Solanum melongena L.) is an important solanaceous crop with high phenotypic diversity and moderate genotypic diversity. Ninety-nine genotypes of eggplant germplasm , landraces and heirloom cultivars) from 32 countries and five continents were evaluated for genetic diversity, population structure, fruit shape, and disease resistance to Phytophthora fruit rot. Fruits from each line were measured for fruit shape and evaluated for resistance to two Phytophthora capsici isolates seven days post inoculation. Only one accession (PI 413784) was completely resistant to both isolates evaluated. Partial resistance to Phytophthora fruit rot was found in accessions from all four eggplant species evaluated in this study. Genetic diversity and population structure were assessed using 22 polymorphic simple sequence repeats (SSRs). The polymorphism information content (PIC) for the population was moderate (0.49) in the population. Genetic analyses using the program STRUCTURE indicated the existence of four genetic clusters within the eggplant collection. Population structure was detected when eggplant lines were grouped by species, continent of origin, country of origin, fruit shape and disease resistance.Eggplant ( Solanum melongena L., is a high-value vegetable commodity in Europe and Asia. China and India are the major producers with 27.7 and 11.9 million tons per year, respectively [2011 FAO]. Eggplant is the third most important solanaceous crop worldwide after potato and tomato, and fourth most important in the U.S. [2011 FAO]. In the U.S., eggplants are a minor crop grown for specialty markets with an approximate production of 62 thousand tons annually [2011 FAO].Cultivated eggplant, S. melongena) originated in Africa and were derived from the closely related Solanum incanum (part of the eggplant complex) Solanum linnaeanumS. incanum and S. linnaeanum can form partially fertile hybrids with S. melongena making them potential sources for desirable traits such as abiotic and biotic disease resistance Unlike most other cultivated solanaceous crops , eggplants are an Old World species. Domestication of the cultivated eggplant is thought to have occurred in Asia as early as 59 B.C. Phytophthora species are capable of causing disease symptoms on eggplants. Infected eggplants can display root and/or fruit symptoms. One causal agent of Phytophthora fruit rot is Phytophthora capsici L., an oomycete that affects multiple solanaceous species including eggplant, pepper, and tomato Phytophthora capsici-induced fruit rot in eggplant, which is the most common Phytophthora-induced symptom in eggplants P. capsiciP. capsici. Partial fruit rot resistance to P. capsici has been identified in other solanaceous species such as peppers and tomatoes . Our objectives were to evaluate a worldwide collection of eggplants for population structure and genetic diversity, and to determine if the population structure is associated with fruit shape or resistance to No specific permits were required for the described field studies, which took place in an experimental field plot at Michigan State University (MSU). This field plot was used by the authors of this paper affiliated to the aforementioned institution  for field trials to evaluate eggplant germplasm and cultivated species.S. melongena), three accessions of S. linnaeanum, one accession of S. gilo, and one accession of S. incanum were obtained from the United States Department of Agriculture Germplasm Resource Information Network (ars-grin.usda.gov), Universidad de Technologia de Valencia, and the INRA  (Ninety-four accessions of eggplants (esearch) . AccessiP. capsici isolates, previously evaluated on pepper, tomato and eggplant, were selected from the long-term collection of Dr. Mary K. Hausbeck at MSU Two virulent P. capsici isolate on V8 agar was placed, mycelium side down onto a non-wounded eggplant fruit surface-disinfested in 10% bleach for 5 min and rinsed with distilled water. Control eggplants were inoculated with a single 6 mm-diameter sterile plug of agar. Plugs were covered with a sterile microcentrifuge tube and affixed into place with petroleum jelly. Eggplants were placed into a humidity chamber consisting of an aluminum pan with a ring of moistened paper towel around the edge, covered with plastic wrap, sealed with tape and kept under constant light at room temperature (25°C). Three fruits (replicates) from each eggplant line were evaluated per isolate. The experiment was performed three times (runs). An experiment replicate included three fruits for each isolate of every eggplant accession evaluated in a completely randomized design (CRD) blocked by isolate. One line (PI 500922) was repeated only one time for a total of two experimental replicates of three fruit per isolate due to poor fruit set. Two control fruits were inoculated with a sterile plug of V8 agar for each line.For inoculation, a single 6 mm-diameter plug from an actively growing Phytophthora isolations were performed on 10% of symptomatic fruits by peeling back the external layer of the fruit and plating three small portions of fruit tissue at the disease margin onto V8 agar plates amended with benomyl, ampicillin, PCNB, and mefenoxam to confirm the causal agent of the symptoms Phytophthora capsici was identified using morphological characteristics according to Waterhouse Eggplant fruits were evaluated for disease severity seven days after inoculation. Fruits were evaluated on the following progressive scale based on the percentage of symptomatic fruit surface to account for differences in fruit size: 0 =  no visible symptoms (resistant (R)), 1 = <25% of the fruit was symptomatic (moderately resistant (MR)), 2 = 25% to <50% (moderately susceptible (MS)), 3 = 50% to <75% (susceptible (S)), 4 = ≥75% symptomatic area (susceptible (S)) . VisibleTen immature fruits of marketable size collected from each line were measured for maximum length (cm) and maximum width (cm) using a hand caliper. Fruit shape was calculated as the ratio of maximum length to midpoint width for each line. Fruit shape ratios were rounded to the nearest whole number. Values between 0 and 1 were considered round, 2–3 were considered oval, 4–5 were semi-elongate and >5 were considered elongate.P = 0.05). Accession by isolate interactions were calculated using the ANOVA slice option of PROC MIXED when P≤0.05. Lines with a consistent disease mean value of ≥2 in each run of the experiment were considered susceptible, with a consistent mean value <2 were termed moderately susceptible, lines with a consistent mean value <1 were moderately resistant, and lines with a mean value  = 0 were resistant. Significant differences for pathogen growth were estimated using the PROC GLIMMIX function of SAS at P = 0.05.Mean values for disease ratings for each accession were estimated using the PROC MEANS function of SAS software v9.3 . Significant differences between disease values (ratings) for eggplant accessions and isolates were estimated using the PROC MIXED function of SAS software. Significant differences were detected between experiment runs and each run was analyzed separately using Fisher's LSD test  according to the manufacturer's instructions. DNA was normalized to 5 ng/ µl using the NanoDrop ND 1000 spectrophotometer and NanoDrop 2.4.7c software .http://primer3.sourceforge.net/) from putative Solanaceae defense-related genes (NCBI http://ncbi.nlm.nih.gov) were tested against a subset of the eggplant collection to identify polymorphic markers. Reactions were performed in 15 µl total volume and contained 1 µl DNA, and 0.15 µl GoTaq , 0.9 µl 25 µM MgCl2, 0.3 µl dNTPs, and 0.6 µl each of forward and reverse primers , with 8.45 µl ddH2O. PCR reactions were performed in a programmable thermal cycler  using the program: initial denaturation, 94°C (3 min) followed by 35 cycles at 94°C (30 s), 60°C (30 s) and 72°C (1 min), with a final extension step of 10 min at 72°C. PCR products were analyzed by electrophoresis in 4% (wt/vol) agarose gel in 1× Tris-borate-EDTA buffer, stained with ethidium bromide (5 ug/ml) for visualization and compared to a 100-bp ladder  to determine amplicon sizes. SSR markers identified as polymorphic in the population were used for genetic diversity, population structure and trait associations.One hundred ninety-two primers from previously published SSR markers  based off of 1000 bootstrap replications.Visualization of the resulting Q  of each accession into predefined categories  was generated using the Population Sorting Tool (PST) in R P<0.0001). No significant differences in disease severity were found between isolates in any replicate of the experiment . The interaction between accessions and isolate was significant for each run  of the experiment. Differences in pathogen growth (absence/presence) and the interaction between pathogen growth and accession were not significant in any replicate (approximately P = 1.0 for each). The majority of the accessions were susceptible at 7 days post inoculation to isolates OP97 (89%) and 12889 (87%), respectively  to isolate OP97. Eggplant lines H15 and PI 441908 were R to isolate 12889 and S and MS to isolate OP97, respectively. Two of the S. linnaeanum lines, PI 388847 and PI 388846, and the S. incanum accession PI 500922 were R or MR to isolate 12889. Lines PI 181896, PI 233916, MM 56, and PI 413783 were MS to isolate 12889. Phytophthora capsici isolates were successfully recovered from diseased fruits and mefenoxam sensitivity was confirmed for each isolate (data not shown).Significant differences between experimental runs indicated the effect of environmental variability during fruit growth and development on fruit disease resistance was high. In each repetition of the experiment, there were significant differences among plant accessions (ectively . Symptomectively . EggplanS. melongena accessions had fruit shape ratios ranging from 1 (round) to 8 (elongate). The wild species evaluated  had a fruit shape ratio of approximately 1 (round) with fruit ≤3 cm (Solanum melongena line PI 413783 had the lowest fruit shape ratio (0.46) and line MM 522 had the