Lates niloticus, is an iconic fish in Africa and is of high commercial importance, both in the species’ native range and where it has been translocated. However, the species is in decline and there is a need to understand its population genetic structure to facilitate sustainable management of the fishery and aquaculture development.Geological evolution of the African continent has been subject to complex processes including uplift, volcanism, desert formation and tectonic rifting. This complex geology has created substantial biogeographical barriers, and coupled with anthropogenic introductions of freshwater fishes, has influenced the genetic diversity, connectivity and sub-structuring of the teleost fauna. Nile perch, Nile perch tissue samples were acquired from two West and four East  African locations. Nineteen polymorphic microsatellite loci were used to study the genetic variation among populations across regions (West and East Africa), as well as between native and introduced environments within East Africa.FCT = 0.26, P = 0.000). STRUCTURE analysis at a broad scale revealed K = 2 clusters, the West African individuals were assigned to one cluster, while all individuals from the East African region, regardless of whether native or introduced, were assigned to another cluster. The distinct genetic clusters identified in the current study between the West and East African Nile perch, appear to have been maintained by presence of biogeographic barriers and restricted gene flow between the two regions. Therefore, any translocations of Nile perch should be carefully considered across the regions of West and East Africa. Further analysis at a regional scale revealed further structuring of up to K = 3 genetic clusters in East African Nile perch. Significantly (P < 0.05) lower genetic diversity based on analysis of allelic richness (AR) was obtained for the two translocated populations of Lake Kyoga (AR= 3.61) and Lake Victoria (AR = 3.52), compared to Nile perch populations from their putative origins of Lakes Albert (AR = 4.12) and Turkana (AR = 4.43). The lower genetic diversity in the translocated populations may be an indication of previous bottlenecks and may also indicate a difficulty for these populations to persist and adapt to climatic changes and anthropogenic pressures that are currently present in the East African region.Results revealed strong and significant genetic structuring among populations across the sampled distribution (divergence across regions,  Advances in the knowledge-base of African freshwater fisheries have been made over the last five decades –3. Like Lates comprises fish from 11 species which occur in both brackish and freshwater, and are widely distributed across Africa, Asia and Australia [Lates niloticus, the Nile Perch, is a freshwater species endemic to African rivers and lakes including the Nile, Chad, Senegal, Niger and Congo River basins. This species also naturally occurs in the East African Rift (EAR) Lakes of Tanganyika, Albert and Turkana. However, more recently the range of Nile perch was expanded through anthropogenic introductions about 50–60 years ago by British colonialists, to include Lakes Nabugabo, Kyoga and Victoria [The genus ustralia . Lates nVictoria , 15–17.Stocking reports for Nile perch are contradictory, but generally indicate that the species was introduced into Lakes Victoria and Kyoga in the 1950s and 1960s , where the species has now become established , 16–21. L. calcarifer, showed at least two genetic subpopulations of Nile perch [While anecdotal observations and official records provide a historical understanding of the pattern of introduction of Nile perch into various lakes within the East African region , there ile perch , 25–27. The current study investigated the population genetic structure of the Nile perch in six populations from Western and Eastern Africa. Unravelling the genetic structure of Nile perch will be useful in understanding how the interactions between biological, physical and geological processes have shaped populations of the species in its natural and introduced range. Information on the broad-scale genetic structure of Nile perch will be critical in achieving successful aquaculture establishments, conservation and management of the wild fisheries.Nile perch were sampled over a wide geographical range in Western and Eastern Africa to capture patterns of both biogeographical and human mediated gene flow of the species in both native and introduced environments. Following animal ethics guidelines, the sampling formed part of RKB’s PhD study approved by the animal ethics committe at James Cook University under approval number A1824. All samples for the study were freshly caught fish acquired from commercial fishers as part of their normal fishing activities and outside protected areas and as such we did not require special permits to get these samples. In Western Africa, two locations were sampled, the Senegal River (n = 18) and Lake Kainji, on the Niger River (n = 30). The samples from Senegal River were acquired from fishers and traders at Richard toll  in Senegal, while our collaborator from the Federal College of Freshwater Fisheries Technology organised the samples from commercial fishers on Lake Kainji. Similarly, the Eastern African samples were acquired from the species’ native habitats of Lakes Albert (n = 48) and Turkana (n = 18), as well as from introduced populations from Lakes Kyoga (n = 38) and Victoria (n = 38). Important to note is that this study was limited to Western and Eastern African populations, as samples could not be obtained from north of Lake Albert (Albert Nile) to the lower parts of the Nile where the habitat gets brackish towards the Mediterranean Sea.2) of the African continent spreading over four countries . The river basin which drains into the Atlantic ocean has been considered as the lifeline of the westernmost part of the Sahel zone of Africa, with agriculture and fishing as the two major economic activities [The Senegal River, covers 1.6%  is important and extensively used for fishing and irrigation in the region [Lake Kainji located in north-western Nigeria is the largest man-made lake in Western Africa formed in 1968 by damming part of the Niger River. The lake covers an area of up to 1,300 kme region .Lake Albert is located at the tip of the eastern arm of the EAR. Lake Albert drains directly into the White Nile, to which it is believed to be the main regulating water source for the Nile responsible for making the Nile a permanent river as opposed to a seasonal river –36. The Lake Kyoga is centrally located in Uganda. Lake Kyoga is connected to Lakes Victoria and Albert by the Victoria Nile River; however, despite the connection the Lakes are separated by a series of cascading falls, the biggest and most significant being the Murchison falls located just before the Victoria Nile drains into Lake Albert. At the top of the falls, the Victoria Nile narrows to a width of about 7 meters and drops by a height of about 120 metres forming a formidable barrier to upstream movement of aquatic animals. Geologically the Murchison falls were formed as a result of riverine retreat from a buried fault line and superposition from deeply lateralised rift surface sediments . SimilarL. niloticus (Nile perch), Oreochromis niloticus (Nile tilapia) and Rastrineobola argentea (Silver fish). Although introduced into Lake Victoria, Nile perch is the primary fished species for both local consumption and export [Lake Victoria is the largest tropical freshwater lake in the world and is home for the majority of Africa’s diverse haplochromine species. Currently three commercial species are heavily fished in the lake; d export . Due to d export , 13.Turkana is a large holomictic endorheic rift valley lake located in the eastern arm of the EAR; the lake is generally considered to be understudied, but biologically and environmentally stable , 38. HisSamples from all the six sampling locations were obtained from commercial line fishers, except for the samples from Lake Victoria which were captured by trawling. From each individual fish, a fin clip was cut from the caudal fin and preserved in 20% dimethyl sulfoxide (DMSO) saturated with sodium chloride salt , 41, or ® Technologies).Total genomic DNA was extracted using the Bioline Isolate II Genomic DNA kit (Bioline) following the manufacturer’s protocol. Briefly; the protocol involved a pre-lysis stage using 180 μl Lysis buffer with 25 μl of protease K incubated at 56°C for 1 hr. A second lysis buffer, G-3 (200 μl) was added and further incubated at 70°C for 10 min. In order to alter the buffer conditions, 210 μl of 100% EtOH were then added prior to two (GW1 and GW2) buffer washes. DNA was then eluted using a warm (70°C) elution buffer and later the eluted DNA was stored at -80°C prior to downstream PCR. DNA quality and quantity were assessed using a ND-1000 Spectrophotometer  and nine markers  were from a sister lcarifer , 44. Forlcarifer  to ensurVisualisation of PCR product was performed on an ABI-3730 instrument (Applied Biosystems) using a 5-standard dye system  at the Georgia Genomics Facility, USA. Alleles were scored using Genemarker 2.4 (Softgenetics) and checked for genotyping errors, stuttering and null alleles in Microchecker 2.2.3 . SuccessHe), observed heterozygosity (Ho), mean number of alleles (Na), allelic richness (AR), rare and private alleles were calculated in GenAlEx 6.41 [FIS, calculated in Genetix v 4.05 [Deviations from Hardy-Weinberg equilibrium (HWE) and locus linkage equilibrium were evaluated using GenAlex 6.41  and FishlEx 6.41 . AdditiolEx 6.41 , 50. Furx v 4.05 .Ne) for each population, were estimated using the linkage disequilibrium method as implemented in NeEstimator v 2 [A heterozygosity excess test for recent demographic history for the Nile perch for each of the six populations was executed in Bottleneck 1.2.02 . Althougator v 2 .FST testing was used to determine whether significant genetic differentiation existed between populations. Following the significant overall FST values obtained (at P < 0.001), pairwise genetic differentiation tests were performed on all the six populations after Weir and Cockerham [FST estimates were all calculated in Arlequin 3.5 [FST were corrected using Bonferroni correction at P < 0.001 [Standard genetic tests were performed to examine the amount of genetic differentiation among the six sites sampled. Using an analysis of molecular variance , the levockerham . AMOVA aquin 3.5  and Genequin 3.5 , with es < 0.001 .FST) and geographic distance (km) matrices were constructed and correlations analysed using a Mantel’s test (1000 permutations) in GenAlex 6.41 [To test whether Nile perch populations conform to an isolation by distance (IBD) model of gene flow, genetic distance (pairwise lex 6.41 . Two IBDR diveRsity package [The Bayesian assignment test implemented in STRUCTURE 2.3.3  was used package . The visLn11 (in samples from Lake Kyoga), Ln23 (samples from Lake Victoria) and Ln31 (samples from lakes Kyoga and Victoria). These deviations from HWE (at P < 0.001 after Bonferroni correction) may suggest a detection of non-random mating within the two introduced populations.Microchecker did not detect any evidence of scoring errors due to stuttering and large allelic dropouts at any of the loci examined. Linkage disequilibrium was observed in only 0.05% (8 out of 172) of loci combinations across all populations, following Bonferroni correction set at P < 0.001 . HoweverLn09 and Ln23, which were monomorphic in the Lake Turkana population, and Lca21 which was also monomorphic in the Lake Kainji (Niger) population. The mean number of alleles (Na) per locus for all six populations was 5.13 ± 0.02 (mean ± SE) and number of alleles in the polymorphic loci ranged from 2–14 alleles  across all 19 loci in the six populations ranged from 2.37 at Lca21 to 9.54 at locus Ln17 . Analysis of AR as a measure of genetic diversity found a significantly (P < 0.05) lower AR among individuals from introduced (Lakes Kyoga and Victoria) populations than in individuals from the putative origins for fish introduced (Lakes Albert and Turkana) in East Africa  and