Sodalis glossinidius and trypanosome infection rates between Glossina palpalis palpalis populations from two Cameroonian foci of human African trypanosomiasis (HAT), Bipindi and Campo. We hypothesized that the geographical isolation of the two foci may have induced independent evolution in the two areas, resulting in the diversification of symbiont genotypes.Previous studies have shown substantial differences in To test this hypothesis, we investigated the symbiont genetic structure using the allelic size variation at four specific microsatellite loci. Classical analysis of molecular variance (AMOVA) and differentiation statistics revealed that most of the genetic diversity was observed among individuals within populations and frequent haplotypes were shared between populations. The structure of genetic diversity varied at different geographical scales, with almost no differentiation within the Campo HAT focus and a low but significant differentiation between the Campo and Bipindi HAT foci.S. glossinidius subpopulations and Glossina species that could favor tsetse fly infections by a given trypanosome species should be further investigated.The data provided new information on the genetic diversity of the secondary symbiont population revealing mild structuring. Possible interactions between  Glossina symbiont, Sodalis glossinidius, and the fly infection by trypanosomes in a south-western region in Cameroon: flies harbouring symbionts had a threefold higher probability of being infected by trypanosomes than flies devoid of symbionts. But the study also showed substantial differences in S. glossinidius and trypanosome infection rates between Glossina populations from two Cameroonian foci of sleeping sickness. We hypothesized that the geographical isolation of the two foci may have induced the independent evolution of each one, leading to the diversification of symbiont genotypes. Microsatellite markers were used and showed that genetic diversity structuring of S. glossinidius varies at different geographical scales with a low but significant differentiation between the Campo and Bipindi HAT foci. This encourages further work on interactions between S. glossinidius subpopulations and Glossina species that could favor tsetse fly infections by a given trypanosome species.Human African trypanosomiasis remains a threat to the poorest people in Africa. The trypanosomes causing the disease are transmitted by tsetse flies. The drugs currently used are unsatisfactory: some are toxic and all are difficult to administer. Furthermore, drug resistance is increasing. Therefore, investigations for novel disease control strategies are urgently needed. Previous analyses showed the association between the presence of  Tsetse flies are medically and agriculturally important vectors that transmit African trypanosomes, the causative agents of sleeping sickness in humans (human African trypanosomiasis – HAT) and Nagana in animals. This debilitating disease still affects a wide range of people in sub-Saharan Africa Glossina and trypanosome species.The biological process leading to transmission of the trypanosomes from one mammalian host to another is complex. Prior to being transmitted, the parasite must first establish in the tsetse fly midgut following an infective blood meal. Then it must mature either in the salivary glands or in the mouthparts, depending on the trypanosome species Sodalis glossinidiusGlossinaS. glossinidius displays active mechanisms of cellular interactions and is an intermediate between free-living and obligate intracellular bacteria evolving toward a specific interaction with GlossinaTsetse flies harbor three different symbiotic microorganisms G. palpalis gambiensis  and G. morsitans morsitans (morsitans group) were shown to harbor genetically distinct populations of S. glossinidiusG. palpalis gambiensis and G. morsitans morsitans transmit preferentially different trypanosomes species. The ability of Trypanosoma brucei gambiense and Trypanosoma brucei brucei to establish in G. palpalis gambiensis midgut was further linked to the presence of S. glossinidius-specific genotypes in the insectarium S. glossinidius rather than a mere presence/absence of the symbiont.Glossina-trypanosome-symbiont partners. The results showed that the trypanosome infection was not randomly distributed between subpopulations of field flies harboring S. glossinidius or free of S. glossinidius. Statistical analyses confirmed the association between the presence of the symbiont and the field flies' infection by trypanosomes: the parasite prevalence was nearly threefold higher within the populations of flies harbouring the symbiont than within those that did not harbour the symbiont Given that flies multiplying in the insectary may undergo specific selective pressures that differ from those in the natural environment, the possibility that field environmental conditions of HAT foci may lead to alternative results could not be excluded. Therefore, an epidemiological investigation was conducted in two HAT foci in the south of Cameroon. This study took into account a large panel of criteria to test the existence of interactions between the three S. glossinidius and trypanosomes, and the different symbiont/parasite associations also showed significant differences between the fly populations from the two HAT foci.The results obtained from fly trapping, the prevalence of Glossina species and may favor fly