highest fruit shape ratio (8).Fruit shape and size varied considerably in the population , Table 2S. melongena fruits from Japan had the highest length:width ratio indicating fruits were slender and elongated. Fruits from Thailand had the lowest fruit shape ratio, indicating fruits were more round. Fruit length and width also varied greatly between countries. Fruits from China were the widest and Japan the narrowest. Fruits from Japan were also the longest and fruits from Thailand were the shortest  genetic differentiation with S phenotypes for isolate OP97. Significant genetic differentiation was also detected between the MS and the S category for isolate 12889. No significant genetic differentiation was found between the R/MR category and the MS categories for either Phytophthora isolate (data not shown). Pairwise Fst differentiation between fruit shape categories was low (0.00) to moderate (0.1)  . Individte (0.1) .S. linnaeanum, S. gilo and S. incanum accessions were placed into genetic Cluster 3, while S. melongena individuals were distributed through each of the clusters (Population structure of the 99 accessions was estimated using the STRUCTURE software and the 22 polymorphic SSRs. Accessions were grouped into four genetic clusters (Ln = - 3381.8). clusters . Seventyclusters . Relatioclusters , countryclusters , speciesclusters  and diseclusters , as someclusters –6. Clustclusters . For botclusters . When grThis study investigated Phytophthora fruit rot resistance, fruit shape, population structure and genetic diversity in a worldwide collection of eggplant. The overall estimate of genetic diversity of the collection was moderate (0.5) in our study, similar to a recent report on eggplant diversity, though this is likely to be an underestimation due to limited sampling P. capsici seen in the field S. incanum, S. gilo, and S. linnaeanum. While further evaluation with more isolates is necessary, PI 413784 appears to be a promising source of host resistance to Phytophthora fruit rot in eggplant.On eggplant, fruit rot is the most common symptom of P. capsici isolates when breeding for resistance. The three wild relatives, S. linnaeanum, S. gilo, and S. incanum, showed partial or isolate-specific resistance to the two isolates evaluated in this study.When categorized by disease resistance  for each isolate, there was significant genetic differentiation among eggplant genotypes infected with isolates OP97 or 12889. Individuals that were R and MR to isolate OP97 were significantly differentiated from individuals that were S. Only S individuals were significantly differentiated from the MS individuals when inoculated with isolate 12889. These results emphasize the importance of utilizing different S. linnaeanum, S. gilo, and S. incanum individuals evaluated were predominantly in genetic Cluster 3. Solanum incanum has long been part of the eggplant complex and ancestral individuals are thought to be one of the progenitors of modern eggplant Solanum linnaeanum is a related species and has only recently been included as a possible progenitor of the modern eggplant with limited crossability S. melongena category, supporting gene movement between S. melongena and its wild relatives, S. incanum and S. linnaeanum. These S. melongena individuals may have been misclassified, but are more likely the result of introgression since the wild species were small fruited and prickly.When grouped by species, Solanum melongena fruit shape, size and color is a byproduct of domestication, selection, and breeding for specific market classes. Phenotypic evaluation of eggplant fruit shape varied greatly among the S. melongena accessions evaluated, while the wild species, S. incanum, S. gilo, and S. linnaeanum, had no variation in fruit shape. Maintaining market class variation may be difficult when incorporating traits like fruit rot resistance, which was most often observed in accessions with a limited size range.Cultivated eggplant, similar to pepper and tomato, is a phenotypically diverse species with varying levels of genotypic diversity S. melongena individuals with elongated fruit shapes and those with round and oval fruit shapes. Individuals with a round fruit shape were also significantly differentiated from semi-elongate fruit shape individuals. These results are consistent with limited breeding among market classes. However, inferred population structure did not correspond with the fruit categories and this may be the result of limited sampling in each geographic location. While only genetic Cluster 3 was not represented in the round or elongate shape category, all other clusters were represented by at least one individual in each category.Significant differences in this population were observed in fruit shape, length and width among eggplant lines when grouped by country of origin, representing different market classes, in this study. Since eggplant has market classes particular to geographic areas, it was expected that population structure categorized by fruit shapes and country of origin would correspond with the inferred genetic clusters. Significant differentiation was seen between When grouped by country and continent, significant population structure and moderate genetic diversity was evident among the categories evaluated. The highest levels of genetic diversity were seen within the continents of Africa and N. America. The highest level of genetic diversity for countries was in the USA. The increased genetic diversity in Africa is likely due to the prevalence and intercrossing of wild and related species, as Africa is the center of origin for several eggplant species. The increased genetic diversity in N. America and the USA may be the result of breeding programs integrating wild relatives and varieties from around the world. The diversity could also be from the movement of Asian and European varieties into the U.S., which may be marketed under different names. Overall differentiation among countries was similar to the differentiation among continents, and future core collections should include individuals from areas with high genetic diversity and genetic differentiation. In particular, genotypes from China were not significantly differentiated from any other country, while genotypes from Thailand, Japan, Spain, Macedonia and the U.S. were frequently significantly differentiated from other countries. Similarly, Asia was not significantly differentiated from populations from Europe, Africa and N. America, while N. America, Europe and Africa were all significantly differentiated from each other. Asia, as a center of diversity and domestication, and in particular genotypes from China, may be more akin to the ancestral population from which these other pools were derived, making them more similar and less differentiated from other eggplants.Cultivated eggplant, compared to other solanaceous species, is an understudied crop with worldwide importance. This study provides an overview of the population structure, genetic diversity and Phytophthora fruit rot resistance of a geographically diverse set of eggplant. The estimates of genetic diversity and the four genetic clusters found in this study are likely to be lower than actual genetic diversity and structure of eggplant due to limited sampling and molecular markers. A previous study using a subset of SSRs in a smaller collection of eggplant was able to identify more allelic variation at each locus


Acipenser brevirostrum, oft considered a phylogenetic relic, is listed as an “endangered species threatened with extinction” in the US and “Vulnerable” on the IUCN Red List. Effective conservation of A. brevirostrum depends on understanding its diversity and evolutionary processes, yet challenges associated with the polyploid nature of its nuclear genome have heretofore limited population genetic analysis to maternally inherited haploid characters. We developed a suite of polysomic microsatellite DNA markers and characterized a sample of 561 shortnose sturgeon collected from major extant populations along the North American Atlantic coast. The 181 alleles observed at 11 loci were scored as binary loci and the data were subjected to multivariate ordination, Bayesian clustering, hierarchical partitioning of variance, and among-population distance metric tests. The methods uncovered moderately high levels of gene diversity suggesting population structuring across and within three metapopulations  that encompass seven demographically discrete and evolutionarily distinct lineages. The predicted groups are consistent with previously described behavioral patterns, especially dispersal and migration, supporting the interpretation that A. brevirostrum exhibit adaptive differences based on watershed. Combined with results of prior genetic  and behavioral studies, the current work suggests that dispersal is an important factor in maintaining genetic diversity in A. brevirostrum and that the basic unit for conservation management is arguably the local population.The shortnose sturgeon,  Sturgeons (Acipenseridae) are one of two living groups of chondrostean fishes; the other group being the paddlefishes (Polydontidae). The fossil record suggests these were dominant fishes of the Permian period ∼200 Myr  and owinHindIII PstI Documenting heritable genetic information is a hallmark of contemporary conservation strategies for delineating management units and has previously been applied to sturgeons where recent molecular systematics studies have called into question the taxonomic foundations of the Acipenseriform classification, which has been historically based on morphological characters Gene duplication and subsequent functional divergence is a fundamental process of adaptive evolution Acipenser brevirostrum is an amphidromous species endemic to the large coastal rivers of eastern North America. This species is distinguished among all the living acipenserids by exhibiting the largest number of chromosomes, 372 A. brevirostrum was listed as an “endangered species” under the US Endangered Species Preservation Act of 1967 and remains so despite