Lake Victoria the lowest He (0.48 ± 0.06). All diversity indices, including the mean expected and observed heterozygosity, for each population across all loci are listed in The overall mean expected and observed heterozygosities (Mean ± SE) were 0.51 ± 0.03 and 0.52 ± 0.02, respectively across all populations. There were no large variations in heterozygosity observed amongst populations, although Lake Albert showed the lowest mean FIS value of– 0.029, while Lake Albert had the highest positive FIS of 0.148. The high FIS within Lake Albert could be a result of sub-structuring of the Nile perch within the lake, and this is consistent with the admixture observed in this Albert population as per the STRUCTURE bar plot. Results of the bottleneck tests did not detect significant deviations in allele frequencies, and in all instances an L-shaped curve was obtained (results not shown). However, with the Wilcoxon test, significant heterozygosity deficiencies were identified within the native populations of Nile perch from Lake Albert and Turkana (P = 0.004) under the IMM model and for Lake Kyoga a heterozygote excess was identified under the IAM model (P = 0.03). The effective population size (Ne) at the lowest frequency of 0.05 ranged from  in Lake Kainji to  in the Senegal River. Effective population size, Ne, in East African populations ranged from Ne = 39.6; 95% CI 28.8–58.8) in Lake Albert to Ne = 79.1; 95% CI 45.5–213) in Lake Victoria. Except for the Senegal population, in all other populations the Ne was finite and restricted.Lake Turkana exhibited the only negative mean FST of 0.26, P < 001) , although the FST were relatively shallow among populations within groups   . GeneticP < 001) . Pairwis< 0.001) .FST indicate major differences in allele frequencies between the East and West African populations. The highest pairwise genetic differentiation was between the Senegal River and the two most geographically distant East African populations of Lake Turkana and Lake Victoria , which were the most geographically distantly sampled populations. The lowest pairwise FST was between Lake Victoria and Albert (FST = 0.08), although it was still significant .The high and significant 2 = 0.86, P = 0.01) .Furthermore, the Mantel’s test revealed a positive and significant correlation (R = 0.01) . This siBayesian STRUCTURE analysis confirmed strong partitioning of genetic varian ce among Nile perch populations with two distinct genetic clusters (K = 2) identified . The indResults showed relational connectivity, reflecting the directional and relative strengths of migration between the six sampled populations of Nile perch. The Nile perch from the Senegal River and Lake Kainji clustered into one genetic group and all the other four populations into another major genetic group. The two major genetic groups exhibited in the divMigrate relative gene flow network diagram , did mirSimilarly, there is a genetic sub-group exhibited among three  of the four East African populations shown by the high gene flow between these populations. However, restricted gene flow was shown to and from the Lake Turkana population to the rest of the East African populations; Turkana is appropriately excluded by geographical distance and historical connectivity. In a way, the relative migration network did show genetic separation of the populations relative to distance, but more so was a surrogate of historical Nile perch translocations that have characterised the species from its native (Lakes Albert and Turkana) to the new introduced (Lake Kyoga and Victoria) environments. Important to note, is that some locations had lower sample numbers (N<30) which may underrepresent the potential diversity in these particular locations. Possible sampling sites affected in the present study may include the Niger River, Senegal River and Lake Turkana.Regional STRUCTURE analysis also revealed strong genetic structuring among the EAR lake populations, with three distinct genetic clusters evident ; the LakFST value obtained between the Senegal and Nigerian individuals sampled in the current study .The Nile perch in Africa was partitioned into two discrete genetic groupings that may require different management strategies; one from West Africa and the other from East Africa. Further structuring within East Africa was revealed, with three discrete populations at a regional scale. The strong genetic structure is a function of restricted levels of gene flow between populations across biogeographical barriers, particularly for the Nile perch in East Africa. Barriers including river flow and reversal, dams and water falls have been identified as being effective in impeding gene flow across connected large water bodies. For instance, to the right of Lake Albert are the Murchison Falls along the Victoria Nile River, a biogeographical barrier separating the Albertine rift system  from Lake Victoria. This barrier has been documented as an effective obstacle in preventing, for instance, Nile perch stocks in Lake Albert from migrating into Lakes Kyoga and Victoria . ImportaFST of 0.26 and strong genetic structuring which is comparable to other freshwater species with similar dispersal and habitat usage [The Nile perch in Africa’s freshwater rivers and lakes was highly differentiated with significant (P < 0.000) global at usage . Becauseat usage . TherefoSyncerus caffer, and Nile tilapia Oreochromis niloticus) in Africa, exhibits a genetic break between the populations of Western and Eastern Africa [West African and East African genetic groupings from the current study are further supported based on a single study carried out in Nile perch from the broader Africa region using a single mitochondrial marker (d-loop region) . That stn Africa , 67.The significant isolation by distance revealed by the Mantel’s test in the current investigation further suggests limited dispersal of the species between the two regions of West and East Africa. The result is consistent with findings in other aquatic species, where patterns of isolation by distance have been found to arise from limited dispersal that decreases with increased distance –74. StroFST, P<0.000). Strong genetic structuring among the East African populations was evident where Lakes Albert and Victoria clustered together (Group 1); and Lake Kyoga with Lake Turkana each were assigned to their own genetic cluster (Group 2 and 3 respectively). Results of the current study showing three genetic groupings differs from an earlier study that identified only two genetic groupings within the East African lakes (25). How the three genetic groupings have evolved in the face of biogeographic events and geological structures is still open to interpretation, but these three genetically distinct Nile perch populations identified for the first time in the current study may be treated as different management units (based on their high level of differentiation and reduced connectivity) for the sustainable exploitation of the species.The genetic structure in East Africa is complex and the current study unraveled up to three differentiated genetic clusters which appeared to be grouped according to lakes. Except for Lakes Victoria and Albert that were relatively undifferentiated from each other, there was comparatively deeper differentiation observed amongst all the other lakes in the East African region , although these populations have become divergent from Lake Turkana individuals  in comparison to their putative original populations (from Lakes Albert and Turkana). These include; heavy fishing pressure coupled with climate and ecological pressures which may in part be responsible for the current declines of Nile perch in Lakes Kyoga and Victoria. Results from the present study are consistent with reports that have shown declining numbers of Nile perch in Lakes Kyoga and Victoria despite the increase in fishing effort of the Nile perch amidst climate and ecological pressures , 88, 89.Although reduced genetic diversity was detected among the translocated populations at the allelic level, the genetic bottleneck was not severe enough to be detected by the bottleneck analysis , 52. ThiKnowledge of genetic structure and relationships revealed in the present study among Nile perch populations will be useful for the effective management and conservation of the species in both its native and introduced environments. Separate management strategies of Nile perch populations from West and East Africa (for both aquaculture development and fisheries restocking) should be considered, following the genetic divergence and differentiation that has occurred over evolutionary time between these two groups. Following restricted gene flow, the two genetic groupings have evolved in isolation, and consequently may have accrued adaptive differences, and any translocations of Nile perch should be carefully considered across the regions of West and East Africa.Programs aimed at restocking these lakes and rivers should seek founder stocks independently within each of the two regions of West and East Africa. As discussed previously, there was complex structuring within East African Nile perch where three distinct genetic stocks were identified, and these seemed to be linked to lakes. Thus, within East Africa, the Nile perch from Lake Albert and Lake Victoria should be treated as a single group, Lake Kyoga as another and Turkana separately. Movement of the Nile perch across lake basins should be discouraged to prevent possible genetic introgression.FST is relatively shallow and could potentially be mixed together without raising concerns of out breeding that may include a reduction in fitness [Furthermore, from an aquaculture perspective, the recognition of the three genetic groupings identified within East Africa, provides a large genetic base upon which genetic diversity can be maximized through potentially mixing the three distinct genetic populations at the start of a Nile perch breeding program for the region. However, it is important to note that even though the populations are distinct,  fitness . The thr fitness , 92.Nile perch in Africa is partitioned into two discrete genetic groupings; one comprising populations from West Africa and the other populations in East Africa. Further structuring within East Africa was revealed, with three discrete populations requiring different management strategies at a regional scale. Although, strong genetic structure is a function of restricted levels of gene flow between populations across biogeographical barriers, in the current study, results indicated that it may also partially be a product of historical anthropogenic introductions. The categorical evidence of decreased genetic diversity exemplified the persistence of founder effects within the translocated populations of Lakes Victoria and Kyoga. The two translocated Nile perch populations of Lakes Kyoga and Victoria were distinct, and ought to be treated separately for sustainable management of the now dwindling fishery in the two new habitats. The use of more robust markers in the current study clearly resolved the genetic structure of Nile perch in East Africa, and the three populations identified will be useful in forming an artificial and diverse base population for selective breeding of the Nile perch. Therefore, findings and information provided by the current study will be critical and useful in achieving successful and comprehensive aquaculture establishments, as well as conservation and management of the wild fisheries for the freshwater Nile perch.S1 TableSignificant deviations following a sequential Bonferroni (at P<0.001) correction are indicated by sig and non-significant by ns.(DOCX)Click here for additional data file.S2 TableSEN- Senegal River, NIG- Lake Kainji, ALB- Lake Albert, KYO- Lake Kyoga, VIC–Lake Victoria and TUR- Lake Turkana. All pairwise comparisons were significant at P<0.05(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 TableAllelic Richness per locus and populationa) Number of alleles sampledb) Nei's estimation of heterozygosity.c) (DOCX)Click here for additional data file.S1 Graph(PDF)Click here for additional data file.S2 Graph(TIF)Click here for additional data file.


