previous workA 628 bp-long sequence of the control region of mtDNA were sequenced from 193 Moroccan individuals, belonging to 6 breeds (access numbers in GenBank: MN229085-MN229277). To enrich the database, the use of these sequences as seeds allowed retrieving from NCBI Blastn tool 652 foreign sequences, distributed in 9 Iberian, 7 Italian, and 10 Oriental breeds. There are two non-native breeds: the Lacaune of Italy and the German Merino. The number of individuals per breed, their origin and their access number are indicated in Suppl. et al.20, and displayed in Suppl. The phylogenetic analysis generated a tree encompassing a total of 845 individuals, rooted by individuals of C and E haplogroups according to Meadows The treatment of the topology information at the level 1, i.e. terminal branches, using Newick-Extra produced the table of affinities between breeds Suppl. . 3, and xy and Da between breeds  was different, the AMOVA revealed significant molecular differences among breeds and within breeds for the 3 groups. Regardless of the compared groups, the main source of variation lay within populations, exceeding 96%. More interestingly, it appeared that there were significant differences between all the combinations of groups, except between Iberian and Italian ones. At a closer look, the difference between Moroccan and Iberian breeds was lower than between Moroccan and Italian ones Suppl. . A.a and Dxy genetic distances provided a confusing view. To test specifically the level of compatibility between the Newick-Extra program and the Network methods, we generated 2 matrices that presented the percentages of connections between each Moroccan breed and the other breeds according to their geographical origin . In contrary to the Newick program for which this counting was straightforward, the count of connections from the network needed to be done manually. For each Moroccan breed, the numbers of individuals belonging to Italian, Iberian, Moroccan  or Oriental  were summed  and altitude. In contrast, the variables m and T min seems to vary in the same direction as most genetic variables except Hd. With regard to breeds, the Moroccan ones and the Iberian Spanish Merino and Churra Badana breeds were located towards high temperature and low rainfall variables. Another set of breeds was clearly associated to high rainfall, e.g. Sarda, Sopravissana, Saloia, Latxa Blonde and Black face. There is another set of three breeds  sharing high values of neutrality test variables. The result of correlation indicated that the variables T min and m were positively and significantly associated to π, Fu and Li F* and D*, and to some extent to D of Tajima  are in the range of c. 6,700–7,400 years B.P., while the southern ones are associated to 7,700 years B.P. for the Blanche de Montagne and 8,400 years B.P. for the D’man  or an over-estimation for the dates of the two calibration ancestors. Using the second method based on the dating of early sheep remains in Italy and Iberia, the expansion date of Moroccan group 1 for example at 7,100 years B.P. is close to archaeological records at c. 7,400. As a result, the data provided by this second method were retained.The different approaches conducted in the present study brought complementary information on the origin of Moroccan sheep breeds. If the Cluster Analysis generated by phylogeny and Newick-Extra method were considered, all the Iberian, Italian and Moroccan breeds are clearly separated from Oriental ones. This suggests that the western Mediterranean populations have undergone a long period of evolution during which numerous mutations have been accumulated. Moreover, it appears that the Moroccan sheep breeds are closer to Portuguese than to Spanish ones and even more so the Italian ones. This view is supported by the network results, where Iberian group is situated between the Italian and Moroccan groups. However, there are two Moroccan groups, a majority group 1 representing around 62% of the genetic background and linked to Iberian populations, and a minority group 2 around 17% having relationships with Italian and Oriental breeds. The individuals situated at the base of A and CE haplogroups, related to Oriental breeds, could be assign to the Moroccan group 2 in reason of their link and ancient origin, making this group up to c. 20% of the total genetic background. The question raised is to determine the significance of these 2 groups in the light of archaeological and expansion time evidences. For this purpose, the method based on mutation rate of control region lead to unrealistic dates, an issue previously discussed by Guo 21, although Sánchez et al.22 argued for a reverse way for some aspects. This view was globally supported by the molecular dating and network approach of the present study, since the Moroccan group 1 expanded later than Iberian populations around 7,100 years B.P. This estimation is close to the indices of domestic sheep remains mentioned at around 7,460 years B.P., at Kaf-That-el-Ghar near Tangier Peninsula4. Concerning human populations, a work dealing with Ifri n’Amr or Moussa  and Kelif el Boroud  near the Atlantic coast, showed that the early Neolithic populations were isolated, in the continuity of the upper Paleolithic ones in relation to Berber settlement, while the upper Neolithic individuals were strongly influenced by Iberian populations21.There is a progressive consensus among archaeologists for an Iberian origin of most features of Neolithic culture in Northwest Africa23. The absence of any relationship with Iberian breeds of haplogroup B strongly suggests that these individuals came to Morocco from Middle East via Africa, maybe from a south Italian – Tunisia route, or from an entirely African route. The connection between Italian and Moroccan individuals of group 2 support the 1st idea. In contrast, the existence of several Moroccan individuals at the root of A and C E haplogroup clades argue for the second solution. Moreover, the dating of Moroccan group 2 expansion around 8,600 years B.P., more ancient than for Italian group, seems to favor this second solution. Unfortunately, the archaeological works are useless until now as the earliest sheep remains