re-assessment in response to a 1994 petition to de-list populations in tributaries to the Gulf of Maine. Of significant note is that of 19 putative population units identified based on the species' perceived strong fidelity to natal rivers The shortnose sturgeon A. brevirostrum has been assessed through nucleotide sequence variation detected in the maternally-inherited mtDNA. This is presumably due to the difficult nature of interpreting allelic data from the functionally polyploid (putatively hexaploid) nuclear genome A. brevirostrum mtDNA CR, analyses of haplotype frequencies at the level of putative individual populations showed significant differences among nearly all river/estuarine systems in which reproduction is known to occur. One prior study A. brevirostrum appear to function in discrete populations, and that relatively low female-mediated gene flow exists between the majority of populations. This implies that effective dispersal among drainages within regions has been sufficient to prevent deep divergence within this species over evolutionary time scales.To date, all published information on phylogeographic- and population-level structuring in Acipenser brevirostrum has been shown to possess the highest number of chromosomes (N = 362–372) among all the Acipenseriformes karyotyped to date in situ hybridization) suggest A. brevirostrum is a hexaploid species A. brevirostrum conservation could identify significant levels of informative genetic variation because certain duplicated loci and repetitive DNA may lack functional constraints, thus allowing rapid accumulation of differentiation in DNA sequences A. brevirostrum. However, no phylogeographic or population informative nuclear markers have been identified for A. brevirostrumA. brevirostrum from a multilocus nuclear DNA perspective, we characterized the inheritance of polysomic microsatellite DNA loci in shortnose sturgeon collected throughout the species' range using loci derived specifically from this species To address this important research need and for the first time, allow an extensive assessment of the phylogeographic structure of Acipenser brevirostrum (n = 561) were sampled from 17 river and estuarine systems representing the species' range (2 fin-clip) taken from soft pelvic fin was stored in 95% absolute ethanol or SDS/urea.While no tissue sampling was collected as part of this study, s' range  by reseaA. brevirostrum in the Potomac and Merrimack Rivers affected categorization of specimens. We chose to treat the Potomac River collection (n = 2) both as of unknown origin and as part of the large Chesapeake Bay-proper collection. The Merrimack River sample consisted of males collected at the same location and time; however, because eggs (n = 4) and embryos (n = 2) were collected in Spring 2009, we considered the Merrimack River collection as a reproducing population. Genomic DNA from ethanol preserved samples was extracted with the Gentra Puregene DNA kit  following the manufacturers guidelines for whole non-mammalian blood and resuspended in TE . Previously extracted genomic DNA from other researchers was extracted using the methods described in The ambiguous reproductive status of A. brevirostrumAbrB438, AbrD10, AbrD114, AbrD135, AbrD141, AbrD193, AbrD236, AbrD332, AbrD345, AbrD379, and AbrD557. Optimized PCR mixes consisted of the following: 100–200 ng of genomic DNA, 1× PCR buffer , 2.0 mM MgCl2, 0.2 mM dNTPs, 0.4 mg/ml Bovine Serum Albumin, 2.0 U Taq DNA polymerase , 0.25 µM forward and 0.5 µM reverse primer, and 0.3 µM fluorescent labeled M13 primer in a total volume of 25 µl. Amplifications were carried out on either PTC-200 or PTC-225 Thermal Cyclers  using the following cycling conditions: initial denaturing at 94°C for 15 min; 29 cycles of 94°C for 1 min; annealing temperature (56–66°C) for 45 sec, 72°C for 45 sec; 5 cycles of 94°C for 1 min; 53°C for 45 sec; 72°C for 45 sec; and final extension at 72°C for 10 min. Fluorescently labeled fragment analysis was performed using an ABI 3130XL Genetic Analyzer and binning of alleles was performed using GENESCAN 2.1 Analysis software and GENOTYPER 3.6 fragment analysis software . Each locus was independently scored, and each amplicon meeting the signal strength conditions specified  and fitting into the appropriate size category  was classified as an allele.A suite of 11 microsatellite loci previously identified from A. brevirostrum, allele dosage could not be reliably estimated from GENESCAN runs preventing the application of standard population genetic diversity statistics that require genotype or allele frequencies for their calculation. Two prior groups A. brevirostrum allelotype matrix using the method of For polyploid individuals, gene duplication, multiple alleles, and the mode of inheritance can lead to practical and statistical complications in allelic identification and interpreting summary and population-level statistics Allelotypes were analyzed for patterns of population ‘genetic’ structure and for regions of genetic discontinuity at both the individual and collection levels. Pair-wise genetic distances of each individual fish to all other individuals  was used by first identifying the “uppermost” hierarchical level of population structure followed by subsequent analysis of each cluster to identify within-cluster structure k = 1 to k = 20 clusters were considered for the 17 collections using a burn-in of 20,000 followed by 50,000 iterations, and 20 independent runs for each k. The optimum number of clusters in the initial phase was identified using Δk as described by Evanno et al. k = 1 to k = C+3 (the number of collections (C) included in the subset plus three), with a burn-in of 20,000 followed by 50,000 iterations, and 20 runs for each k. In the within-cluster analyses, k also was determined using the Evanno et al. method A second individual-based analysis designed to infer population structure among collections and identify genetic discontinuity in the allelotype matrix was performed using the model-based clustering method of the program STRUCTURE 2.3.2 PT, for assessing the underlying structure contained in the allelotype matrix. Jaccard's similarity coefficient A. brevirostrum using PAST. The test statistic, R  and its significance were computed by permutation of group membership, with 10,000 replicates. R values were presumed to be proportional to genetic distance. AMOVA ΦPT calculated in GenAlEx using the binary data was considered as a coefficient of dissimilarity PT values and significance levels of the variance components , based on 10,000 permutations, were measured for all collections. The relationship between the ecological-based (Jaccard's) distance and the population genetic distance (ΦPT) matrices for all 17 collections was statistically assessed with a matrix regression analysis Direct comparisons were made of two pair-wise population-scale distance measures, Jaccard's and ΦPT for the amount of genetic differentiation at these selectively neutral microsatellite loci according to S. Wright's classical relationship ST, ΦPT, into this equation is, to our knowledge, untested.Because a small number of effective migrants can have a profound effect on a small population, as the effects of drift can be large in the absence of balancing gene exchange, estimation of the number of migrants rather than simply the rate A. brevirostrum was inferred by analysis of the pair-wise population ΦPT matrix using two methods. The non-metric multidimensional scaling  of ng NMDS,  option oA. brevirostrum, two comparisons were made to visualize patterns among the 14 collections common to both studies. Ordination of the nDNA ΦPT and mtDNA ΦST matrices was independently performed using the MDS option of PAST. A separate analysis of the relationship between the ΦST and ΦPT pair-wise distance matrices was statistically assessed with the Mantel matrix regression analysis To compare the genetic variation observed for nDNA (this study) with mtDNA Maximum likelihood assignment tests were used to determine the likelihood of each individual's multilocus allelotype being found in the collection from which it was sampled using the program AFLPOP 1.1 n = 3) to 152 . Estimated heterozygosity (assuming Hardy-Weinberg equilibrium) ranged from 10.4% (Cape Fear) to 19.3% (Hudson). A lower estimate of heterozygosity was observed among southeastern populations compared to mid-Atlantic and northeastern populations.A total of 181 alleles were observed at the 11 loci resulting in a binary character matrix of 181 loci (columns) by 561 individuals (rows) (summarized in Northeast - including five rivers from the Gulf of Maine (GOM), namely Saint John River, Canada; Penobscot, Kennebec, Androscoggin and Merrimack rivers; 2) Mid-Atlantic - including the Connecticut, Hudson, and Delaware rivers, and the Chesapeake Bay proper; and 3) Southeast – including the Cape Fear River, Winyah Bay, the Santee-Cooper, Edisto, Savannah, Ogeechee, Altamaha rivers, and Lake Marion. The number of inferred clusters (k) determined by STRUCTURE for the initial  analysis was three, corresponding to the Northeast, Mid-Atlantic, and Southeast regional groupings identified by PCO  existing among the three regions, a sequential method of inferring the number of clusters was employed to identify within-cluster structure  and differentiated from Connecticut and Hudson River samples  indicating the presence of multiple populations displaying statistically significant differences in allele frequencies throughout the range of A. brevirostrum. Given the range and magnitude (0.040–0.249) of the ΦPT values in question, the non-significant findings associated with this collection can likely be attributed to the inadequate sample size. The six remaining non-significant ΦPT values were observed among geographically proximal collections within the Northeast and Southeast groupings (clusters). Pair-wise ΦPT values were greatest among collections compared among the three major clusters and lowest among collections within these groupings. Low ΦPT estimates were observed among collections in the Northeast (average 