Fredericella sultana) or are released as buoyant stages (Cristatella mucedo) and that live primarily in either lotic (F. sultana) or lentic environments (C. mucedo). Describing population genetic structure in multiple sites characterized by varying degrees of hydrological connectivity within each of three (or four) UK regions enabled us to test the following hypotheses: (1) genetic diversity and gene flow will be more influenced by hydrological connectivity in populations of C. mucedo ; (2) populations of F. sultana will be characterized by greater genetic divergence than those of C. mucedo ; and (3) genetic variation will be greatest in F. sultana . We found that hydrological connectivity enhanced genetic diversity and gene flow among C. mucedo populations but not in F. sultana while higher overall measures of clonal diversity and greater genetic divergence characterized populations of F. sultana. We suggest that genetic divergence over time within F. sultana populations reflects a general constraint of releasing propagules that might eventually be swept to sea when taxa inhabit running waters. In contrast, taxa that primarily inhabit lakes and ponds may colonize across hydrologically connected regions, establishing genetically related populations. Our study contributes more nuanced views about drivers of population genetic structures in passively dispersing freshwater invertebrates as outlined by the Monopolization Hypothesis  by highlighting how a range of demographic and evolutionary processes reflect life‐history attributes of benthic colonial invertebrates (bryozoans) and cyclically parthenogenetic zooplankton. In addition, growing evidence that genetic divergence may commonly characterize populations of diverse groups of riverine taxa suggests that organisms inhabiting lotic systems may be particularly challenged by environmental change. Such change may predispose riverine populations to extinction as a result of genetic divergence combined with limited dispersal and gene flow.Variation in dispersal capacity may influence population genetic variation and relatedness of freshwater animals thus demonstrating how life‐history traits influence patterns and processes that in turn influence biodiversity. The majority of studies have focused on the consequences of dispersal variation in taxa inhabiting riverine systems whose dendritic nature and upstream/downstream gradients facilitate characterizing populations along networks. We undertook extensive, large‐scale investigations of the impacts of hydrological connectivity on population genetic variation in two freshwater bryozoan species whose dispersive propagules (statoblasts) are either attached to surfaces  or are released as buoyant stages (Cristatella mucedo). Characterizing population genetic structure in multiple sites within each of three (or four) UK regions enabled us to test hypotheses regarding how dispersal variation and hydrological connectivity impact genetic variation in colonial freshwater invertebrates that primarily inhabit lotic (F. sultana) or lentic (C. mucedo) habitats. We found that hydrological connectivity enhanced genetic diversity and gene flow in and among C. mucedo populations but not in F. sultana.We undertook extensive, large‐scale investigations of two freshwater bryozoans species whose dispersive propagules (statoblasts) are either attached to surfaces ( Natural drivers of such heterogeneity include flooding, drought, landslides, and the gradual infilling of ponds and lakes with sediments. Additional anthropogenic drivers include canalization, dams, and cultural eutrophication. These various processes produce an array of habitats characterized by varying successional stages as organisms respond to the availability of suitable habitats across the “hydroscape.” For long term persistence, a key feature of aquatic organisms is thus the ability to colonize new sites when their local habitats degrade. Such colonization may confer resilience to local populations by introducing genetic variation that prevents extinction resulting from, for example, genetic drift, population bottlenecks, disease, or environmental change  or drifting in currents (hydrochory) to reach new sites  and lives in still water environments, and the bryozoan Fredericella sultana which inhabits flowing water systems and produces statoblasts that remain attached  than in lotic populations of F. sultana .Patterns of genetic diversity and gene flow will be more influenced by hydrological connectivity in lentic populations of C. mucedo will be characterized by less genetic divergence among sites than F. sultana.As a consequence of its relatively high dispersal capacity, F. sultana will demonstrate greater overall genetic variation than C. mucedo in keeping with a propensity for genetic divergence linked with its relatively low dispersal potential.As outlined above, previous studies are biased toward comparing variation in dispersal capacity and its impacts on genetic diversity in populations of taxa in lotic habitats and particularly of insects and fish. More targeted research that addresses dispersal capacity, pathways and consequences for a range of taxa inhabiting lotic and lentic systems should substantially contribute to our general understanding of how aquatic biodiversity is generated and maintained. Accordingly, the aim of this study is to test hypotheses about how differences in dispersal potential and hydrological connectivity will generally influence patterns of genetic variation and divergence in colonial invertebrates that primarily inhabit either lotic or lentic habitats. In particular, we focus on the bryozoan d Figure . PreviouC. mucedo and F. sultana.By addressing these hypotheses, our study provides novel evidence regarding the general impact of hydrological connectivity on genetic diversity of invertebrates that vary in dispersal capacity in lotic and lentic environments and the evolutionary consequences of dispersal variation. Our results also provide the first evidence that hydrochory can contribute to the dispersal of freshwater bryozoans. These issues in turn have implications for how rapidly changing freshwater environments may impact the persistence and conservation of freshwater organisms that vary in dispersal capacity. Below we provide further relevant background information on the biology of 1.1Freshwater bryozoans (Phylum Bryozoa: Class Phylactolaemata) are common residents of both lotic and lentic freshwater environments  in large numbers at the end of the summer growing season  and hydrochory but such dispersal has not been demonstrated. Abd‐Elfattah, El‐Matbouli, and Kumar (F. sultana statoblasts ingested by carp (but not brown trout) were viable and thus provide some proof‐of‐concept that fish may effect dispersal. However, no records of F. sultana statoblasts in wild caught birds or fish were encountered during preparation of a recent review by one of us . Within each region, we aimed to collect some 15 populations of each species from a series of sites characterized by differences in hydrological connectivity as detailed below. We note that it is impossible to dictate exactly where bryozoan populations will be found as they can be highly patchy over space and time , collections were not restricted to populations from the same catchment Populations of the two species were assigned to somewhat different categories of hydrological connectivity in view of differences in dispersal capacities and distributions as described below.To test generality of our results, we collected bryozoans from the following three regions of the UK: Norfolk, Cumbria, Greater Glasgow. The regions can be broadly characterized as lowland, agricultural (Norfolk), upland (Cumbria), and urban . Single populations collected from satellite lakes with hydrological connection to Upper Lough Erne (via channels and a further satellite lake) were considered to be hydrologically connected . Isolated sites had no apparent hydrological connections to other water bodies . We note that DHC and HC categories as used here are not entirely consistent with those used for populations in other regions (where more than one catchment was sampled). However, relative trends in connectivity are expected to be similar with greater connectivity of populations within the Upper Lough Erne and a lower degree of connectivity with satellite lakes connected by channels to the Upper Lough Erne.For F. sultana, populations were collected from sites assigned to two categories of hydrological connectivity: directly hydrologically connected (DHC) and isolated (I)  or interconnected lake systems. In this case, we included data for populations from different catchments . However, dispersal of F. sultana via hydrochory should be much reduced in this array of isolated sites relative to that among the DHC sites.In the case of ) Figure . Just asC. mucedo were collected after passing the mud samples through sieves with mesh sizes of 1.7 mm and 630 μm. The material retained after processing was examined with a stereomicroscope to identify and collect whole and undamaged statoblasts. Statoblasts were then preserved in absolute ethanol and stored at −20°C until DNA extraction. Branches of F. sultana were collected from independent colonies growing variously on hard surfaces  and were placed in individual 50‐ml falcon tubes filled with water from the collecting site. Following their collection, the branches (henceforth referred to as colonies) were observed with a stereomicroscope to confirm colonies were alive  and were then preserved in absolute ethanol and stored at −20°C until DNA extraction.Mud was collected by wading into shallow regions of water bodies and using a ladle mounted on a pole, or from a boat using an Ekman grab. Statoblasts of Our general sampling strategy was to collect some 30 samples per site for multilocus genotyping of each bryozoan species.2.2Genomic DNA (gDNA) for both species was isolated using the HotShot protocol , and genotype peaks were scored using Peakscanner 2.0 (Applied Biosystems). Allele calling was performed with FlexiBin v2 , (b) population clonal diversity , and (c) a genotypic richness index , allelic richness (RS), observed (HO), expected heterozygosity (HE), inbreeding coefficient (FIS), population differentiation (FST), and deviation from Hardy–Weinberg equilibrium (HWE) were calculated using FSTAT 2.9.4 .The mean number of alleles  and hence as a rate of gene flow. Migrate‐n  and the historical migration rate, M . A Brownian motion model was used, and mutation was considered constant over the set of 10 microsatellite markers per species. The MCMC procedure consisted of one long chain with 500,000 recorded genealogies for each locus, with 10,000 genealogies discarded as burn‐in.Several approaches were used to determine whether genetic diversity within sites is influenced by hydrological connectivity. We determined if identical clones occurred in multiple sites by comparing MLGs obtained from GenClone2.0  on samples within landscapes to examine a potential role of hydrological connectivity in segregating sites. We then employed a discriminant analysis of principle components  were simulated for C. mucedo and between 1 and 34 clusters (K) for F. sultana. Each K was tested five times. The online software STRUCTURE HARVESTER 0.6.92  to gain evidence for genetic divergence by determining whether FST values were correlated with geographical distance among sampling sites (according to Isolation by Distance theory). For each dataset, a semi‐matrix of pairwise genetic distances (FST) between samples was created and regressed against the corresponding shortest linear distances between samples with a permutation test of 10,000 iterations.We employed Mantel tests (using Genepop (ISOLDE) Rousset, ; http://FST‐based test was carried out in Arlequin  and was repeated under various sets of combinations to examine associations with hydrological connectivity and regions. A preliminary test was conducted to identify outlier loci using the whole dataset. Pairwise tests were performed between samples collected from different “populations” as revealed by DAPC and STRUCTURE analyses  an HE) during a strong event of reduction in population size. The expected heterozygosity at mutation–drift equilibrium (Heq) derives from the number of observed alleles. Hence, a rapid decline in alleles results in HE being larger than Heq, producing a heterozygosity excess  and a two‐phase mutation model (TPM) with 95% single‐step mutations and 5% multistep mutations. An additional test implemented by BOTTLENECK 1.2 based on allele frequency shift (Shift‐Mode test) was used to further substantiate the occurrence of a recent bottleneck. Samples significant for all the three