out of Morocco are dated around 7,000–7,500 years B.P. in Sinai Peninsula24. If our calculations were not overestimated due to calibration bias, we think that new investigations should be conducted in the south of Morocco, and of course in other areas of Africa, in order to detect very early Neolithic sheep remains. This idea does not appear to be unrealistic, since there is a lack of archaeological sites for the period around 8,000 years B.P., especially in the south of Atlas region3.About the origin of Moroccan group 2, the possibility of an ancient settlement of population from a route different from the Gibraltar strait was explored as for goat populationset al.25 established that between 15,000 and 14,000 years B.P. , the Taforalt’s population, in the Mediterranean coast of Morocco, presented affinities with the population of Levant and to sub-Saharan Africans. However, the studies on the genetics of Sahelian sheep are still scarce28.It is interesting to draw a parallel with the genetics of ancient humans. Van de Loosdrecht st century Roman Columella in chapter 729, who indicated that the selection of fine wool sheep was conducted using individuals from Tarento region in South East Italy. Moreover, the 13 Oriental individuals found in the Moroccan group 1 could be due to recent trade events toward Turkey and beyond. Following the successive population movements of the founders on Moroccan territory, the animals have progressively differentiated into local populations and then breeds. As shown previously, the four breeds of the North, between 6,700 and 7,400 years B.P. have expanded later than the two breeds settled on south of Atlas, between 7,700 years B.P. (Blanche de Montagne) and 8,400 years B.P. (D’man).This dating suggests that some D’man ancestors would have reach Morocco via another route than Gibraltar strait, maybe from Africa. However, the origin of this last breed remains still obscure and new data on Sahelian breed could enlighten the situation. The four northern ones from Sardi in the West to Beni-Guil in the East were well genetically differentiated with less than 9% of connection. In contrast, the two southern ones, Blanche de Montagne and D’man had strong and recent links with Sardi and Timahdite breeds. The strong proportion of connections between the D’man and the other breeds were also found from the study based on SNPs over Northwest African sheep breeds12. It can be assumed that the D’man and the Blanche de Montagne, being small sized breeds10, were crossed with the biggest Sardi and Timahdite individuals to increase their meat production. Otherwise, the D’man being well known for its high prolificacy30, was crossed to Sardi, Timahdite and Blanche de Montagne breeds to improve their reproductive characteristics.Besides the early founders of the two Moroccan groups, there were 20 individuals (around 10%) and 14 (around 7%) showing very similar sequences to Italian and Iberian groups, respectively, suggesting late introduction events southward. Conversely, the Moroccan group 1 contained 9 Italian individuals that are interpreted as a late event of importation from Morocco to Italy, maybe at the Roman’s epoch: these individuals had stayed a long time in the Moroccan territory, acquiring their genetic identity. Similarly, the Italian group included all the individuals of Spanish Merinos, a view supported by the text of 13 decreased the nucleotide diversity at the level of Moroccan breeds. At the level of European and Moroccan breeds, this correlation is not true, but the low temperatures (T min and m) induced a selection signature (Fu and Li F* and D* statistics) for the breeds settled in high lands above 650 m A.S.L . The influence of these altitudes on low temperatures had also suggested a decrease of nucleotide diversity.The European genetic component of Moroccan breeds, mostly Iberian, is actually highly dominant compared to the native “Berber” one. Given the climatic contrast between the two areas from each side of Gibraltar strait, it was expected that the drought and the high temperatures observed in Morocco would induce a selection pressure on the sheep flocks. Effectively, the shortage of rainfall and low vegetal productivity expressed by QMuch effort has been made in the recent years to document the transition between the Paleolithic and Neolithic cultures in Morocco. The results presented here evidenced two successive waves of domestic sheep arrival on the Moroccan territory, corresponding to this transition: the most ancient one could be associated to the first Berbers around 8,600 years B.P., still representing 20% for the Moroccan genetic background, maybe as a response to the aridification of Northern Sahara at the end of the “African humid period”. The second one was the most important with 62% of this same background, and reached Morocco around 7100 years B.P. from Iberia. Two further independent importations of individuals from Iberia and Italy were recorded, less or equal to 10% of the genetic diversity each, but hardly datable due to their weak numbers. Archaeologists have shown an increase in exchanges with Iberia since 7,500 years B.P., in good agreement with the dating conducted on the expansion of most Moroccan breeds. However, for the special case of the D’man breed, the calculated expansion exceeded the period of human movements between Morocco and European continent, suggesting non-European route for the introduction of this breed ancestors. The human genetic researches argued for a continuity of local populations from broadly 15,000 to 7,000 years B.P., more or less related to Middle East, South and East African ones, and a sudden change of population since 5,000 years B.P., corresponding to a transition from hunting-gathering to agricultural practices.Blood sampling was performed by veterinarians of the ANOC  during routine medical care or follow up, such that no ethical authorization was required. Breeders and ANOC consented to the blood samplings and their utilization for scientific purpose.A total of 193 blood samples were taken from sheep belonging to six Moroccan sheep breeds: Sardi (n = 33), Timahdite (n = 37), Boujaad (n = 31), Beni-Guil (n = 31), D’man (n = 29), and