0.06) and Southeast (0.047) clusters. Moderately high ΦPT estimates were observed among the four collections in the mid-Atlantic cluster (averaging 0.077) although the value between the Delaware River and Chesapeake Bay collections was 0.018. Considering the hierarchical AMOVA results (A. brevirostrum were consistently supported: 1) St. John River in Canada, 2) three Maine rivers, 3) Merrimack River, 4) Connecticut River, 5) Hudson River, 6) Delaware River/Chesapeake Bay; and 7) southeast .At the population level, pair-wise Φ results  in lightPT values. This correspondence was further reflected in the Mantel matrix regression analysis of the relationship between ΦPT and Jaccard's distance measures, which indicated a strong, nearly absolute, correlation between the two distance metrics .Non-parametric analysis of similarity (ANOSIM) of Jaccard's  distance resulted in a similar pattern of genetic differentiation in the pairwise R values (PT matrix (data not shown) revealed a congruent pattern among the 17 collections with that observed with the individual-based PCO and in the STRUCTURE analysis (Multivariate (NMDS) analysis of the underlying structure contained in the population-level pair-wise Φanalysis  resultinPT matrix also was depicted with an unrooted neighbor-joining (N-J) tree  among the three major groupings of collections was strongly supported in the backbone of the phenogram and the evolutionary distinctiveness of the populations or groups of populations was confirmed by high bootstrap support, in particular, the absolute (100%) bootstrap support distinguishing the southeast clade from all other collections, and the high bootstrap support for clades containing the Northeast (98%) and Mid-Atlantic (89%) collections. The high degree of genetic similarity observed with other analyses among the Penobscot, Androscoggin and Kennebec Rivers collections and between the Delaware River and Chesapeake Bay collections (99% bootstrap support) was confirmed with high bootstrap support for these pairings.The underlying genetic structure of the Φ-J) tree , which iA. brevirostrum migrants per generation, emN (based on ΦPT), estimated between all pairs of collections  of the genetic variation occurred among collections (river/estuary populations) and 84% (P<0.001) was attributed to differentiation within collections. Of 11 hierarchical AMOVA analyses (P<0.001) of the genetic variation occurring among groupings, 3% (P<0.001) occurring among populations within groupings, and 80% of the genetic variation was due to variation within collections. All four models, based on the subclusters identified by the PCO and STRUCTURE analyses, were variations either including or omitting the Saint John and Merrimack Rivers as distinct populations. All attempts to manipulate the southeast grouping of populations resulted in a decrease in variation among grouping components and an increase in the amount of within population variation.Quantitative estimates of hierarchical gene diversity among all collections also identified statistically significant genetic structuring; 16% , assignment to each collection was statistically greater than would be expected by chance (P<0.05). When the 17 rivers were pooled by the geographic regions identified in The average correct assignment to collection of origin was 58.6% and ranged from 0% (Cape Fear River) to 97.8% (Connecticut River) . With thPT (this study) and ΦST  of pair-wise distance metrics, performed matrix regression analyses, estimated the evolutionary history among populations via phylogenetic algorithm (phenogram), conducted hierarchical partitioning of variation (AMOVA), and assessed the success of likelihood assignment testing. Notably, the results of all statistical approaches used to assess phylogeographic structure resulted in consistent findings.This study represents the first report of nuclear DNA variation in the higher-order polyploid Acipenser species such as A. brevirostrum are considered to be evolutionarily static and are often referred to as phylogenetic relics. Intraspecific examination of the nuclear genome has revealed the presence of considerable allelic diversity and differentiation that appears to be reflective of the actions of various evolutionary processes. Phylogeographically, these findings suggest the presence of similar levels of genetic diversity and variation among the collections punctuated with a series of genetic discontinuities of varying “depth” across the species' range that could indicate demographic independence, regional adaptation, and reflect vicariant geographic events. Populations sampled within these regional groupings exhibited shallow but statistically significant differentiation, which were congruent with theoretical estimates of gene flow. Moreover, patterns of population relatedness were consistent with the observations of Owing to their morphological similarity to lower Jurassic (∼200 MYBP) Acipenseriformes A. brevirostrum range, six lineages are relevant in conservation considerations. In addition to support for recognition of these zones of discontinuity in clustering, the phylogeographic analysis, AMOVAs, and assignment testing, all suggested there are low levels of theoretical gene exchange between collections on either side of these genetic discontinuities.Upon inspection of the patterns of allelotypic variation at the individual and population scales in the nDNA two major (“deep”) zones of genetic discontinuity are inferred from all analyses conducted 1) separating the Northeastern and Mid-Atlantic collections and 2) delineating the Mid-Atlantic and Southeastern populations. Moreover, narrower  zones of genetic discontinuity were evident among Saint John, Merrimack and the three Maine rivers in the Northeast region, and between the Connecticut and Hudson River collections and between Hudson River and an apparent Delaware River/Chesapeake Bay metapopulation (unconfirmed) within the mid-Atlantic region. This implies there are seven demographically and evolutionary distinct lineages across the range and that within the United States portion of the A. brevirostrum. Parker A. brevirostrum were locally adapted to particular rivers. These researchers demonstrated differences in the innate dispersal patterns in early life stages of A. brevirostrum from Connecticut River versus sturgeon of Savannah River origin, suggesting that A. brevirostrum are likely behaviorally adapted to unique features of their watershed. Parker and Kynard A. brevirostrum from different rivers can have different migration strategies. Similar adaptive differences have been inferred for other sturgeon species including Acipenser fulvescens (Wolf and Menominee rivers [Kynard and Parker unpublished data]), A. transmontanus , we are faced with the need to specify evolutionarily relevant thresholds for deciding statistical significance of haplotype or allelotype frequencies. Ultimately, the approach that may offer maximum flexibility in reaching recovery goals may be to allocate (or nest) biological units that are most likely to respond to management within regions. The following two sections examine specific cases and the suggestion that a metapopulation approach may serve best to illustrate the structure of gene flow in this species.The phylogeographic and genomically-congruent patterns predicting genetic structuring of A. brevirostrum population's age-to-reproduction is different than other GOM sturgeon, 2) it is the northernmost reproducing population, and 3) it experiences differences in control of exploitation, management of habitat, and conservation status.Differentiation among the Northeast populations is on the whole less than observed for the Mid-Atlantic populations but greater than among the Southeast populations. Within the Northeast group, the Saint John River, Canada collection constitutes a population that is appreciably differentiated from the GOM collections at a number of levels. Foremost, the degree of gene differentiation between Saint John River and the GOM is “shallow”. This “shallowness” of may be attributed to the fact that these populations are relatively young (due to recent deglaciation of the region) as compared to more southerly distributed populations based on the observed levels of divergence. In addition, designation as a distinct management unit may be warranted because 1) the Saint John River A. brevirostrum exhibited patterns of nDNA variation that suggest the collection is genetically differentiated from the other GOM collections. Such an interpretation should be made with caution because the sample consisted of 22 males collected at the same location and time, and because the level of differentiation is not as great as that observed among other collections proposed as distinct evolutionary lineages. Choosing to consider this collection as a distinct evolutionary lineage would serve the precautionary principle and conserve biodiversity.The GOM collections analyzed in this study included a recently obtained sampling from the Merrimack River. These A. brevirostrum in the Potomac  Maine rivers , 2) Delaware River and Chesapeake Bay proper, and 3) the Southeast assemblage . Population biology theory predicts that lower dispersal and associated gene flow leads to decreased genetic diversity in small isolated populations, which generates adverse consequences for fitness, and subsequently for demographic stability. Given recent tagging data suggesting A. brevirostrum migrate to adjacent rivers to a greater extent than previously believed A. brevirostrum populations.In addition to the demographically discrete and evolutionarily significant lineages identified for A. brevirostrum nuclear genome to discriminate evolutionarily significant lineages of management relevance. The variation detected was highly phylogeographically congruent with the inferences based on the mtDNA control region  that likely delineate seven demographically discrete and evolutionarily significant lineages, each with differing adaptive potential for this species. These zones of inferred genetic discontinuity represented deeper levels