tests were considered as bottlenecked. Samples with <4 MLGs were removed because the sample size was too small for processing by BOTTLENECK 1.2.We tested for genetic signatures derived from recent population bottlenecks using a method implemented in BOTTLENECK 1.2  remained significant. Null alleles were detected in 15 of 330 tests for C. mucedo with 2 of 330 (0.6%) remaining significant after the Brookfield correction. Because the Brookfield correction improved both datasets and no loci showed a systematic presence of null alleles, both corrected datasets were used for subsequent statistical analyses.Allele dropout and stuttering were consistently absent. Null alleles were detected in 45 of 270 tests for 3.2Cristatella mucedo and F. sultana were characterized by 269 and 337 unique clones, respectively, across all sites and regions. Six of the 34 populations of F. sultana were monoclonal while all 48 populations of C. mucedo presented >1 MLG . Nevertheless, average clonal diversity (NC) and average genotype richness (R) within sites were significantly greater for F. sultana , observed heterozygosity (HO), number of alleles (NA), and inbreeding (FIS) showed relatively similar ranges across populations of both C. mucedo and F. sultana (Table FIS was significantly larger than expected under HWE (Hardy–Weinberg equilibrium) in 3 of 48 C. mucedo populations  and in 4 of 34 F. sultana populations  and the overall test for HWE incorporating all populations was also significant (p < .001), indicating a general lack of conformation to HWE in both species. For F. sultana, there was strong evidence for linkage disequilibrium (LD) between loci in two cases . Tests were carried out excluding linked loci from the dataset in order to evaluate their impact on characterizing clonal and genetic variation. No impact was observed when excluding the linked loci Fs04, Fs12TKU, and Fs13 and therefore, these loci were retained in the final analysis. There was no evidence for linkage disequilibrium among populations of C. mucedo.Patterns of expected heterozygosity . Three cases of shared clones were found for F. sultana  and richness  being reduced in isolated relative to connected sites  while FST was significantly larger in HC and isolated sites than in DHC sites  values (derived from Migrate‐n) were significantly  larger in DHC sites than in HC and isolated sites for C. mucedo , Cumbria , and Northern Ireland , Cumbria , or Glasgow  were broadly consistent with the above results. DHC, HC and isolated p > .05) between DHC, HC, and I systems in C. mucedo. The proportions of migrants in populations from DHC sites varied between 0.25 (EST) and 1 ; in DH sites from 0.14 (LOL and BAS) to 1 (BTF and DRO); and in I sites between 0  and 1 (BAR and MOK). In the case of F. sultana, there was a nonsignificant trend for numbers of migrants to be larger in populations from isolated sites than in DHC sites . The proportions of migrants in populations from DHC sites varied from 0.14 (WIM) to 1 ; in I systems varied between 0.18 (GLA) and 1 .The assignment test with GENECLASS showed a gradual but nonsignificant decrease in the number of migrants  and richness  in Cumbria relative to Norfolk and GG‐NI . There was no evidence of regional variation in other estimates of genetic diversity or migration in C. mucedo and F. sultana populations  Norfolk populations, (b) Cumbria populations, and (c) Glasgow and Northern Ireland populations  Norfolk populations, (b) Cumbria populations, and (c) Glasgow populations  = −10,203.18 ± 1.94) that were characterized by the Norfolk populations and the remaining material  = −9,585.90 ± 3.70; Figure F. sultana material  = −12,222.26 ± 3.19; Figure The general discriminant analysis of principle components (DAPC) resolved l Figure . An addil Figure  identifiC. mucedo . The proportion of migrants in Norfolk populations varied between 0 (SEL) and 1 (BTF and WRO), in Cumbria from 0.14 (LOL and BAS) to 1 (MOK), in Glasgow from 0.14 (CRR) to 1 , and in Northern Ireland from 0 (BRY and CGN) to 1 (DRO). There was a trend for the proportion of migrants in F. sultana populations to be larger in Norfolk and in Glasgow than in Cumbria . The proportion of migrants in Norfolk populations varied between 0.18 (GLA) and 1 , in Cumbria from 0.14 (WIM) to 1 (YEW), and in Glasgow from 0.31 (UNC) to 1 (BLW and LUW).The assignment test with GENECLASS indicated that the number of migrants did not vary with regions in 3.5p > .05) between geographic distance and FST values for both C. mucedo and F. sultana populations within Norfolk, Glasgow, and Northern Ireland  were significant (p < .05) but none remained significant after the Bonferroni correction for multiple tests  of F. sultana were characterized by a sufficient number of MLGs (≥4) to conduct BOTTLENECK 1.2 tests. Thirteen of 26 tests (50%) and 20 of 34 tests (58.8%) provided evidence for recent bottlenecks in F. sultana and C. mucedo, respectively  than for F. sultana .Thirty‐four of 48 populations (70.8%) of ly Table . A highe44.1C. mucedo are influenced by hydrological connectivity. Thus, shared multilocus genotypes (MLGs) were disproportionately detected among populations in directly hydrologically connected (DHC) sites (14 of 15 cases). Furthermore, populations in 10 of the 11 DHC sites were characterized by ongoing gene flow while there was no evidence for gene flow among populations in the 26 sites that were not directly hydrologically connected. There were also greater numbers of clones, alleles, and migrants in populations from DHC sites and higher values for clonal richness and expected heterozygosity in these populations. Discriminant analysis of principal components (DAPC) separated populations according to hydrological connectivity in Cumbria and Norfolk. The failure of DAPC to separate populations from Greater Glasgow and Northern Ireland according to our measures of hydrological connectivity suggests other complicating processes. For example, sites in Northern Ireland can be periodically connected during occasional seasonal flooding . However, we would expect that hydrochory could be achieved across much greater distances given the extensive distances associated with some hydrologically connected systems and diapause through the entire winter period of C. mucedo statoblasts. Dispersal by other means may also contribute to the observed patterns. For example, statoblasts may be disseminated by animal vectors that either require hydrological connectivity  to reach distant sites or that may be prone to follow water courses . Notably C. mucedo statoblasts are often observed in digestive tracts of fish and waterbirds and in waterbird feces, and they have been shown to hatch after passage through vertebrate digestive tracts  and once between sites in different but relatively close river catchments in Norfolk (the Rivers Stiffkey and Wensum whose watersheds and associated ditch networks are in places separated by <2 km). Hydrological connectivity had no impact on any measures of population genetic diversity or gene flow. These results suggest that piptoblasts are effective at routinely remaining within the vicinity where they were produced, contributing to persistence within local populations and limiting downstream dispersal that could invariably lead to the sea. It also suggests that, despite a propensity for the tubular, branching colonies to fragment and re‐attach to surfaces  of Cumbrian C. mucedo populations and positive correlations between FST and geographic distance for both species in Cumbria are somewhat consistent with either older upland populations in more varied sites or impacts of selection in the other regions. Finally, both DAPC and STRUCTURE analyses resolved all F. sultana populations according to their respective regions of collection.Patterns of genetic diversity in 4.2C. mucedo and F. sultana modulate genetic variation within and among benthic invertebrate populations, and thus, how life‐history traits may be linked with genetic divergence and the potential for local selection. Evidence for a relatively greater impact of these processes in F. sultana populations includes a greater number of loci potentially under selection (5 vs. 1 outlier out of 10 loci) and more cases of linkage disequilibrium between loci (2 vs. 0) for F. sultana and C. mucedo, respectively. The inference of greater genetic divergence in F. sultana is further supported by the larger number of unique clones detected overall , as well as the greater average clonal diversity and greater average genotype richness values within sites for F. sultana relative to C. mucedo samples.Our results provide evidence that varying dispersal capacities of C. mucedo populations with evidence of bottlenecking than in F. sultana . This is in keeping with substantial evidence for metapopulation dynamics in C. mucedo in the form of extinction and colonization .There was a somewhat higher proportion of Okamura,  and geneOkamura, . RelativF. sultana and bottlenecks in C. mucedo populations. Further independent evidence in support of the contrasting patterns of genetic variation in these two species is obtained from analyses of mtDNA variation in populations from Switzerland and the UK  and migratory waterbirds  enhances survivorship. This suggests that dormancy variation can influence postrecruitment dynamics at least in some bryozoan systems. However, the extent that these surface‐associated propagules can effectively act like a zooplankton “egg bank,” enhancing persistence of locally adapted genotypes and thwarting colonization, requires further study.Our results for allaghan  found thF. sultana), (b) ongoing gene flow and relatively low genetic differentiation among populations associated with high dispersal capacity (C. mucedo), and (c) genetic differentiation and local adaptation in populations despite high dispersal capacity due to rapid population growth, large propagule banks and persistent founder effects .In summary, our two bryozoan systems and cyclically parthenogenetic zooplankton collectively illustrate a range of demographic and evolutionary processes reflecting particular life‐history attributes of aquatic invertebrates. These outcomes include the following: (a) genetic divergence among populations in association with low dispersal capacity (4.4F. sultana populations. Such retention is likely to reduce displacement downstream and is characteristic of sponge gemmules, bryozoan piptoblasts and sessoblasts, and embryos of Hydra. Propagule retention may thus be a critical life‐history trait that enables many attached forms to inhabit running water systems rather than being swept to the sea. Exceptions that prove the rule include glochidia larvae that are released by bivalves to hitchhike on fish in order to colonize new sites. Due to the constraints imposed by flow and displacement, river habitats may particularly promote population divergence of inhabitants that lack mobility in general, or at critical periods in their life history. Indeed, this scenario of constraint may fundamentally explain the numerous subspecies, strains, and lineages of salmonids whose spawned eggs are retained in specific stretches of particular rivers in the Pacific coast of North America (e.g., Johnson, Kemp, & Thorgaard, Our study provides evidence that propagule retention promotes genetic divergence among F. sultana, may thus be most impacted by the many changing conditions that now commonly challenge our biota, such as habitat loss, agricultural intensification, climate change, and invasive species. In contrast, taxa with high dispersal capacity and regular gene flow, such as C. mucedo and zooplankton, may locate favorable environments and, in the case of zooplankton, may rapidly adapt to new conditions. These considerations imply that conservation of river inhabitants is of particular concern in our changing world.Spatial and temporal patterns of dispersal and colonization will contribute to biodiversity and species distributions and abundances. In turn, variation in life‐history traits, such as dispersal capacity and population growth rates, can determine the extent that populations diverge and may therefore resist invasion by externally sourced colonists. Taxa associated with running water habitats and with limited gene flow among populations, such as None declared.Cristatella mucedo statoblasts from Northern Ireland sediments.This research was designed by BO, who also contributed in the collection of samples, interpretation of results and development of the manuscript. PR contributed in the collection of samples, DNA extractions, genotyping, computational analyses, and the development of the manuscript. EP helped in collecting  Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.