Blanche de Montagne (n = 32). The blood samples were taken by an authorized person. The individuals were collected from farm breeders in the breeding areas of different breeds and supervised by the “Association Nationale Ovine et Caprine” ANOC, in order to ensure that they correspond to native breeds without introgression with foreign commercial or other local breeds.31. Details about PCR amplification and sequencing are reported in Suppl. Specimens were chosen according to their genealogy so that they are unrelated. Blood samples were taken from the jugular vein and collected in ethylene diamine tetra acetic (EDTA) acid tubes and frozen at −20 °C until extraction. Total genomic DNA was extracted from blood samples using an alkaline lysis methodWe built a database of control region (CR) sequences including 193 from Moroccan and 652 from non-Moroccan breeds. This database was constructed by exploring the NCBI gene bank using BLASTn algorithm. Taking each Moroccan sequence as seed, we consistently selected at least the top six ranks per blasted sequence. Then, we looked at the following ranks to possibly retain those who had the same score as the sixth. Among all the breeds obtained, we retained those that showed more than three individuals, belonging to Mediterranean, African, and Near East regions. Then, we expanded the database by including 25 sequences or more per breed if available. The sequences selected were then recorded with their access number and the reference to their breed , the haplotype diversity (Hd), the nucleotide diversity (π)34, the number of segregation sites (S), the number of singletons (Sg) and the number of parsimoniously informative sites (P). In addition, four neutrality tests were performed: Fu and Li F*, Fu and Li D*35, Fu Fs36 and D of Tajima37 using DNAsp for each Moroccan, Iberian and Italian breed. The number of segregation sites (S), the singletons (Sg) as well as the parsimoniously informative sites (P) were calculated for each breed by the “Polymorphic sites” option, while the remaining diversity indices were calculated by the “DNA Polymorphism” option. To calculate these previous parameters for each of the four groups within haplogroup B, the sequence numbers of Iberian and Italian ones were adjusted by a random method to the number recorded in Moroccan group 1, i.e. 119, to avoid a sampling size effect. The haplogroup frequencies were deduced from the phylogenetic analysis, and the haplogroup diversity from Shannon index calculated using PAST software v.2.9738.In order to determine the genetic variation within populations, several diversity parameters were calculated with the DNAsp v5 programa (the corrected average pair-wise differences) and Dxy (the average number of pair-wise differences)34 were calculated by DNAsp v533. The obtained results were highlighted with the help of “Non-Metric Multidimensional Scaling Plot” and Cluster Analysis with User distance using PAST software v.2.9738. Connectivities as well as evolutionary relationships between different individuals were also analyzed through the construction of a phylogenetic tree by the Neighbor-Joining method, using the NETWORK software v.5.0.0.1 (available on the website: http://www.fluxus-engineering.com/). The Median-Joining Network39 is an haplotype network of all individuals. To reduce the large data set, the “Star Contraction” option was used. In order to simplify the graphical representation of the constructed network, the optional “MP calculation” procedure according to Neighbor-Joining method was used to remove unnecessary vectors and median links and to avoid reticulations. In addition, the length of the branches of some individuals were also modified manually, when necessary, to improve the visualization of results.To estimate the divergence between populations (breeds), the genetic distances D32. The best model according to the lowest Bayesian Information Criterion corresponded to Hasegawa-Kishino-Yano model40, including Gamma distribution (5 discrete categories) and Invariant sites (G + I). The gap treatment was done by eliminating segments that had less than 95% presence . The robustness of branches was tested using 500 bootstrap replicates. The tree was redrawn using Seaview v.441.A phylogenetic analysis was carried out in order to reveal the affinities between Moroccan breeds and themselves and also to other breeds in the world. The phylogenetic tree was constructed using the Maximum Likelihood (ML) method implemented in MEGA software v.10.0As expected, the phylogenetic tree produced several sets of individuals corresponding to each haplogroup, preventing to evidence the relationships between breeds. The use of the conventional genetic distance indices produced the same effects. To overcome this problem, we developed a new phylogeny-derived method called Newick-Extra Suppl. .38.Relationships between the studied breeds (asymmetric matrix generated by Newick-Extra) were visualized through the Non-Metric Dimensional Scaling Plot (NMDS) and Cluster analysis using Spearman’s Rho as distance metrics, using PAST software v.2.9742. Sources of variation were considered in a single model: among groups, among breeds – within groups – and within breeds. Mismatch distribution was used for each European and Moroccan breed in order to address eventual demographic expansion events, by computing the τ parameter. This last one has shown to be useful to date historical expansion of human44, wild45, and domestic animals, e.g. goats46. To test the validity of an expansion hypothesis, the observed mismatch distribution was compared to the simulation values generated by coalescent algorithm using the probability associated to the sum of square deviation (SSD)44. In order to calculate the expansion time of breeds, two strategies were conducted. The first one used the formula τ = 2μt, where μ is the mutation rate for the whole considered segment and t is time in generations47. To calculate the mutation rate (μ) of the 628 pb segment, a data set was built, including two individuals of Ovis aries, O. ammon, O. vignei, per haplogroup and one for O. orientalis, O. dalli and O. nivicola. nivicola  was computed to test significant differences between