of differentiation and a higher degree of reproductive isolation than that typically attributed to population-level differentiation; groups of populations of evolutionary significance which may warrant distinct conservation considerations. Perhaps more notable than the delineation of evolutionarily significant lineages, was the identification of at least one putative metapopulation within each of the three major regional groupings of populations, a finding that is encouraging as migration may help stave localized extinctions. It should be noted that while an increased level of gene flow is present within these putative metapopulations, a demographic connection has yet to be established and documentation of such a link should be considered a high priority research need. Moreover, many of the populations within an evolutionarily significant lineage were genetically differentiated to some degree and each geographic population is subjected to differing threats A. brevirostrum is arguably the individual  population.This study presents evidence that sufficient levels of genetic diversity are present in the A. brevirostrum populations should be a prominent recovery goal; the allelotypic patterns observed in this study provide guidance by facilitating understanding of where and how such efforts could be attempted. For example, the best available information suggests that individual rivers and estuaries along the North Carolina coast do not currently support reproducing populations of A. brevirostrum. If the distance to North Carolina (or elsewhere) rivers that could support a reproducing population exceeds the vagility of sturgeon inhabiting the southeast or Delaware River/Chesapeake Bay metapopulations, targeted translocations or restorative supplementation may represent plausible restoration strategies. In contrast, rivers geographically south of the Altamaha River historically occupied by shortnose sturgeon , no longer support reproducing A. brevirostrum populations. The shoreline distances of these rivers to the Altamaha River are similar to that observed among the major rivers comprising the Southeast metapopulation. Given the recent findings of moderate physical migration A. brevirostrum inhabiting many of the rivers in the Southeast metapopulation, it seems logical that if one or more of these southernmost rivers provided suitable spawning and rearing habitat, A. brevirostrum would have effectively colonized this region. Therefore, habitat characterization and/or restoration in these southernmost rivers could facilitate range expansion.Assuming conservation of local populations within a metapopulation is the acceptable focus, restoration of effective connectivity among currently fragmented A. brevirostrum have been shown to be moderately high in both nuclear (this study) and mitochondrial  between Jaccard and ΦAcipenser brevirostrum) surveyed at 11 polysomic microsatellite DNA loci.(TIF)Click here for additional data file.Figure S2ST matrix for 14 Atlantic coast collections of shortnose sturgeon   for the same collections surveyed at 11 polysomic microsatellite DNA loci.) and the nuclear DNA Φ The correlation coefficient (r) for this analysis was 0.84 (P<0.0001).(TIF)Click here for additional data file.Table S1Pair-wise R values  and Bonferroni-corrected probability values  from the non-parametric Analysis of Similarity (ANOSIM) (Clark 1993) on Jaccard's distance metric (1-Jaccard similarity) measured among 17 collections of shortnose sturgeon (Acipenser brevirostrum) surveyed at 11 polysomic microsatellite loci.(DOC)Click here for additional data file.Table S2Hierarchical structuring of genetic variation was measured for numerous combinations of shortnose sturgeon (Acipenser brevirostrum) collections using analysis of molecular variance (AMOVA). Significance levels of the variance components were based on 1000 permutations. Abbreviations are as follows: NE = Northeast regional grouping includes Saint John River (SJ), Canada, Penobscot, Kennebec, Androscoggin and Merrimack rivers; Mid-Atlantic regional grouping includes the Connecticut (CT), Hudson (H), and Delaware (DE) rivers, and the Chesapeake Bay proper (CB); and the 3) SE = Southeast regional grouping includes the Cape Fear River (CF), Winyah Bay (WB), Santee-Cooper (S-C), Edisto (E), Savannah (S), Ogeechee (O), and Altamaha (ALT) rivers, and Lake Marion (LM).(DOC)Click here for additional data file.Table S3Assignment to collection of origin for 17 shortnose sturgeon (Acipenser brevirostrum) collections surveyed at 11 polysomic microsatellite DNA markers. Mis-assigned individuals are distributed horizontally.(DOC)Click here for additional data file.Table S4Assignment to proposed grouping (five groupings model) in shortnose sturgeon (Acipenser brevirostrum) surveyed at 11 polysomic microsatellite DNA markers. The overall correct assignment rate to proposed grouping was 99.1% (522/527). Mis-assigned individuals are distributed vertically. Northeast regional grouping includes Saint John River (SJ), Canada, Penobscot, Kennebec, Androscoggin and Merrimack rivers; and the Southeast regional grouping includes the Cape Fear River (CF), Winyah Bay (WB), Santee-Cooper (S-C), Edisto (E), Savannah (S), Ogeechee (O), and Altamaha (ALT) rivers, and Lake Marion (LM).(DOC)Click here for additional data file.


We tested for fine scale, regional, and historic patterns of genetic structure. As expected, significant differentiation was detected among rivers within drainages and among drainages. At finer scales, an unexpected lack of within-river genetic structure among fragmented sandy microhabitats suggests that stratified dispersal resulting from unstable sand bar habitat degradation  may preclude substantial genetic differentiation within rivers. Among-drainage genetic structure indicates that postglacial (14 kya) drainage connectivity continues to influence contemporary genetic structure among Eastern Sand Darter populations in southern Ontario. These results provide an unexpected contrast to other benthic riverine fish in the Great Lakes drainage and suggest that habitat-specific fishes, such as the Eastern Sand Darter, can evolve dispersal strategies that overcome fragmented and temporally unstable habitats.Quantifying spatial genetic structure can reveal the relative influences of contemporary and historic factors underlying localized and regional patterns of genetic diversity and gene flow – important considerations for the development of effective conservation efforts. Using 10 polymorphic microsatellite loci, we characterize genetic variation among populations across the range of the Eastern Sand Darter  Specialized microhabitat dependence presents a formidable challenge to species conservation in changing environments. For some species, the coupling of microhabitat specialization with increased habitat degradation and fragmentation can initiate or accelerate declines in population size and, ultimately, local extirpation. Microhabitat specialization provides an extreme example of local adaptation and raises questions about mechanisms that allow the persistence of such specialized life histories in variable environments. Characterization of genetic structure and gene flow among fragmented habitats can yield important information for the conservation of such microhabitat-dependent species. Specifically, a hierarchical analysis can reveal the relative importance of large-scale historical processes  and more contemporary fine-scale processes  in shaping overall patterns of genetic variation Wiens .Molecular genetic methods can provide nonlethal means to successfully characterize many aspects of ecosystem processes and population connectivity for species at risk, including landscape effects on genetic substructure  is a small benthic riverine fish federally listed as threatened in Canada and listed as threatened in many states in its American distribution , and the Ausable River (Lake St. Clair drainage).The Eastern Sand Darter  characterize contemporary population connectivity through analyses of genetic structure and dispersal and (2) determine the relative influence of historic (postglaciation) colonization patterns versus current connectivity processes on drainage-level genetic structure. In general, we expect high genetic structure among sand bar sites for A. pellucida, even at small spatial scales, due to the species’ restricted distribution to sandy substrate habitats. Additionally, we expect high levels of genetic divergence among regions as a result of population isolation and decline  and an additional five loci from other darter species  probes, captured with streptavidin-coated beads , and enriched using polymerase chain reactions (PCR). The resulting enriched DNA library was inserted into TOPO vectors and transformed into One Shot competent Escherichia coli cells . Clone inserts were amplified using M13 universal forward and reverse primers and sequenced at Genome Quebec Innovation Centre . Microsatellite primer pairs were designed and optimized for polymorphism and ease of PCR amplification. PCR amplification of all ten microsatellite loci used in this study was performed in 12.75 μL reactions containing approximately 50–100 ng template DNA, 0.25 μL of 0.5 μmol/L dye-labeled forward primer, 0.25 μL of 0.5 μmol/L reverse primer, 200 μmol/L of each dNTP, various concentrations of MgCl2 , with significance determined by jackknifing across all loci at the 95% confidence interval in FSTAT (Goudet AR), number of alleles (A), observed heterozygosity (HO), expected heterozygosity (HE), inbreeding coefficient (FIS) were also calculated in FSTAT.Genetic differentiation was estimated by calculating pairwise T Goudet . AllelicFST/(1 − FST); Slatkin A. pellucida prefers shallow, sandy habitats so hydrological distances were determined at the drainage scale using two methods: shallow water restriction  and open-water dispersal .Rivers with more than three sampling sites were tested for adherence to an isolation by distance (IBD) model of migration–drift equilibrium. IBD was determined using the association between linearized