Passer domesticus) and the Spanish sparrow (Passer hispaniolensis) provide a striking example. Throughout their range of sympatry, these two species do not regularly interbreed; however, a stabilized hybrid form  exists on the Italian Peninsula and on several Mediterranean islands. The spatial distribution pattern on the Eurasian continent strongly contrasts the situation in North Africa, where house sparrows and Spanish sparrows occur in close vicinity of phenotypically intermediate populations across a broad mosaic hybrid zone. In this study, we investigate patterns of divergence and admixture among the two parental species, stabilized and nonstabilized hybrid populations in Italy and Algeria based on a mitochondrial marker, a sex chromosomal marker, and 12 microsatellite loci. In Algeria, despite strong spatial and temporal separation of urban early‐breeding house sparrows and hybrids and rural late‐breeding Spanish sparrows, we found strong genetic admixture of mitochondrial and nuclear markers across all study populations and phenotypes. That pattern of admixture in the North African hybrid zone is strikingly different from i) the Iberian area of sympatry where we observed only weak asymmetrical introgression of Spanish sparrow nuclear alleles into local house sparrow populations and ii) the very homogenous Italian sparrow population where the mitogenome of one parent (P. domesticus) and the Z‐chromosomal marker of the other parent (P. hispaniolensis) are fixed. The North African sparrow hybrids provide a further example of enhanced hybridization along with recent urbanization and anthropogenic land‐use changes in a mosaic landscape.Under different environmental conditions, hybridization between the same species might result in different patterns of genetic admixture. Particularly, species pairs with large distribution ranges and long evolutionary history may have experienced several independent hybridization events over time in different zones of overlap. In birds, the diverse hybrid populations of the house sparrow ( In this study, we investigate patterns of gene flow between the house sparrow, the Spanish sparrow, and their phenotypical hybrids on two study sites in Algeria. Unlike in European areas of sympatry, nuclear markers suggest strong genetic admixture across Algerian populations and across phenotypes. Hybridization may increase genetic and/or phenotypic variability within short evolutionary timescales, leading to superior adaptation of hybrids in different contexts such as novel ecological niches , a stabilized homoploid hybrid form that resulted from interbreeding of the house sparrow (Passer domesticus) and the Spanish sparrow (Passer hispaniolensis)  to analyze patterns of genetic admixture in the North African hybrid zone. Based on the current state of knowledge outlined above, we predict that,CHD1Z alleles; Belkacem et al., Phenotypic hybrids in Algerian mixed populations should be identifiable as genetic hybrids. Furthermore, these should show similar variation of the three genetic marker systems as the Italian hybrid form  in large breeding colonies, whereas only a few detached farmhouses provide nesting sites for house sparrows and hybrids  to 1 (P. hispaniolensis), for example, a male with five traits showing the typical house sparrow phenotype and one trait being intermediate scored 0.0833  and microsatellites  from each other and from the admixed Italian hybrid form  and the Z‐chromosomal marker of the other parent (P. hispaniolensis) are fixed  sequences from 197 of our samples were already available to us from Belkacem et al. . DNA extraction, standard primer combinations, PCR, and sequencing protocols followed Belkacem et al.  and CHD1Z‐R . Labeled PCR fragments were run on a 16‐column ABI 3130xl capillary sequencer (Applied Biosystems).We amplified 2.5ND2 and CHD1Z) were aligned manually using MEGA 5.1  were translated into protein sequences with MEGA 5.1 in order to control for stop codons and, thus, to exclude numts  as a potential source of error. Minimum‐spanning networks of ND2 haplotypes and CHD1Z alleles were reconstructed with TCS v. 1.2 with gaps treated as fifth state . To avoid drawbacks due to haplodiploidy of the Z‐chromosome .We also used Arlequin 3.5 to calculate diversity indices (such as haplotype and nucleotide diversity for 2.6Tm) and to maximize spacing between markers with overlapping fluorescence spectra  following the manufacturer's instructions. To increase the number of possible reactions, the protocol was modified as follows: The reaction setup for each multiplex PCR had a total volume of 10 μl containing 5 μl Multiplex Master Mix , 3 μl RNase‐free water, 1 μl of each respective primer mix, and 1 μl DNA template (<200 ng DNA/reaction).We designed a new multiplex microsatellite protocol for 13 microsatellite loci using published information about the microsatellite primers Table . To miniA total of 323 samples were genotyped for 13 unlinked microsatellite loci. Locus Pdo27 had to be excluded from further analyses due to ambiguities during allele scoring; thus, 12 microsatellite loci remained to estimate population genetic parameters. Alleles were scored manually using GeneMapper 4.0 (Applied Biosystems) using a modified method proposed by Amos et al.  and Guicn > 5. We also used Arlequin 3.5 to calculate locus‐specific observed and expected heterozygosities  for each sample population, and pairwise FST (fixation index) values among populations and to test for departure from Hardy–Weinberg equilibrium (HWE) and for linkage disequilibrium (LD). We performed an exact test of HWE based on a Monte Carlo Markov chain (MCMC) length of 106 repetitions and 105 dememorization steps. To test whether loci were in LD, the number of permutations was set on 104. LD and HWE tests were adjusted with sequential Bonferroni correction to minimize the chance of a type one error  with ten runs for each value of K. We performed 106 iterations per run with a burn‐in period of 5 × 105 steps. For further processing of the STRUCTURE output, we used STRUCTURE HARVESTER , we used the approach by Evanno, Regnaut, and Goudet  and 95% probability intervals (PI) of q inferred from STRUCTURE output  as the most plausible population structure when using the complete data set (n = 323) under the admixture–frequency‐correlated model. The house sparrow population from Nepal was separated from all other populations, whereas Western Palearctic house sparrows, Spanish sparrows, and Italian sparrows were not distinguished as separate clusters (plot not shown). However, the result for k = 3 from the same run yielded a biological meaningful scenario: Again, the Himalayan house sparrow population appeared as a separate cluster, but Western Palearctic populations of house sparrows  and Spanish sparrows  were distinguished as a second and third cluster . Most other populations showed a signal of genetic admixture. Based on the results above, we performed a second STRUCTURE analysis excluding the Nepal house sparrow population. Using this reduced Western Palearctic data set (n = 301) under the admixture–frequency‐correlated model, the two parental species were recovered as distinct genetic clusters with k = 2 as the most plausible population structure  with q < 0.1 in microsatellite analysis .In most house sparrow populations, the s Figure  and S5. P. hispaniolensis mtDNA lineage and the CHD1Z alleles 3–11 were 100% fixed and all individuals were assigned to the Spanish sparrow cluster with q > 0.90 in microsatellite analysis. These characteristics were met in the Spanish sparrow populations from Egypt, Central Asia, southern Spain , and Fuerteventura . We therefore considered this a scenario of weak admixture in the Iberian house sparrow population  represented more than 50% of the local individual genotypes  compared to other Eurasian populations of P. domesticus and P. hispaniolensis  and compared to Italian populations of P. italiae   compared to Eurasian P. domesticus and P. hispaniolensis populations  and P. italiae populations   and P. hispaniolensis  showed least (or low) genetic diversity indices: house sparrows from Nepal  and Spanish sparrows from Fuerteventura  were not significantly different from 0 for most pairwise comparisons of admixed urban populations at Djelfa and Hassi El Euch with most other study populations of all three species . Remarkably, pairwise FST values for intraspecific comparisons of Himalayan and Western Palearctic house sparrow populations were higher (0.109 < FST < 0.124) than those for interspecific comparisons between Western Palearctic house sparrows and Spanish sparrows . None of the birds from that crop field population had gray in the crown: Most birds showed the characteristic brown crown of P. hispaniolensis, although some birds had an aberrant phenotype with a black central crown patch  for population genetic studies relate to their particular mechanism of mutation . In northwest Africa, genetically admixed house sparrow populations might have originated from (a) local hybridization with Moroccan Spanish sparrows or (b) ancestral polymorphism, because colonization of North Africa by the house sparrow was suggested to have occurred via the Strait of Gibraltar from Iberian source populations .Second, genetic admixture was not only found in phenotypic house sparrows from the Algerian contact area but also for the Moroccan house sparrow population compatibility between genomes of parental species, that is, postzygotic reproductive barriers that strengthen over divergence time. Instead, additional factors can be important such as prezygotic reproductive barriers  or environment‐dependent selection regimes acting on parental species and their hybrids. If the complex interplay between these factors varies over time and space, spatial patterns in secondary contact can be very different and range from sharply defined narrow hybrid zones to patchy distributions of parental species and hybrid populations in mosaic hybrid zones Curry, . In factPostglacial range expansion of the house sparrow was presumably strongly associated with the rise of human agriculture and civilization and adaptation to novel habitat in an anthropogenic environment (Sætre et al., None declared.MP, AAB, and HS designed the theoretical and methodical framework. HW and OG performed laboratory work and most statistical analyses. MP and MV performed further statistics. GG and MLV carried out fieldwork and collected samples in Italy, AAB in Algeria, DC in Spain, and JM in Nepal and other regions of Asia. MW provided further samples. MP drafted the manuscript. All authors read the draft and contributed to the discussion and completion of the final manuscript. Click here for additional data file. Click here for additional data file.