geographical breed groups, using ARLEQUIN v.3.519 was extended to the Moroccan Blanche de Montagne, Iberian and Italian studied breeds. The Oriental animals were not considered because they were distantly related to Moroccan breeds. Briefly, the geographic perimeter of the breeding area of a given breed was obtained through bibliographic data: anoc.ma, agraria.org, sprega.com and confelac.org for Moroccan, Italian, Portuguese and Spanish breeds, respectively. Concerning the Spanish Merino breed, information was taken from Lancioni et al.51. The following climatic parameters of at least 10 localities distributed in the breeding area were recorded from climate-data.org: Alti: altitude (m), m: minima mean of the coldest month (°C), M: maxima mean of the hottest month (°C), R: annual rainfall (mm), T max: annual maxima mean (°C), T min: annual minima mean (°C) for the period 1984 to 2014, and Q3 = 52. For each breed, the means of these parameters recorded for the different localities were calculated.The aim was to define the optimal climatic conditions for the breeds in order to detect eventual sources of natural selection on genetic variations. The methods developed by Petit and Boujenane53 was considered with three modalities . The non-autochthon breeds (German Merino and Lacaune) were removed from the lists of Iberian and Italian breeds, respectively.To test whether the regions showed significant differences in the climatic parameters, a one-way ANOVA conducted using SYSTAT vers. 1238. Prior to the calculations, non-normal distributions were transformed . However, D of Tajima and the Shannon index were non-transformable.The relationships between genetic and climatic data were studied using principal component analysis and correlation analysis implemented in PAST software v.2.97Supplementary information.


Aristeus antennatus in the seven most important fishing grounds of the Western Mediterranean Sea, were assessed using twelve microsatellite loci during 2 consecutive years (2016 and 2017), in a total of 1403 adult individuals. A high level of geographical connectivity among groups was observed in the two studied years. In fact, no significant geographical differentiation was found in 2016 , whereas it was indicated in 2017 . This small divergence in 2017 was not attributed to the distance among locations nor to the effect of the Ibiza Channel. Significant allele frequency changes were found at local level between the 2 years , mainly due to Blanes’ fishing ground. Larval dispersal from the North to the South through the main superficial current supports the high level of connectivity pattern found. The temporal genetic instability detected in the Blanes’ fishing ground could be explained by oceanographic temporary features. Our findings evidence only one biological unit in the study region and establish the baseline for an inter-federal management plan of A. antennatus.Temporal variability of the genetic structure and connectivity patterns of the blue and red shrimp  Aristeus antennatus  has a wide distribution in the Western basin of the Mediterranean Sea1, where it is the most eurybathic species, with a depth range from 80 to nearly 3000 m and a peak of abundance between 400 and 800 m3. Aristeus antennatus mainly inhabits the muddy bottoms of the upper and middle slope where it is commercially exploited, but it is found also at the lower slope, non-exploited by fishery4. This species has temporal movements between the open slope and the margins of submarine canyons4. Mature males and females form mating aggregations on the middle slope during the reproductive period4, which takes place in late spring and summer5. On the other hand, even though pelagic larval duration is still unknown in A. antennatus, all larval stages found in a few number of studies were in the upper layers of the water column [6 and references therein].The blue and red shrimp 8 in the Catalan-Levantine coast, the Balearic Islands and Alborán Sea , being considered as one of the most important marine resources9 included in the priority species list for action by General Fisheries Commission for the Mediterranean Sea10. Nevertheless, the lack of an integral management plan for the species along the North-western Mediterranean coast11 has implied that the management of A. antennatus has been applied locally13 without taking into account the genetic diversity and connectivity among populations and without identifying the conservation units, which should be required for a sustainable management of the species14.In the Western Mediterranean coast, the species has been intensively exploited by trawling fishery between depths of 400–800 mA. antennatus in the Mediterranean Sea, but they pointed out the Western Mediterranean Sea as a single genetic unit17. Thus, in a fine-scale study, where the differentiation could be relatively small, more highly polymorphic molecular markers are required, such as microsatellites18. Although a set of eight microsatellite loci failed to establish any population structure in the blue and red shrimp in the Central Mediterranean Sea19, microsatellites have successfully been used to identify genetic stock in other marine penaeoid species, such as Penaeus monodon20 and Farfantepenaeus notialis21. The presence of oceanographic discontinuities has been identified as one of the main factors determining the genetic differentiation among populations in marine species, but dispersal by oceanic currents promotes connectivity among them24. Along the Mediterranean coast of the Iberian Peninsula, the main surface current is the Northern Current, which flows south-westward to at least the Ibiza Channel25, where a part of the flow crosses this channel towards the Alborán Sea, whereas most recirculates north-eastward to the Balearic Islands, forming the Balearic current26. The Ibiza Channel  have been used to identify population substructure of A. antennatus at regional scale have been reported30. It is particularly interesting to determine whether these changes are related to spatial and temporal variation on the genetic structure of A. antennatus populations, because a federal interest of integral management has been recently stated31 following the