genetic differentiation  generations, allele frequencies correlated, and admixture allowed. The number of genetic clusters was allowed to range from K = 1 (range-wide panmixia) to the total number of rivers plus one (K = 17). Runs were compiled using full searches in CLUMPP1.1.2 , and the Monmonier's maximum difference algorithm identified which of the borders between neighboring populations exhibited anomalous genetic divergence relative to spatial separation.Analysis of molecular variance (AMOVA) was used to hierarchically partition genetic variation within each drainage into three levels: among rivers, among sites within rivers, and within sites using ARLEQUIN. We also identified the number of population genetic clusters using the Bayesian-based clustering program STRUCTURE 2.3.4  linkage disequilibrium was determined for five of 390 possible locus-by-locus combinations over all the sites, with no two loci identified in linkage disequilibrium for more than one site indicating that our marker loci likely are not linked.A total of 1051 individuals were collected from 39 sites in 16 rivers across the entire species range from June 2010 to November 2011 , although some (<10%) between-site FST values were substantial and significant  compared to the other drainages . This pattern persisted even when geographic distances were corrected to 1000 km .Within-river pairwise ant Table. The paiant Table; this isges Table. Global R2 = 0.61, P = 0.039); however, no significant IBD was found in the Hocking River. Low FST values among all sites in the Thames and Grand rivers resulted in a lack of IBD correlation for both rivers . Mantel tests of IBD among rivers within each drainage showed that both the Ohio drainage  and Great Lakes drainage  had significant IBD  nor St. Lawrence drainage  adhered to an IBD pattern of divergence.Due to limited numbers of within-river sample sites, IBD was only assessed in three rivers  in the Great Lakes drainage and one river (Hocking River) in the Ohio drainage. Significant within-river IBD was found for the Maumee River (P = 0.002), Great Lakes drainage , St. Lawrence drainage . However, the among-river genetic variance component was 15–20 times higher in the three drainages: Ohio drainage , Great Lakes drainage , St. Lawrence drainage . The highest proportion of genetic variance in all analyses was attributed to the within-sites component: Ohio drainage , Great Lakes drainage , St. Lawrence drainage . The Wabash drainage was excluded from the AMOVA analysis because the within-river sample sites were not replicated  with population delineation at chiefly the river level  genetic variance: Ohio drainage , as opposed to contemporary connectivity alone . The ΔAICc between the two AMOVAs was 12.6, highly supportive for the historic model (AICc = 2763) versus the contemporary model (AICc = 2686) of genetic variation. Both AMOVA analyses revealed substantial and very similar components of the genetic variance attributed to within-river variation .AMOVA results for both historic and contemporary connectivity models yielded highly significant models; however, a greater proportion of the among-group genetic variance was explained when the groups reflected the historic connection between the Wabash River and Great Lakes drainages . However, our analyses clearly indicate substantial movement of individuals among the sampled habitats. The lack of genetic structure within the sampled rivers likely reflects species-specific dispersal that counteracts patchy habitat distribution.Freshwater fish species inhabiting formerly glaciated regions commonly exhibit genetic signatures that reflect the influence of historical glacial refugia and recolonization patterns  may contribute to the lack of within-river genetic structure and this, in turn, would act to buffer individual sand bar populations from genetic drift effects and loss of genetic diversity , suggesting that open-lake habitat does not represent a major barrier to the dispersal–drift equilibrium for Etheostoma caeruleum, Haponski et al. A. pellucida: The first occurred between the Thames and Sydenham rivers, and the other two were among rivers in the Wabash drainage. Low genetic differentiation between the Thames and Sydenham river populations can be explained by either dispersal between the spatially close river mouths in the shallow Lake St. Clair or headwater connections from natural floods or anthropogenic fish movement. The genetic similarity between these two rivers most likely reflects a headwater connection or human-mediated transfer, as suggested for Greenside Darter  were covered most recently by the Wisconsinan continental ice sheet , 12,000–13,500 years ago, and subsequently through Lampsilis Lake (present-day St. Lawrence River), 8500–10,000 years ago  The genetic divergence of Trautman . FollowiA. pellucida populations throughout the species range. Based on the pattern of among-river genetic divergence, supplementation and reintroduction plans for extirpated systems with currently suitable habitat (Dextrase et al. Our study emphasizes the blending of contemporary and historic influences on the genetic structure of 10.5061/dryad.q2d3v.Microsatellite genotype data available on Dryad: DOI None declared.


Scyliorhinus canicula), across European seas. The results show significant genetic differences among most of the Mediterranean sample collections, but no significant structure among Atlantic shelf areas. The data suggest the Mediterranean populations are likely to have persisted in a stable and structured environment during Pleistocene sea-level changes. Conversely, the Northeast Atlantic populations would have experienced major changes in habitat availability during glacial cycles, driving patterns of population reduction and expansion. The data also provide evidence of male-biased dispersal and female philopatry over large spatial scales, implying complex sex-determined differences in the behaviour of elasmobranchs. On the basis of this evidence, we suggest that patterns of connectivity are determined by trends of past habitat stability that provides opportunity for local adaptation in species exhibiting philopatric behaviour, implying that resilience of populations to fisheries and other stressors may differ across the range of species.Elasmobranchs represent important components of marine ecosystems, but they can be vulnerable to overexploitation. This has driven investigations into the population genetic structure of large-bodied pelagic sharks, but relatively little is known of population structure in smaller demersal taxa, which are perhaps more representative of the biodiversity of the group. This study explores spatial population genetic structure of the small-spotted catshark ( They Scyliorhinus canicula L., 1758) is a relatively small demersal species belonging to one of the largest families of sharks, the Scyliorhinidae. It is generally considered to be the most abundant catshark in European shelf seas [S. canicula is still targeted for consumption today [The effects of marine overexploitation have been clearly shown by severe declines in many shark species . Understelf seas  and occuelf seas . It is aelf seas ,20. In telf seas , and it elf seas . In the elf seas , and S. on today . Recent on today . For exaon today .Scyliorhinus canicula has a range of traits associated with a low dispersal potential, including internal fertilization and deposition of demersal eggs. In addition, mark–recapture studies suggest adults do not generally make long migrations [S. canicula noted significant morphological differences in dentition between west African, Mediterranean and west European populations, indicating the west African group could represent a distinct taxon [Investigations of elasmobranch population structure focusing on wide-ranging pelagic sharks have often revealed genetic differentiation over broad inter- or intra-oceanic scale ,28. By cgrations . These fgrations ,25. Indegrations ,33. Simict taxon .Documented sex-biased dispersal and philopatry have also been shown to have serious effects on elasmobranch population structure. Female sharks generally make far greater investment in reproduction than males, potentially leading to discrepancies between optimal male- and female-fitness strategies, and generating sex-specific differences in behaviour ,35. EvidMolecular data have the power to infer historic processes, such as population expansions or contractions, locations of refugia and patterns of recolonization . InferenS. canicula collected across European seas. A particular focus is made of the Atlantic–Mediterranean transition, as it is often considered to be an important phylogeographic break. We also use these data to test for sex-biased differences in dispersal and philopatry. We discuss the results in the light of published work on the behavioural ecology of the species, and highlight the conservation and management implications.Here, we test for population genetic structure among populations of 3.3.1n=45; 2008, n=26; 2010, n=39). A minority of samples were also collected at landing or at fish markets, most notably all those from Portugal, Sardinia and Crete. Genomic DNA was isolated from S. canicula using the Wizard technique .Tissue samples were collected between 2007 and 2011 , primarily from research cruises throughout Europe and the Mediterranean  section of the mitochondrial DNA (mtDNA) control region was amplified using the newly designed primers ScyD1pF (ATGACATGGCCCACATATCC) and Scan2R (TTCTCTTCTCAAGACCGGGTA), using PCR conditions described in Griffiths et al. . PCR prov. 7.0.9  and aliglustalX2 .3.4FST values between the sampling periods in Arlequin 3.11 [icrochecker [ervus 3.0 [FIS), probability of conformity to the expectations of Hardy–Weinberg equilibrium (HWE) and linkage disequilibrium (LD) which were calculated in Genepop 4.0.7 [Patterns of temporal variation at the Western Channel site were examined by calculating pairwise uin 3.11 . Microchochecker  was usedrvus 3.0 , except op 4.0.7 . Allelicop 4.0.7 .n=1 and Norway n=4; FST values was conducted in Arlequin using 10 000 randomizations, and 95% CI were estimated across loci in GDA 1.0 [enAlEx 6.5 [enAlEx.Sample collections with inappropriately small sample sizes for estimating allelic frequencies from microsatellite loci  and the Mediterranean .A hierarchical analysis of molecular variance (AMOVA) was performed in A3.5tructure v. 2.3.4 [K, varied between 1 and 12 in each run. The ΔK method of Evanno et al. [K. In the analysis of each cluster, plots of the absolute values of ln Pr(X|K) and ΔK were generated by Structure Harvester [The complete microsatellite dataset was analysed in the software Sv. 2.3.4 . A ‘hierv. 