Anopheles coluzzii populations in four localities of Benin.Changes in the natural habitats of insect groups are determined the genetic polymorphisms between individuals. The objective of this study was to establish the genetic structure of the Anopheles mosquitoes collected with the allelic and genotypic frequencies of kdr gene determined. The multiple comparison Chi square test for proportions was performed with R version 3.3.3. Next, the observed heterozygosity, expected heterozygosity, and indices of fixation, and genetic differentiation were estimated. Finally, the Hardy–Weinberg equilibrium (EHW) was determined to assess whether panmixia exists in the different populations of mosquitoes of the agroecological zones under study.Insecticide surveys and larval sampling were conducted on 4 study localities, including Cotonou, Ketou, Zagnanado, and Sô-Ava. Molecular characterizations were performed on the Anopheles coluzzii was strongly represented across all study localities. The L1014F allele was observed in the localities of Kétou, Cotonou and Zagnanado. Likewise, insecticide selection pressure of homozygous resistant individuals (L1014F/L1014F) was significantly higher in Kétou, Cotonou and Zagnanado . Surprisingly in Sô-Ava, a relatively high frequency of the L1014F allele despite the reported absence of pesticide use was observed. All mosquito populations were found to be deficient in heterozygosity across the study sites (FIS< 0). No genetic differentiation (FST< 0) was observed in the localities of Zagnanado and Kétou.Carbamates, pyrethroids, organophosphorus and organochlorines use have been reported in all localities except Sô-Ava. The survey on the use of insecticides showed that insecticide selection pressures differ across the investigated localities. It would be desirable to rotate or apply formulations of combined products with different modes of action. Doing so would enable a better management of resistant homozygous individuals, and mitigate the resistance effect of commonly used insecticides. Vector-borne diseases are among the main causes of morbidity and mortality in humans and animals . Out of In the fight against malaria, two prevention methods are used: long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS) . On the An. gambiae  and large when it is between . This differentiation is very large if its value is greater than 0.25. The multiple comparison Chi square test for proportions was performed with R version 3.3.3.Several analytical approaches were used to establish the genetic structure of populations. The genetic constitution of each population is determined by calculating the allelic and genotypic frequencies from the ockerham  and Robeockerham , implemeby Hartl . DependiSixty farmers and household managers were asked about the pesticides they use. Sociological surveys have revealed that several classes of pesticides have been used in Zagnanado, Kétou and Cotonou. Those products have various commercial names, including Glyphosate 480, Herbfini, Herbex tra, Glycel, Finish, Force Up, Atrazine, Paraphosis, Fariapermefos EC, Lambda super, Pentagon, Para plus. Figure Pesticides identified during the surveys are of the classes of pyrethroids , organophosphorus compounds , organochlorines (endosulfan) and carbamates (Furadan). The proportions of each family of pesticides surveyed in the localities under study are shown in Fig. As it can be seen in Fig. An. gambiae complex were sampled and the results are presented in Table An. coluzzii, An. gambiae or rarely their hybrids. Regardless of the locality, An. coluzzii was the majority species and An. gambiae was poorly represented. The An. gambiae/An.coluzzii hybrids were found only in Cotonou in statistically insignificant proportion. The relatively small number of An. gambiae obtained across the localities did not allow a robust statistical comparison of the proportions by the localities. For this reason, the study of the genetic structure was carried out only with An. coluzzii.The various species of the L1014 of the kdr gene, the genotypes of An. coluzzii in each locality were determined and the different results are presented in Table L1014F/L1014F) were strongly represented across the localities with values ranging from 55.294 to 92.572%. The highest rate was obtained in Cotonou (92.572%) while the lowest was reported in Sô-Ava (52.941%). In both heterozygotes (L1014F/L1014L) and sensitive homozygotes (L1014L/L1014L), the tendency is reversed with the highest proportions reported in Sô-Ava and the lowest in Cotonou. In each locality, the resistant homozygotes are highly represented, followed by the heterozygotes while the wild type homozygotes are poorly represented. From the Table L1014F/L1014F) in Cotonou, Sô-Ava, Zagnanado and Ketou . A detailed pairwise comparison showed a significant difference in Cotonou-Sô-Ava, Cotonou-Kétou, Sô-Ava- Ketou, Sô-Ava- Ketou, Sô-Ava- Zagnanado , but there is no significant difference between Cotonou-Zagnanado  and Zagnanado-Kétou . Similar results were observed with the heterozygous individuals (L1014L/L1014F) across all localities. However, no significant difference was observed across municipalities in susceptible homozygous individuals (L1014L/L1014L).Using the site An. coluzzii population of Cotonou, Kétou and Zagnanado as it can easily be seen in Table An. coluzzii population of Sô-Ava . In order to confirm the EHW in Sô-Ava, the contingency test of χ2 at ddl = 1 was performed to allow us sufficient robustness and confidence on the conclusions to be drawn. For this purpose, the theoretically expected number of each genotype have been calculated. These numbers are 44.3964, 34.2071, 6.3964 for genotypes L1014F/L1014F, L1014F/L1014L, L1014L/L1014L, respectively.On the one hand, the equilibrium of Hardy–Weinberg (EHW) has not been observed in the 2 = [(45 − 44.3964)2/ + (33 − 34.2071)2/(34.2071) + (7 − 6.3964)2/(6.3964)] = 0.1077. The probability for ddl = 1 associated with this value of χ2 is 0.7427 which is both contained in the domain of validity and very close to that (0.7882) displayed by the p-value.χ(Ho) and expected heterozygoties (He), fixation index (FIS), genetic differentiations (FST), in An. coluzzii populations across the localities.Table An. coluzzii populations except for Sô-Ava. No genetic differentiation was observed in the An. coluzzii populations of Zagnanado and Ketou (FST< 0). Nevertheless, this differentiation was moderate in Cotonou.The observed heterozygosity was lower than that expected in the populations. EHW has not been observed in most populations. The heterozygosity deficit was widely shared among the The surveys conducted on the utilization of insecticide identified four classes of pesticides in the localities with variable use against mosquito vectors and other pests. Except for Sô-Ava, pyrethroids and organophosphates are the mostly used classes of pesticides. This is not surprising since previous studies have shown in Benin that these two classes of insecticides are predominantly used in agriculture against crop pests, and also in public health in the fight against malaria-carrying insects . The stuAnopheles coluzzii is strongly represented across all study localities. Corbel et al. [An. coluzzii is the predominant species (> 98%) in southern Benin. In Cotonou and Sô-Ava, the constant presence of water favours the availability of breeding sites and the rapid reproduction of mosquitoes. In this study, molecular identification of the collected mosquito samples clearly shows that the different species live in sympathy with a predominance of An. coluzzii, thus confirming the same observation reported by other studies [An. coluzzii develops in polluted waters and in permanent and semi-permanent breeding sites [An. coluzzii, while temporary breeding sites are colonized by An. gambiae. In Kétou and Zagnanado, the predominance of An. coluzzii still reflects the high distribution of this species in southern Benin as reported in previous studies [An. gambiae species was poorly represented while its twin sister species An. coluzzii was dominant in all localities. The few numbers of An. gambiae collected could only be related to the collection method used (larvae collection) due to difficult access to the majority of An. gambiae breeding sites. Adult collections generally show a more diverse species composition than those observed in larvae collections.l et al.  reported studies –45. Mosqng sites . This obng sites  have rep studies . The domL1014F allele of the kdr gene observed in Sô-Ava despite the reported absence of pesticide use remains to be carefully investigated. The proximity of Cotonou to Sô-Ava may explain the relatively high pesticide selection of resistant mosquitoes in Sô-Ava. In fact, resistant mosquitoes could migrate from Cotonou to colonize the surroundings of Sô-Ava. For example, Nkya et al. [kdr gene is associated with an overproduction of cytochrome P450 gene products (CYP9J4 and CYP6P1) and alpha esterases AGAP006227 in a region where insecticide pressure is low but very close to an agricultural area in Kilimanjaro, Tanzania where the use of these synthetic substances is intense. It appears that this pattern of joint metabolic actions is conducive to the survival of the mosquito, and is one that is shared by populations of mosquitoes of the same lineage. Further Biochemical investigations on the detoxification of toxic chemicals are essential in populations of An. coluzzii. In the case of Sô-Ava, the use of long-lasting insecticidal nets (LLINs) with other unknown factors potentially favour the selection of resistant individuals in this locality. Pesticides use in vector control is considered to be the main cause of resistance emergence in mosquito populations [The relatively high frequency of a et al.  showed tulations .L1014F allele was observed. Yadouleton et al. [In Cotonou, a high frequency of n et al.  showed tn et al. . Other sn et al. –53.L1014F allele was also observed in Ketou and Zagnanado. The main crops in these areas were found to be cotton and rice, respectively [An. gambiae has become a major concern for the success of malaria control in the last decade [A high frequency of ectively , 32. Proectively –56. In fectively . This prt decade , 58. Thit decade , 60. At L1014F allele was observed, which was observed by the high frequency of the L1014F/L1014F genotypes. Several studies have shown in Benin, in the sub-region and throughout Africa that its resistance has been associated with high pesticide use. For example, in Burkina Faso, the work of Dabire et al. [kdr mutations. Similarly, resistance to DDT and pyrethroids from An. gambiae s.s. populations in urban and agro-industrial environments has been observed in southern Cameroon [In the municipalities of Cotonou, Zagnanado and Kétou, a high selection of the e et al.  showed tCameroon .An. coluzzii exposed to insecticides and, in some cases, an almost total loss of molecular polymorphism was observed, with a frequency of the resistant allele L1014F exceeding 95%. In light of the results of this study and in the current environment, the selective value of resistant homozygotes L1014F/L1014F is likely higher than that of heterozygotes L1014F/L1014L which is in turn, higher than that of sensitive homozygotes L1014L/L1014L. In the current context, studies on the ecological adaptation of disease vectors need to be deepened in order to provide further clarification on mosquito ethology.At this stage, the genetic structure suggests that directional selection seems to be predominant in the populations of Anopheles population. This situation can explain the relatively high p-values (Table The EHW was observed in Sô-Ava. Since this locality is a lake city with a permanent presence of water and a strong wind action, it is not excluded that these factors mitigate or inhibit the effect of insecticides in the es Table , suggestL1014F allele even if other factors may be involved. This observation would result in the effect of pesticides on mosquitoes which eliminate susceptible individuals leaving first-generation resistant individuals inbreed because of their limited population size. The resulting offspring