Food and Agriculture Organization recommendations32.Temporal changes in the distribution along the water column, in the diet and the food consumption rates, in the abundance and fishery landings of A. antennatus among the most important fishing grounds in the North-western Mediterranean Sea, from the Gulf of Lions (France) to Cabo de Palos (Spain), using twelve microsatellite loci. We also analysed the influence of the Ibiza Channel as oceanographic barrier to the dispersion of individuals.The main aim of this study was to describe the temporal stability of the genetic structure and connectivity patterns of After the quality filtering, where individuals with less than eight genotyped loci were eliminated, 1386 out of 1403 individuals were used for further analysis Table .Table 2GNA) ranged from 2  to 23 (locus Aa138 in BLA16 and locus Aa681 in VIG17) (Supplementary Table AR) ranged from 8.5 (ROS16) to 9.0 (SPO16) in 2016, and from 8.8 (DEN17) to 9.3 (PDS17 and PAL17) in 2017  ranged from 0.440 (VIG16) to 0.486 (PAL16) among samples collected in 2016, and from 0.431 (DEN17) to 0.498 (VIG17) among those in 2017. The expected heterozygosity (HE) was higher than HO and ranged from 0.629 (DEN16) to 0.639 (PAL16 and SPO16) in 2016, and from 0.620 (VIG17) to 0.643 (PAL17) in 2017 (Table Aa1255 and Aa1061 in PAL16 after Bonferroni correction (p < 0.0001) but not in other locations. Therefore, all loci were included in further analyses.Observed heterozygosity  occurred at 10 out of 12 loci at least in one sample, related to deficiency of heterozygote individuals  nor among the 2017 ones , indicating no signal of isolation by distance. For the two temporal collections, migration rates among samples suggested a high level of connectivity, but not related to the geographical proximity among locations  . Thus, no oceanographic groupings were tested further for 2016 collection. Otherwise, in the 2017 collection a small but significant divergence was detected among samples . Nevertheless, hierarchical analyses did not give support to the Ibiza Channel as a barrier to A. antennatus dispersal . However, at the local level, significant differentiation between 2016 and 2017 was restricted to the Blanes’ fishing ground   were in accordance with recent studies using the same set of microsatellite loci . The mean number of alleles per locus and sample , probably due to the sample size in our study almost doubling the one in those previous studies. Higher diversity levels were observed in Italian and Algerian A. antennatus samples using eight distinct loci . Nevertheless, our results were similar to those reported in other shrimp species, such as Acanthephyra pelagica from the Atlantic coast of Canada , the deep-sea shrimp Rimicaris hybisae from the Caribbean Sea  and Melicertus kerathurus from the Mediterranean Sea and the North-eastern Atlantic Ocean .Our mean observed and expected heterozygosities  and microsatellites diversity19. In a recent study using the same set of microsatellite loci, genetic differentiation between the Alborán Sea and the remainder Western Mediterranean Basin was detected34. However, their results did not point out any significant differentiation among the only three samples of the Spanish Mediterranean coast, whereas our results indicated some degree of differentiation in the 2017 temporal collection , where each modal size class is related to an age class45. Our samples are mainly composed of 2-year-old individuals as showed by their mean CL  and Cap de Creus canyon (Roses) where water flow has cyclonic vorticity, in Blanes canyon an anticyclonic eddy has been detected, which can cause a northward direction against the main southward water circulation50. Indeed, Clavel-Henry et al.48 applying different biophysical models in order to estimate potential connections between North-western Mediterranean fishing grounds of A. antennatus, confirmed that singular features in submarine canyons could have an important role in the species dispersal. For instance, in the open slope at the south of Blanes canyon a northward circulation of particles was detected due to the anticyclonic eddy in Blanes area48. Another explanation could be the cascading event, an intense dense shelf water mass formed in the Gulf of Lions and the subsequent downslope cascade, which occurs periodically during dry and cold winters30. Cascading events have been related to an increase in A. antennatus recruitment inside the North-western Mediterranean submarine canyons in the following years after a cascading event51, including Blanes canyon52. Nevertheless, this could be ruled out not only because the last cascading event (2012–2013) was some years before our collections54, but also because it did not have a significant effect in the Blanes canyon55. Additionally, the anthropogenic effects on submarine canyons have been deeply discussed. The bottom trawling activities have a direct effect on the ecological dynamics of the submarine canyons, causing physical disturbances which are variable over time and changes in density and composition of different meiofaunal species, as noticed in Blanes canyon55.Special attention should be given to the role of submarine canyons, taking into account the fact that they are considered to serve as nursery, refuge and recruitment grounds for A. antennatus from the Gulf of Lions (France) to Cabo de Palos (Spain). From a fisheries perspective, we advise considering A. antennatus in the North-western Mediterranean coast as one biological unit for stock assessment, and promoting coordinated fishery management plans. Taking the genetic information into account for future management advice would be of significant help to design an effective inter-federal management policy, and therefore a long term sustainability of the fishery resource would be ensured22.In conclusion, despite the temporal differentiation found in Blanes’ fishing ground, in the present study we identified a high level of genetic connectivity of A. antennatus fisheries information and the interest of the Spanish Government to start an integrated management plan31. A total of 1403 adults were collected at seven Mediterranean locations (fishing grounds): Port de la