2.3.4  with mulo et al.  was applarvester .3.6rlequin. Rarefied haplotype richness, standardized to a sample size of 22, was calculated using Contrib 1.02 [rlequin, using both the genetic distance-based ΦST and the frequency-based mtFST, with significance of differences estimated with 10 000 permutations. A hierarchical AMOVA was performed in Arlequin, using the same scenarios as implemented with the nuclear markers. Tamura and Nei’s [enAlEx. Mantel tests were used to look for significant correlations between genetic distances and geographical distances, also in GenAlEx.Summary statistics were primarily calculated using And Nei’s  genetic nd Nei’s  and visu3.7etwork 4.6.1.1 [D [Fs [rlequin [HKY+Γ+I mutation model was selected and the Markov chain Monte Carlo (MCMC) was set between 50 and 200 million iterations, depending on length required for convergence. A fixed clock was set using a divergence rate of 0.361% estimated from homologous control sequences from S. canicula and Scyliorhinus stellaris  and the divergence date (22 Ma) from Sorenson et al. [A median joining network was used to investigate genealogical relationships between mtDNA haplotypes with N 4.6.1.1 . Tajima’6.1.1 [D  and Fu’s1 [D [Fs  tests we[rlequin , with si[rlequin . Bayesia[rlequin , and plon et al. . This isn et al. , 0.67% [n et al. ). It is 3.8et al. [t-tests in R 2.15.1 were used to compare mtFST calculated using mtDNA haplotype frequencies and FST calculated using microsatellite allele frequencies.Initially, to assess differences between male and female dispersal a test comparing pairwise genetic distances between populations for microsatellite and mitochondrial data was employed. Specifically, with male-biased dispersal, lower genetic distances were expected to be present in biparentally inherited (microsatellite) markers than in maternally inherited  markers. Following Daly-Engel et al. , paired FIS), fixation index (FST), degree of relatedness, mean assignment indices (mAIc) and variance of the assignment indices (vAIc) between the philopatric and dispersing groups were used [FIS in the most dispersing sex, while also leading to correspondingly lower FST and relatedness values. The assignment index statistic indicates the probability of a genotype occurring in a population, and unequal levels of gene flow between males and females could lead to negative values of the mAIc in the most dispersing sex (as the distribution is centred on zero), while also increasing the corresponding vAIc.Tests for sex-biased dispersal based on the microsatellite data alone were conducted in FSTAT. Samples from Sardinia were removed from analyses as no information on sex was available. Five methods based on differences on the inbreeding coefficient (ere used . In prin4.4.1FST ranged from less than 0.000 to 0.006 and none were significant at the 99% CI) and all individuals were grouped together into a single sample collection. The mean observed heterozygosity across all populations was 0.632, varying between 0.571  and 0.659 (North Sea). The mean expected heterozygosity across populations was 0.615, varying between 0.562  and 0.661 . Pairwise FST values ranged from −0.005 to 0.070 and were significant for all combinations that included the Mediterranean sampling sites, while genetic differentiation was absent among all Northeast Atlantic populations, after Bonferroni correction . There was a significant association between genetic and geographical distance across all samples . This was also observed within the Atlantic , but not within the Mediterranean . Hierarchical AMOVA . The first cluster included the samples from the Atlantic, with all individuals demonstrating admixture coefficients showing more than 80% membership to this cluster, reflecting clear common ancestry. Similarly, individuals from Sardinia, Crete and the Adriatic also demonstrated admixture coefficients showing more than 80% membership to a second cluster, suggesting clear division between these two clusters. The Mallorca samples demonstrated some degree of admixture between these groups, exhibiting coefficients with an average 73% membership to the first Atlantic-dominated cluster and 27% membership to the second Mediterranean-dominated cluster. Subsequent hierarchical analysis of subsets of the data identified additional clusters corresponding to sampling locations within the Mediterranean, namely Crete, the Adriatic, Sardinia and a Mallorcan cluster that also incorporated the single individual from Tenerife. No evidence of genetic subdivision was found across the Atlantic samples.Analysis of the complete microsatellite dataset in S4.3ΦST=0.308, p<0.001). Pairwise ΦST values ranged from −0.146 to 0.600, and were similar to the mtFST values . Haplotype diversity ranged from 0.589 (Crete) to 0.815 (Western Channel), while rarefied haplotype richness ranged from 3.749 (Crete) to 8.322 . Values of nucleotide diversity were low, ranging from 0.002  to 0.004 . There was a significant association between genetic and geographical distance across all sample collections , but not within the Atlantic , or within the Mediterranean . Hierarchical AMOVA  indicates slowly declining population sizes within the Mediterranean, with evidence of population growth in the past 100 000–500 000 years (with the exception of the Sardinian sample that shows a recent decline in effective population size). This contrasts with the Northeast Atlantic, in which the samples generally demonstrate a more sustained period of much greater population increase, dating back over 500 000–1 250 000 years.Demographic analyses showed that the Atlantic sample collections had unimodal distributions with negative Tajima’s 4.5FST was significantly greater than FST across the sampling area .The mtDNA data identified two main groups of haplotypes; the first included the highest frequency haplotypes, central to the network that were predominantly associated with samples from the Northeast Atlantic and the Balearic Islands. The second includes haplotypes unique to specimens found in central and eastern parts of the Mediterranean . Furtheren found ,78,79. Hcanicula , where vion used . Additio [et al.  grouped Amblyraja radiata) in the north Atlantic over comparable scales [The pattern of highly divided population structure across the Mediterranean contrasts very sharply with results from the Northeast Atlantic. Regardless of the markers used, or the analytical approach, there was no evidence of significant genetic differences between sample collections originating from this region. Individuals from Norway and Africa demonstrated microsatellite alleles and haplotypes that were generally common across the Atlantic, suggesting little evidence of population structure, even at the most extreme latitudinal ranges considered. This lack of genetic differences across the Atlantic waters does not conform to expectations from typical life-history characteristics of small, demersal elasmobranchs that suggest low dispersal potential . Neverthe scales ,82. Perhe scales ,84.b), and microsatellite markers indicating a more intermediate position between the Atlantic and the Mediterranean groups (a). This result is consistent with the Balearic Islands representing an important region of secondary contact. There is strong evidence that many marine species have invaded the western Mediterranean through the Strait of Gibraltar since the last glacial maximum, bringing previously allopatric lineages into contact [While nuclear and mtDNA data generally exhibited highly concordant results, differing signals of population structure were identified around Mallorca, with mtDNA suggesting a close similarity with the Atlantic group b, and min groups a. This r contact ,85–87. T5.1There have been long-standing hypotheses suggesting that the Strait of Gibraltar  or the Almeria–Oran Front represents phylogeographic barriers that shape the biogeographical patterns of marine Atlantic–Mediterranean organisms (reviewed in ). A numbS. canicula. The Atlantic populations typically showed patterns of population expansion, while the Mediterranean appeared much more stable. This could be explained by the long-term stability and the consistent existence of suitable habitat in the Mediterranean during the Pleistocene glacial and interglacial cycles, as has been observed for other species [S. canicula during climatically driven sea-level fluctuations, allowing its persistence [The Mediterranean and the Northeast Atlantic share a history of inter-connectedness and have a large number of species in common. However, some evidence of contrasting demographic signatures was detected between the Atlantic and the Mediterranean populations of  species ,96. Manysistence . Recolonsistence –45,98,99sistence , where l5.2S. canicula. This supports growing evidence of sex-based differences in dispersal and behaviour in elasmobranchs more widely [FST values, regardless of differences in sex-biased dispersal. Therefore, the recent approach of Daly-Engel et al. [Despite obvious congruence in patterns of population structure identified between the mtDNA and microsatellite data tables  and 5, ge widely ,77. Howel et al.  was usedl et al. . If popul et al. .In order to overcome the limitations with comparisons of mtDNA and nuclear DNA, an additional suite of tests for sex-biased dispersal that focus on the microsatellite markers alone was conducted. These tests have a number of assumptions, including non-overlapping generations, equal sample size, juvenile dispersal and equal sex ratios  that areThe apparent male-biased dispersal supports a long history of work demonstrating strong sex-based differences in the behaviour of the small-spotted catshark. It was the first elasmobranch species for which systematic analyses of unequal sex ratios in trawl catches provided clear evidence for unisexual aggregations ,106, wit5.3S. canicula to form multiple stocks within its distributional range. This has important implications for sustainable management, as effective conservation measures may need to be implemented at the level of the demographic unit to ensure long-term stock viability in the face of exploitation [The assessment and management of shark stocks is not well established, in part, because many characteristics such as dispersal or migratory behaviour are not fully understood . Additiooitation . This isoitation , but for