will transmit almost exclusively the resistance allele to their descendants and, therefore, ensure the expansion of the resistance genetic mutation within the populations. Several studies have documented on the African continent, the role of chemical use in the development and spread of resistance. Such studies carried out in Benin [The fixation index calculated in the localities of Cotonou, Kétou and Zagnanado shows that all populations display a gap in panmixia. Heterozygous deficiency is generally observed in populations of these communities. It has been shown in population genetics that the causes of such an equilibrium gap of Hardy–Weinberg can be the Wahlund effect, inbreeding, genetic drift, selection against heterozygotes or their combination. Based on the results of the sociological surveys, it is tempting to believe in a situation of strong selection ofthe in Benin , Camerooin Benin , Sudan [in Benin  and otheNo genetic differentiation was observed in the populations of mosquitoes of Zagnanado and Kétou. With these two localities being geographically close, this finding may be related to an apparent homogeneity of biotic and abiotic factors that could create genetic differentiation between individuals, and to some extent to the resolution power of the type of marker used. Moderate genetic differentiation in the population of mosquitoes of Cotonou may be rooted in the extrinsic factors capable of exerting, among others, insecticide pressures on the individuals of this population.An. coluzzii. The use of LLINs could also contribute to the selection of resistant individuals [Genotypic differentiation within the different populations of the localities studied shows that genotypes are distributed similarly to Sô-Ava despite the fact the use of any class of pesticides was registered in that community. In one way or another, this observation suggests that there are several other unidentified factors which are involved in the distribution of ividuals .The survey on the use of insecticides showed that insecticide selection pressures differ across the investigated localities. Although vector control programmes depend exceptionally on the use of residual insecticides, they should be preceded by a study of the genetic structure of vector populations because it depends essentially on the ecology of the environment and the genetic pool of vector populations. It would be desirable to rotate or apply formulations of combined products with different modes of action. Doing so would enable a better management of resistant homozygous individuals, and mitigate the resistance effect of commonly used insecticides. Further investigations using other methodological approaches such as microsatellites and nucleotide sequencing of appropriate genes providing a higher resolution, are necessary. Such deeper investigations may provide a better evaluation of the genetic differentiation within and between populations of disease vectors and assess the impact of the environment on their genetic structure and diversity.


Siphamia tubifer, and its luminous bacterial symbiont, Photobacterium mandapamensis in the Okinawa Islands, Japan. Using restriction site-associated sequencing (RAD-Seq) methods, we show that populations of the facultative light organ symbiont of S. tubifer exhibit genetic structure at fine spatial scales of tens of kilometers despite the absence of physical barriers to dispersal and in contrast to populations of the host fish. These results suggest that the host’s behavioral ecology and environmental interactions between host and symbiont help to structure symbiont populations in the region, consequently fostering the specificity of the association between host generations. Our approach also revealed several symbiont genes that were divergent between host populations, including hfq and a homolog of varS, both of which play a role in host association in Vibrio cholerae. Overall, this study highlights the important role that a host animal can play in structuring the distribution of its bacterial symbiont, particularly in highly connected marine environments, thereby promoting specificity of the symbiosis between host generations.All organisms depend on symbiotic associations with bacteria for their success, yet how these interspecific interactions influence the population structure, ecology, and evolution of microbial symbionts is not well understood. Additionally, patterns of genetic variation in interacting species can reveal ecological traits that are important to gene flow and co-evolution. In this study, we define patterns of spatial and temporal genetic variation of a coral reef fish,  In open marine environments, where there are few physical barriers to dispersal, microbes are expected to exist as well-mixed populations, supporting the classic panmictic view in microbial biogeography that “everything is everywhere, but the environment selects” . HoweverThe evolution of a symbiotic association requires that the specific partnership between host and symbiont remains stable over host generations. Such specificity is maintained in vertically transmitted symbioses via the intimate transfer of symbiont from parent to offspring and commonly leads to co-speciation . In contSiphamia tubifer, inhabits shallow coral reefs in the Indo-Pacific and has known life history attributes  methods , generating 100 bp single-end sequence reads.The whole, intact light organ, composed of fish tissue and containing the luminous bacterial symbiont population, was aseptically removed from each fish specimen and individually preserved in RNAlater methods , using tprocess_radtags command in Stacks v1.35 . Individuals with fewer than 100,000 total sequence reads or a mean depth of coverage per stack less than 100x were discarded from the analysis as they were considered to be outliers with respect to evenness of coverage across the individual sequence libraries  across all individuals. Additional missing data filters were also tested but did not have a notable effect on the analysis  were performed on the Bray-Curtis dissimilarity matrices calculated from the presence or absence of these haplotypes across all light organs in each dataset. Significant differences in genetic differentiation were confirmed by a permutational multivariate analysis of variance (PERMANOVA) on the genetic dissimilarity matrices using the  “vegan”  package  “vegan”  using sa “vegan”  were theSiphamia tubifer sequence reads were assembled de novo in Stacks and genotypes were assigned to each individual fish. Specifically, the first single nucleotide polymorphism (SNP) within each of the 8,637 loci present in 70% of individuals in each population with a minor allele frequency of 5% or greater were analyzed for each corresponding host fish. We selected these data filters based on previous RAD-Seq studies of other marine organisms  using the “adegenet”  additional PERMANOVAs were performed on the genetic dissimilarity matrices as described above for the symbiotic bacteria. Mantel tests with 1,000 permutations were also performed on the dissimilarity matrices calculated for each dataset  to test for correlations between the genetic distances of the host fish to that of its light organ symbionts with the “vegan” package  and lowest (<1%) scores along first two axes of variation were identified and of these, 24 outlier haplotypes were further selected as potential outliers driving the observed genetic divergence between symbiont populations based on their large differences in CAP scores relative to all other haplotypes  using the  package  in R wasplotypes . For eac  around Okinawa Island and up to 140 km  between Okinawa Island and Kume Island. The host fish exhibited no evidence of genetic structure at either scale sampled in 2013 and 2014  from fish sampled in 2014 revealed a signature of genetic structure for populations of the symbiotic bacteria in contrast to the host fish (n = 465) from light organs sampled in 2013 at the more local geographic scale of two to tens of kilometers suggest that the symbiotic bacteria are more genetically homogeneous around Okinawa Island .Using a ddRAD-Seq approach, we identified a total of 601 bacterial loci for the light organ symbionts of s genome  and thusost fish , and wasost fish . This stost fish , which fost fish . Howevera Island , althouga Island . FurtherWe identified putative loci driving the observed pattern of genetic divergence between populations of the symbiotic bacteria in 2014 and identified 24 outlier haplotypes from 22 loci. These haplotypes were highly correlated, either positively or negatively, with the primary axis of genetic variance differentiating the Kume Island and Okinawa Island populations . All 22 To determine the extent to which light organ symbiont populations were stable over time, we also analyzed the genetic variance of bacteria from fish light organs collected from one location Sesoko,  in threeP. mandapamensis populations between reefs, we also examined symbiont diversity within individual light organs. Across all 282 S. tubifer specimens examined, an average of six (±1.6 SD) distinct bacterial genotypes were detected within a light organ   , indicatS. tubifer, a symbiotically luminous fish commonly found on coral reefs in the Okinawa Islands. This approach together with RAD-Seq methods allowed us to obtain deeply sampled, fine-scale genomic data capable of distinguishing populations of this bacterium from various locations sampled in the region, and in the absence of obvious barriers to dispersal. Although RAD-Seq methods have become increasingly popular in recent years . Additional documents such as the STACKS catalogs and haplotype files generated will be made available by the authors, without undue reservation, to any qualified researcher.The raw Illumina sequences from this study are accessible at NCBI SRA Accession No. The animal study was reviewed and approved by The University of Michigan’s Institutional Animal Care and Use Committee.AG conceived and designed the study, performed the laboratory work, and analyzed the data. AG and PD performed the fieldwork and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


AFLPs) to investigate effects of population size, geographic isolation, immigration, and gene flow on genetic structure, divergence, and diversity in populations of Tetrix subulata pygmy grasshoppers (Orthoptera: Tetrigidae) from 20 sampling locations in southern Sweden. Analyses of 1564 AFLP markers revealed low to moderate levels of genetic diversity  within and significant divergence among sampling localities. This suggests that evolution of functional traits in response to divergent selection is possible and that gene flow is restricted. Genetic diversity increased with population size and with increasing proportion of long‐winged phenotypes (a proxy of recent immigration) across populations on the island of Öland, but not on the mainland. Our data further suggested that the open water separating Öland from the mainland acts as a dispersal barrier that restricts migration and leads to genetic divergence among regions. Isolation by distance was evident for short interpopulation distances on the mainland, but gradually disappeared as populations separated by longer distances were included. Results illustrate that integrating ecological and molecular data is key to identifying drivers of population genetic structure in natural populations. Our findings also underscore the importance of landscape structure and spatial sampling scheme for conclusions regarding the role of gene flow and isolation by distance.Genetic diversity within and among populations and species is influenced by complex demographic and evolutionary processes. Despite extensive research, there is no consensus regarding how landscape structure, spatial distribution, gene flow, and population dynamics impact genetic composition of natural populations. Here, we used amplified fragment length polymorphisms ( It has long been recognized that the effects of stochastic events and selection depend on the spatial and temporal scales of environmental variation relative to the mobility, behavior, dispersal capacity, and life span