Selva, Roses, Palamós, Blanes, Vilanova i la Geltrú, Dénia, and Santa Pola (Table 56.Sampling was coordinated by the CONECTA-GEN project Roldán 2014). Locations were chosen because of the large quantity of historical 4. LocatiAa138, Aa1255, Aa956, Aa496b, Aa123, Aa681, Aa667, Aa1444, Aa751, Aa818, Aa1061, and Aa119557. Microsatellite loci were amplified using approximately 30 ng of template DNA in 10 μl final volume, following the methodology described in Planella et al.33. PCR products were analysed in an ABI PRISM 3730xl automatic sequencer  at the sequencing unit of the University of Santiago de Compostela (Campus Lugo). Allele scoring was performed with GeneMapper 4.0 software , using the LIZ 500 (Applied Biosystems) as size standard.A set of twelve polymorphic microsatellite loci split into three multiplex PCRs and one singleplex PCR were used: NA) and the allelic richness (AR) calculated using FSTAT v. 2.9.358, and the average among loci of the observed and expected heterozygosity  using Genepop v. 4.659. Deviations of observed genotype proportions from expected under HWE were summarized using the inbreeding coefficient FIS60 and its significance was statistically checked using the exact probability test of Guo and Thompson61 implemented in Genepop v. 4.659. Genotypic linkage disequilibrium between all pairs of loci was tested with Genepop v. 4.659, using a Fisher’s exact test with 10,000 dememorizations and 5000 iterations per batch. The Bonferroni correction was used to adjust significance levels for multiple simultaneous comparisons. The presence of null alleles was assessed with the software Micro-Checker v. 2.2.362 by performing 1000 randomizations.Genetic diversity within each sample was described by the mean number of alleles per locus (FST) were calculated using the software Arlequin v. 3.563 with 10,000 permutations to test significance. We applied the Bonferroni correction for multiple comparisons. FST values corrected for the presence of null alleles and its significance were estimated using the FreeNA software64. Hierarchical analyses of molecular variance (AMOVA65) as implemented in Arlequin v.3.563 were performed to assess oceanographic and temporal divergence among samples. The oceanographic grouping considers the Ibiza Channel as an oceanographic barrier isolating fishing areas located northward and southward  where each run consisted of a burn-in period length of 50,000 iterations followed by 200,000 Markov chain Monte Carlo iterations. The most likely number of K was selected according to the Evanno’s method67 implemented in STRUCTURE HARVESTER68.Pairwise genetic differentiation between samples  and 1000 replicates. We explored a range of different values for FST by varying the number of generation of drift (t). The percentage of significant outcomes was interpreted as the power of the tests for detecting the defined level of genetic divergence. The effective population size (Ne) was estimated for each sample using the bias-corrected version of the method based on linkage disequilibrium71 as implemented in NeEstimator v. 2.172. Ten out of the fourteen samples had an infinite Ne, so the selected Ne to assess the statistical power of our loci to detect genetic differentiation was 10,000.The software POWSIM v. 4.1FST/(1 − FST)73. The geographical distances were measured as the shortest coastline between locations in kilometres. The statistical significance was assessed by Mantel tests with 10,000 permutations using the software NTSYSpc v. 2.174.Isolation by distance (IBD) among fishing grounds was assessed from the correlation between genetic and geographical distance matrices among studied locations. Pairwise genetic differentiation was linearized as 75, the relative direction and magnitude of migration between samples for each temporal collection were summarized as a network from the estimated effective number of migrants (Nm).Using divMigrate-onlineSupplementary Information.


Salvelinus fontinalis). In Nova Scotia, Canada, hatchery brook trout have been stocked since the early 1900s, and despite continued stocking efforts, populations have suffered declines in recent decades. Before this study, the genetic structure of brook trout populations in the province was unknown; however, given the potential negative consequences associated with hatchery stocking, it is possible that hatchery programs have adversely affected the genetic integrity of wild populations. To assess the influence of hatchery supplementation on wild populations, we genotyped wild brook trout from 12 river systems and hatchery brook trout from two major hatcheries using 100 microsatellite loci. Genetic analyses of wild trout revealed extensive population genetic structure among and within river systems and significant isolation‐by‐distance. Hatchery stocks were genetically distinct from wild populations, and most populations showed limited to no evidence of hatchery introgression (<5% hatchery ancestry). Only a single location had a substantial number of hatchery‐derived trout and was located in the only river where a local strain is used for supplementation. The amount of hatchery stocking within a watershed did not influence the level of hatchery introgression. Neutral genetic structure of wild populations was influenced by geography with some influence of climate and stocking indices. Overall, our study suggests that long‐term stocking has not significantly affected the genetic integrity of wild trout populations, highlighting the variable outcomes of stocking and the need to evaluate the consequences on a case‐by‐case basis.Many populations of freshwater fishes are threatened with losses, and increasingly, the release of hatchery individuals is one strategy being implemented to support wild populations. However, stocking of hatchery individuals may pose long‐term threats to wild populations, particularly if genetic interactions occur between wild and hatchery individuals. One highly prized sport fish that has been heavily stocked throughout its range is the brook trout ( The thirty‐three legacy loci included 5 loci from brook trout S. fontinalis . Loci were also examined for missing data; however, all loci were genotyped in >70% of