In contrast, no such differences are observed for the European taurine ancestry (P=0.1357). Excluding European introgressed animals, low and nonsignificant genetic differentiation and isolation by distance are observed among sub-locations . Following a short population expansion, a major reduction in effective population size (Ne) is observed from approximately 240 years ago to present time. Our results support ancient zebu × AT admixture in the EASZ population, subsequently shaped by selection and/or genetic drift, followed by a more recent exotic European cattle introgression.The Kenyan East African zebu cattle are valuable and widely used genetic resources. Previous studies using microsatellite loci revealed the complex history of these populations with the presence of taurine and zebu genetic backgrounds. Here, we estimate at genome-wide level the genetic composition and population structure of the East African Shorthorn Zebu (EASZ) of western Kenya. A total of 548 EASZ from 20 sub-locations were genotyped using the Illumina BovineSNP50 v. 1 beadchip. STRUCTURE analysis reveals admixture with Asian zebu, African and European taurine cattle. The EASZ were separated into three categories: substantial (⩾12.5%), moderate (1.56%<X<12.5%) and non-introgressed (⩽1.56%) according to the European taurine genetic proportion. The non-European taurine introgressed animals ( Bos taurus (Bos indicus) originated in the Indian subcontinent and migrated into Africa more recently (B. primigenius africanus (now extinct) contributed to the genetic stock of African domestic cattle remains unknown , the latter sometimes referred to as sanga . In KenyIn more recent times, a wave of ‘exotic' taurine cattle introductions has set off following governmental and international livestock development agendas aimed at improving livestock productivity  are on the verge of extinction in East Africa , but theThe recent availability of genome-wide scan tools are offering new opportunities for genetic characterization  of cattlIn this study, we characterize at genome-wide level the genetic diversity and architecture of 548 EASZ, an indigenous cattle population from Kenya. We present evidences for ancient zebu × taurine admixture, population bottleneck and expansion as well as the presence of recent and ongoing exotic taurine introgression. No significant genetic differentiation in non-European introgressed animals is observed across the studied sites. Moreover, our study further provides insight in the usefulness and limitations of low-density single-nucleotide polymorphisms (SNPs) chips toward understanding the genomic architecture and history of indigenous African tropical cattle.A total of 548 calves were sampled from 20 different randomly selected sub-locations that traverse four distinct ecological zones in Western and Nyanza provinces of Kenya . Upon rewww.cbcb.umd.edu/research/bos_taurus_assembly.shtml). Genotyping of the 548 calves was carried out at the USDA-ARS bovine functional  and GeneSeek (http://www.neogen.com/geneseek/) laboratories. An additional 158 reference animals representing two European taurine breeds (Holstein n=64 and Jersey n=28), one AT breed (N'dama n=25), one Asian zebu breed (Nelore n=21) and one East African admixed breed  breeds were drawn from The Illumina BovineSNP50 v. 1 beadchip  includes 56 947 SNPs comprising 54 436 autosomal SNPs, 1341X chromosome SNPs and 1170 unmapped SNPs .Quality control was carried out using  program . The chen=54 436) were screened. Autosomal SNPs with call rates of <90% were excluded. Unless stated, no minor allele frequency threshold (to avoid exclusion of SNPs that maybe informative in a single breed or population) and Hardy and Weinberg Equilibrium (as introgression may result in loci not in Hardy and Weinberg equilibrium) criteria, were applied. A total of 6151 SNPs failed the inclusion criterion, leaving a total 48 285 SNP for analysis. A random subset of 45 000 SNPs was used for STRUCTURE, principal component analysis, genetic relatedness and genetic differentiation analyses. No random sampling of SNPs was performed for linkage disequilibrium (LD) and effective population size calculations. Genotyping data has been deposited in Dryad  and reference breeds (n=158) totaling 706 animals (data set 1), (ii) non-European introgressed calves (n=425) and reference breeds (n=158) giving a total of 583 animals (data set 2) and (iii) non-European introgressed calves (n=425) (data set 3). The Ward clustering method  and the non-European introgressed calves (data set 3) using  v 1.3.1  to read envalues . Both paenvalues .Fst statistics were calculated using the R-based hierfstat 0.04–6 package  were used for the estimation of genetic differentiation between sub-locations in relation to geographic distance =[1/(1+4Nec)]+(1/n). Where E (r2) is the expected LD, Ne is the effective population size, c is the recombination frequency, n is the chromosome size or twice the sample size, c was calculated using the Haldane's mapping function  drawn from the cleaned data set of 48 285 markers. These particular analyses were run using the moderate and substantial calf categories consisting of 123 calves and 158 animals representing the five reference breeds . Ancestral genome proportions were generated from the STRUCTURE runs and used in the subsequent linear correlation analyses carried out between markers sets in R  within EASZ calves (n=548) and reference breeds are indicated in Ho of 0.25±0.02 s.d. with no significant differences across sub-locations (P>0.001).Out of 48 285 autosomal SNPs remaining in the data set following quality control, 11 269 markers were monomorphic across the EASZ population. The mean observed heterozygozity    . Princip=5 (n=3) . For K=4ckground . In addickground . K=5 divriesian) .K=3 model. Based on three observed clusters, we defined three categories of calves: calves with ⩾12.5% European taurine background , calves with between 1.56 and 12.5% European taurine introgression (category 2 representing the ‘moderate' European taurine introgressed sample set n=94) and calves with ⩽1.56% European taurine background (category 3 representing the ‘non-European' introgressed sample set n=425). The 123 introgressed calves, representing the moderate and substantial categories, are found within 12 sub-locations in the northern and central regions of the study area  of data set 2 reveals a homogenous admixed EASZ population (P<0.0001) was also observed.STRUCTURE analysis . Considering only EASZ calves with moderate and substantial European taurine introgression, no significant difference is observed in European taurine ancestry proportion across chromosomes (P=0.136).Strong positive correlations for the AT and Asian zebu ancestry between EASZ and Sheko are observed across chromosomes (K=3), which distinguishes the same calves  after removing the moderate and substantial European introgressed calves. Mantel test between pairwise sub-locations genetic differentiation ) and geographic distances was not significant  for the pure calves category  was obtained for the 15–25K pairwise marker comparison for the European background (r=0.92 (P<0.0001) was obtained for the 35–45K pairwise marker comparisons  and using only calves with moderate and substantial European taurine introgression. We use linear correlation between sets of randomly selected number of markers using STRUCTURE's output of inferred genetic membership proportions at stimated . As expeckground , and forckground . Howeverparisons .r2 over 0.1–0.5) has been suggested as a way to reduce data redundancy in STRUCTURE analysis and ascertainment bias in the estimation of diversity. Approximately 44% of the neighboring marker pairs linkage were above the lower limit (r2=0.1) and only 11% above the higher limit (r2=0.5) within the non-introgressed calves cohort. The proportion of marker pairs in LD approximates the genome coverage of the chip, for example, in mapping applications. Taking into account the marker gaps, these proportions equal to coverage of 40 and 8%, with the respective r2 limits. The median for neighboring marker pairs was 0.075. Therefore, no LD-based pruning was undertaken.Pruning neighboring markers in strong LD  is approximately a quarter of those generally observed in cattle populations (Decrease in LD as a function of distance between markers has been reported in several cattle population or breeds (for example, r2<0.2 , reachesulations . It is sThere are several algorithms for inferring population size from LD and it remains unclear which method is the most appropriate one (for example, Historical cattle generation lengths, prior the intensive breeding system, are expected to be longer than 4 years . We assuThe BovineSNP50 v. 1 beadchip used in this study allowed us to estimate the ancestry proportions of the three main cattle lineages within the EASZ. The largest proportion of the markers included on this beadchip was selected for informativeness in European taurine breeds . This asLivestock Revolution' (The history of East African cattle is complex. It was closely intermingled with the history of local human communities (Data available from the Dryad Digital Repository: doi:10.5061/dryad.bc598.