of the organism  describes the accumulation of local genetic differences under geographically restricted dispersal, and an expectation under the IBD hypothesis is that neutral genetic differentiation will increase with increasing geographic distance Slatkin, . HoweverEvaluating how dispersal influences population structure is complicated further by the fact that dispersal does not always translate into gene flow. This is because immigrants may die before they reproduce, behavioral differences between individuals from different populations may function as mating barriers, and out‐breeding depression associated with admixture of genotypes from different locally adapted populations may result in nonviable offspring  pygmy grasshoppers   Figure  are suit) Figure , and theT. subulata normally move only a few meters per day , or that represent older populations that have been much influenced by recent immigration (and that might show signatures associated with admixture).Pygmy grasshoppers are promiscuous  population dynamics make pygmy grasshoppers an interesting model system for studies of population genetic structure. Our goals in this study were to use amplified fragment length polymorphism (AFLP) data , diurnal, ground‐dwelling, and widely distributed insect that inhabits biomes ranging from tropical rainforests to arctic regions of Europe, Asia, and much of North America south to Mexico , in which 442 marked T. subulata were released and recaptured by two people on five occasions, capture probability (40%) did not vary over time periods despite that weather conditions were different  of these were recaptured 4 days later when the area was searched by three people.Capture probability is similar across studies of n = 19 locations) and search time (n = 17 locations) in addition to the total number of grasshoppers collected. Total number of grasshoppers collected increased with increasing number of people involved in the search  but was not significantly associated with search time . It also was not necessary to adjust for search time because the questions addressed concerned population size rather than population density. There was a strong correlation between total number of grasshoppers collected and number of grasshoppers collected per person involved in the search , indicating that total number of grasshoppers collected is a reliable surrogate for population size.Humans that search for pygmy grasshoppers tend to behave in accordance with optimal foraging theory. The cumulative number of grasshoppers collected increases asymptotically with search time. When several people search at the same area, the asymptote is reached faster but the cumulative number of grasshoppers collected does not increase. This is likely because the number and density of grasshoppers remaining in an area decreases in an exponential decaying manner over time, and individuals tend to stop searching when detection rate drops below a critical level. Thus, giving up density is almost invariable, but giving up time decreases when more people are searching, such that total search effort is comparable across sampling sites and/or occasions. For subsets of the 20 sampling locations, we recorded the number of people involved  with population size remained qualitatively unchanged when we used the restricted data set and treated number of grasshoppers collected per person as a proxy for population size to control for differences in search effort.F1,18 = 43.41, r = .84, n = 20 samples, p < .0001, analysis based on data for 658 males and 1815 females reported in Berggren et al. . Furthermore, results from previous capture–mark–recapture studies show that capture probability is independent of both sex , or that represent older populations that have been much influenced by recent immigration (and that might show signatures associated with admixture).The proportion of long‐winged individuals at each site was used as a proxy for immigration rate. Earlier work  individuals for DNA extraction . Three negative controls and nine positive controls were included on each plate. PCR reactions were diluted 1:8 of which 2 μl was sent to Uppsala Genome Center for fragment analysis using capillary electrophoresis on an ABI3730XL DNA Analyzer (Applied Biosystems).Analysis of AFLP was carried out as described by Vos  and cleaned by removing chromatogram files with chromatograms of poor quality. Chromatograms were visualized and evaluated using GeneMapper 5.0 (Applied Biosystems), and peak heights from a total of 1,970 polymorphic sites were extracted for further analysis. Peak heights were normalized and converted to a binary presence–absence matrix of genotypes with a locus selection threshold of 200 and a phenotype calling threshold of 10 using AFLPscore , and our results and conclusions are therefore not influenced by any difficulties associated with transferring AFLP information across laboratories.2.4IS value of 0  was estimated using 5,000 permutations implemented in AFLPsurv .Genetic differentiation between sampling localities  were correlated with the geographic distances (log km) that separated populations, we used a Mantel test with 10,000 randomizations implemented in ARLEQUIN version 3.5 , that is, whether the genetic differences between populations  using Jaccard distance, which calculates the dissimilarity between asymmetric binary variables, implemented in the vegan package from the software R Studio V.0.98.501  ranged from 1 to 10, and each K was run 15 times using a burn‐in period of 10,000 iterations followed by 20,000 Markov chain Monte Carlo (MCMC) repetitions without using sample locations as local priors  that best fitted the distribution in the data using the Evanno method  were sequentially removed from the model, starting with the highest order three‐way interaction. Eta‐squared, η2, was calculated to estimate local effect size  across all localities   Table . Nearly,v) Table .K = 3); the mean likelihood values reached a plateau after K = 3  indicated that the individuals included in our data set clustered into three populations K = ; the meaFST = 0.12, p < .001).Results of the nested AMOVA provided statistically significant evidence for spatial population structure Table . NestingFST were not positively correlated with the geographic distances separating populations  when calculated over all mainland sites, correlations calculated over more closely located mainland sites (<50 km) were clearly positive  were not lower than estimates based on comparisons between two large populations , indicating that the results of our analyses of population differentiation were not influenced by population size.Pairwise Nm) indicated that migration rate between populations (mean = 8) ranged from 1.7 to 2,500 individuals per generation (Table Estimated gene flow (3.3Much (83%) of the total variation among populations in the level of within‐population genetic diversity, estimated by Hj, could be accounted for by geographic region together with our estimates of population size and immigration rate Table . As expeF1,6 = 24.46, p = .0026).The effect on genetic diversity of immigration rate, as estimated by the incidence of the long‐winged phenotype, depended on region Table . The sig4FST value of 0.25) 20 Tetrix subulata pygmy grasshopper populations sampled in southern Sweden. The signature of divergence was particularly strong between populations from the mainland and populations from the island of Öland situated 5 km off the Swedish east coast in the Baltic Sea, but significant differentiation was evident also within the two regions. The overall genetic structure seen in these grasshoppers can be accounted for by a combination of landscape structure and habitat configuration, together with effects of immigration and (re)colonizations on gene flow, and effects of drift associated with small population sizes, as discussed below.Our analyses of 1,564 polymorphic AFLP markers demonstrate low to moderate genetic diversity  within and significant differentiation among  increased overall with increasing population size. This suggests that rare alleles may have been lost from smaller populations due to the eroding effects of random genetic drift, in concordance with predictions from neutral genetic theory colonizations, and admixture on genetic diversity within subpopulations have been less investigated. We found that the level of genetic diversity within populations increased with increasing proportion of long‐winged phenotypes across populations on Öland, but no such association was evident on the Swedish mainland. Importantly, this differential pattern remained unchanged when the most isolated populations on the mainland were excluded from the analyses such that the comparison was made among mainland and insular groups of populations separated by comparable geographic distances. These results thus seem to indicate that the positive effects of gene flow from immigration and admixture on diversity were stronger on Öland. Previous studies show that That the level of genetic diversity within populations was not associated with the incidence of long‐winged phenotypes across mainland populations might reflect the combined outcome of different processes (immigration and admixture vs. founder events) with opposing effects on genetic diversity.It is less likely that the relationship between genetic diversity and long‐winged phenotypes on Öland was influenced to any important degree by founder events, because one would normally hypothesize populations that have been recently established by a few founder individuals to have lower (not higher) genetic diversity  in the Baltic proper. The open water separating Öland from the mainland was hypothesized to act as a dispersal barrier, reducing gene flow and contributing to large‐scale genetic differentiation. That some of the mainland populations that were close to the island (A57 and A58) grouped with the Öland populations along axis 2 in Figure The two clusters on Öland might represent different colonization events separated in time or descendants to colonizers that originated from geographically and genetically different source populations. That populations belonging to the two different clusters on Öland existed relatively close to each other might be due to behavioral reproductive isolation or reflect negative fitness effects of interpopulation hybridization and genetic admixture  indicating genetic divergence among populations from different sampling localities within each of the two regions. Subpopulations are generally considered to be greatly genetically differentiated if they exhibit differentiation indexes (FST) in the range of 0.25  were clearly positive  that separates Öland from the Swedish mainland has restricted gene flow and leads to genetic divergence among geographic regions. The positive association between genetic divergence and geographic separation expected under the isolation‐by‐distance hypothesis was only evident over short interpopulation distances (<50 km) on the mainland and gradually disappeared as populations separated by longer distances (up to 550 km) were included.In conclusion, this study provides an empirical example that integrating ecological and molecular data are key to identifying processes that influence population genetic structure and diversity in natural populations. Our results specifically demonstrate that even crude estimates of census population size can have high predictive power regarding the level of genetic diversity and evolutionary potential. Our findings also illustrate the potential of using the incidence of flight‐capable phenotypes as a proxy for immigration in investigations of genetic structure in wing‐polymorphic species. Finally, the overall results regarding population differentiation further underscore the importance of landscape structure and spatial sampling scheme for conclusions regarding the role of gene flow and isolation by distance.None declared.Data on sampling locations, sex, wing morph, and AFLP genotypes of individuals used in the analyses will be made available at Dryad Digital Repository following acceptance of the manuscript. Click here for additional data file.