individuals, and thus, no loci were removed from the analyses. After filtering, a total of 1,520 wild trout and 209 hatchery trout were used for genetic analyses in our study. For wild brook trout sites, all loci had 2 or more alleles with a mean of 7.66 alleles per locus . In addition, across all wild sites and loci comparisons , only 1% of comparisons showed significant deviations  from Hardy–Weinberg equilibrium (HWE) using the R package diveRsity , observed and expected heterozygosity (Ho and He), and effective population size (Ne). Mean estimates of inbreeding and heterozygosity were calculated with 95% confidence intervals using the R package diveRsity  of 0.01, and estimates were calculated with corresponding confidence intervals adjusted using the jackknife method  in Arlequin v3.5.2.2  using Google Earth and these values were added to distance estimates. The relationship between genetic and geographic distance was examined using mantel.randtest function with 1,000 permutations in the R package ade4  was performed using the R package Jombart, . PCoA waFST) was calculated between all sites with corresponding significance . To determine significant genetic divergence between sites, we used an adjusted (Bonferroni) alpha level of 0.0014 to account for multiple comparisons among the 35 sampling locations.For all subsequent analyses, hatchery samples were included in the analyses to compare genetic differences between hatchery and wild trout. Using Arlequin v3.5.2.2  estimates to assess best K . To generate these two simulated hatchery populations, we used freqbasedsim_AlleleSample function in the R package parallelstructure , CLUMPP  across runs for each wild site. In cases where two hatchery sources were used, the mean membership proportions to each hatchery source were summed to determine total hatchery membership. Proportion of hatchery membership for each site was considered a measure of hatchery introgression for the site.Using simulated and centered datasets for both the Fraser's Mills and Margaree hatcheries, we next used two approaches to investigate evidence of introgression. First, STRUCTURE v2.3.4 . Records begin in 1976, and stocking information includes number and size of stocked fish, location, date, stocking objective , and hatchery/strain origin. We compiled brook trout stocking data for all water bodies within the 12 systems sampled in our study. For each river system, we calculated the total number of fish stocked, number of stocking events, mean number and weight (grams) of fish stocked per event, number of fish stocked in fall and spring programs, proportion of adults stocked, and the first and last year in records  on a given date, and LCD is the least‐cost distance between the sampling site R and the stocking site i,dS. For each site, LCD was calculated based on the straight‐line distance (kilometers) between the sampling site and the nearest stocking site within that river. Following this equation, total propagule pressure is based on the sum of propagule pressure from all stocking events.Detailed hatchery stocking records were obtained from the province of Nova Scotia (source: 2) around each site. Human population density  was used as a proxy for habitat disturbance  around each site to capture human activity in the surrounding region.In addition to stocking data, we also calculated the average human density (persons/km2.5.2http://worldclim.org/version2) at a spatial resolution of 2.5 arc minutes  was performed using the R package 2.5.4Q‐value from STRUCTURE analysis) for each site and was log‐transformed and used as the response in the model.In addition to determining how environmental and anthropogenic variables correlate with genetic structure, we also used multiple regression to investigate whether factors such as environmental and anthropogenic variables (same as above) were associated with the level of hatchery introgression. The environmental variables used in the model included principal components of bioclimatic variables  as well as elevation. Anthropogenic variables included stocking principal components (stocking PCs 1–2) and human population density. The full model included these seven variables, and variance inflation factors were all below 5 , suggesting generally low collinearity among variables. The level of introgression was determined by the proportion of hatchery membership  were generally higher for sites in the eastern part of the province (Cape Breton Island), where Angus Farquhars Brook (Ang) in Baddeck River had the highest expected heterozygosity estimates (He = 0.40), but the majority of sites in the province  did not differ significantly in heterozygosity from Angus Farquhars Brook. Only one site had significantly lower observed heterozygosity relative to all other sites, and this was Greens Brook (GR) in Saint Mary's River .Estimates of genetic diversity in wild trout populations are presented in Table FIS) suggested no significant deviations from random mating in the majority of sites. Only two sites showed significant evidence of inbreeding based on bootstrapped 95% confidence intervals, and this included the Upper Medway (UM) system and Cooks Brook (CO) in the LaHave system  was also generally higher in this part of the province. Harris Brook (HA) in the Baddeck River had the largest positive Ne , although two other sites had negative Ne values, which can be indicative of large population sizes . The site with the smallest Ne was Glencoe Brook (GL) in East River Pictou (Ne = 10.1 [9.2–11.1]).In addition to higher heterozygosity estimates in the eastern part of the province, effective population size  separated individuals from sites in the eastern and western part of the province along the first PCo axis Figure a. IntermUsing both hatchery and wild samples in the PCoA Figure b, hatcheFST) between sites were significant for the majority of pairwise comparisons, including sites within the same river system , sites in River Denys , and sites in Baddeck . All sites were significantly divergent from sites in other river systems except for one site in Baddeck , which was not significantly divergent from all sites in the River Denys systems (FST < −0.007). Low genetic divergence between River Denys and Baddeck sites could indicate migratory anadromous trout, as some of these locations are in lower reaches of the systems. Both hatchery samples (Fraser's Mills and Margaree) were significantly genetically divergent from most wild brook trout sampling sites, with one exception. The Margaree hatchery was not genetically divergent from samples that had evidence of fin erosion  in Lake O'Law Brook in Margaree River (FST = 0.007). Samples without fin erosion from the same location were genetically divergent from the Margaree hatchery (FST = 0.01). Fraser's Mills was significantly divergent from all wild sites. Although Fraser's Mills hatchery strains were combined for these analyses, genetic divergence (FST) between the hatchery strains and sample sizes are provided in Table Estimates of genetic divergence , whereas samples without fin erosion showed moderate levels of hatchery membership . Following Harbicht et al.  or less than 10% , respectively. For Lake O'Law samples with fin erosion, 52% (n = 16/31) were pure hatchery trout and only two trout were pure wild origin. Conversely, for samples without fin erosion, 69% (n = 38/55) were pure wild trout and only three trout were pure hatchery. Overall, aside from Lake O'Law, hatchery introgression was low in wild populations.To assess introgression, STRUCTURE runs were performed within each river system with centered hatchery populations see . Evidenct et al. , we consi.e., no F1s) or pure hatchery trout were found at wild sites. While not all sites could be examined using NEWHYBRIDS given reduced discriminatory power of loci in some locations, we found that sites were primarily (>75%) comprised of pure wild trout with evidence of potential F2 hybrids and backcross‐wild individuals at some sites  on the first RDA axis and stocking  on the second RDA axis related to genetic structure of brook trout populations . The stocking variable contributing to stocking PC2 is the proportion of adults stocked in a system, and this variable showed a positive relationship with genetic population structure associated with PCo axis 2 that separated populations along RDA2 , bioclimatic and stocking data were collapsed into principal components (PCs) as many showed high correlations with each other , stocking PC2 (p = .001), and bioclimatic PC2 (p = .001) on genetic structure of brook trout  and stocking PC2 (p = .004) on brook trout genetic structure . The significant variables (bioclimatic PC2 and stocking PC2) tended to be associated with population structure along the second axis (PCo axis 2). Consistent with the first RDA , stocking PC2  showed a positive relationship with genetic population structure (PCo axis 2) when GR was removed  in our analyses was divergent from all other sites, and thus, we performed the same analyses as above with this site excluded in case this one site skewed our analyses. Without Greens Brook (GR), dbRDA revealed a significant effect of geography (hydrologic distance from the westernmost site) . Nonetheless, we note that elevation approached statistical significance in the model . However, given that sites in Lake O'Law had much higher levels of introgression relative to all other sites, we removed Lake O'Law and reran the analysis. Re‐analysis again revealed that no variables significantly explained levels of hatchery introgression in wild brook trout populations .Multiple regression analysis revealed that no anthropogenic or environmental variables significantly explained levels of hatchery introgression in wild brook trout populations  may thus explain why rates of introgression and hatchery survival are higher as the locally derived strain and wild sourced individuals may be better adapted to the environment compared to non‐native strains. In addition, at the Lake O'Law site, samples were collected in 2017, and recent stocking efforts in 2016 in the Margaree system  may have contributed to the large number of hatchery fish collected in Lake O'Law Brook. However, a nearby upstream site, Portree Brook, contained only a single hatchery trout and over 81% pure wild trout despite comparable proximity to stocking sites. Portree Brook was sampled in 2018; thus, it is possible that higher numbers of hatchery trout may have been found at this location in the previous year, dispersal of hatchery trout is limited to certain regions of the watershed, or this location is less accessible to stocking.In animal captive‐breeding programs, the goal of many programs is to reduce the amount of generations in captivity , and all sites where Fraser's Mills were stocked had low levels of introgression. The exact genetic origin of Fraser's Mills trout is unknown, but several strains at the hatchery appear to be genetically distinct from each other . High levels of genetic structure are not unexpected, as brook trout ranks among the most highly structured animal species  greater than 50, and only one site may be of particular concern given its low Ne estimate. Ne for Glencoe Brook (GL in East River Pictou) was 10.1 (95% CI: 9.2–11.1), and Ne estimates below 50 individuals may be a concern because, in small populations, the effects of genetic drift will outweigh the influence of selection making it difficult for such populations to respond to environmental change  generally showed higher (but not significant) levels of diversity. Some of these locations may harbor migratory anadromous trout, which may contribute to increased genetic diversity in the region (Ferchaud et al., In conclusion, our study found that hatchery introgression is limited in Nova Scotia brook trout, with the exception of one location where a local strain is used for hatchery stocking. We acknowledge that the limited hatchery introgression found here made it difficult to address one of our goals of identifying environmental and anthropogenic variables that influence levels of hatchery introgression. While other studies have found evidence of hatchery introgression in brook trout populations, our results are comparable to other studies conducted in riverine systems where limited introgression is often found (Jones et al., We have no conflict of interest to report. Click here for additional data file.