infection by a given trypanosome species in different ways.We hypothesized that the geographical isolation of the two foci may have induced independent evolution of fly and symbiont populations in each area, resulting in a diversification of symbiont genotypes. If so, it may be assumed that such genotypes may interact differently with S. glossinidius and the distribution of the different genotypes within the fly populations of the different sampling areas. Following preliminary assays, microsatellite markers seemed to be well suited to perform this analysis.The foregoing assumption necessitated a large-scale analysis of the genetic diversity of S. glossinidius, from Glossina palpalis palpalis flies sampled in Campo and Bipindi, two sleeping sickness foci in Cameroon. The aim was to analyze their genetic diversity in order to determine the genetic structure of Sodalis and to study possible rates of gene flow at various spatial scales of Cameroonian S. glossinidius populations.To the best of our knowledge, the present study is the first large-scale genetic population investigation on the tsetse fly symbiont, Glossina palpalis palpalis flies were collected in two HAT foci (Bipindi and Campo) situated in the Ocean Division of the South Region of Cameroon. The Campo focus  presents several biotopes . The Bipindi focus  has a typical forest bioecological environment including equatorial forest and farmland along roads and around villages. Both foci contain highly diversified wild fauna The Bipindi focus has been known since 1920 Campo is located on the Atlantic coast and extends along the Ntem River Glossina species were first identified and then sorted into teneral and non-teneral flies, according to morphological criteria Entomological surveys were conducted in 2007 in Bipindi and Campo. The geographical positions of the sampling sites were determined using the global positioning system. Tsetse flies were captured using pyramidal traps g, 15 min), the pellet was rinsed with 70% ethanol, air-dried, and resuspended in distilled sterile water. The DNA samples were stored at −20°C until PCR amplification.DNA was extracted from tsetse fly midguts using cetyl trimethyl ammonium bromide (CTAB) as described by Navajas et al. http://frodo.wi.mit.edu/primer3/)  , Table 2For each of the four couples of primers, one of the primers was 5′ end labelled with an infrared dye (IRD700 or IRD800) for sizing the PCR products with an automatic sequencer. Primers were synthesized by MWG .S. glossinidius was performed on midgut-extracted DNA, as previously described S. glossinidius were further processed for S. glossinidius microsatellite genotyping. The method was adapted from Farikou et al. Specific primers amplifying tsetse fly mitochondrial DNA were used to control the quality of the extracted DNA, as previously described 2, 0.2 mM dNTPs (QBiogene), 4 pmol of each primer, 0.6 U Taq DNA polymerase (QBiogene), and 3 µl of fivefold diluted DNA in a 20-µl reaction volume. Amplifications were carried out as follows: 3 min at 94°C for initial denaturation, 35 cycles of denaturation at 94°C, annealing at 58°C for the marker ADNg5/2 and 55°C for the markers ADNg15/16, ADNg12/13, and ADNg21/22, and extension at 72°C for 30 s. The final cycle was followed by an additional 10 min at 72°C to complete polymerization.The amplification reaction mixture consisted of 10 mM Tris–HCl (pH 8.3), 50 mM KCl, 1.5 mM MgClS. glossinidius, and not host (fly) DNA. In order to assess whether the amplicons corresponded to S. glossinidius microsatellites and to determine the number of microsatellite repeats, the different alleles were cloned into PGEM-T Easy . For each different allele, one recombinant plasmid was then sequenced (GenBank accession numbers JN032317–JN032335) and compared with the reference sequence of S. glossinidiusPrimer sets for each locus were tested to ensure that they specifically amplified After specific amplification, infrared dye-labeled (IRD700 or IRD 800) PCR products were diluted to 1/5, 1/10, or 1/50 in loading buffer , pH 8.0, and 1 mg/ml bromophenol blue). Then they were denatured for 3 min at 95°C, and transferred to ice before loading. The sample-loading volume was 1.22 µl. Each mixture was separated, in a 1- to 2-h run at 1500 V, on a 6.5% (wt/vol) Long Ranger polyacrylamide gel, using 1× Tris-borate-EDTA buffer , on a two-dye, model 4300 LI-COR-automated DNA sequencer.Infrared images of the patterns were analyzed using the semiautomated scoring program Quantar . Measurement of allele length on polyacrylamide gels was automated using molecular size markers.Finally, genetic diversity was analyzed on 244 samples carrying the symbiont. Of these, 131 were from the Campo focus, corresponding to three villages: 38 from Akak village , 33 from Campo Beach/Ipono , and 60 from Mabiogo . 113 samples were from the Bipindi focus: Ebimimbang village .HE) were estimated for each locus and each population using ARLEQUIN software, version 3.5.1 HEh) RH), computed using the CONTRIB 1.02 program RH) is defined as the expected number of different haplotypes found in each population using a standardized sample size fixed as the smallest available number of genotyped individuals.The populations corresponded to the four villages. The mean number of alleles per locus, the allele frequencies, and the level of heterozygosity  based on haplotype frequencies CTF, which can be interpreted as the relative divergence between HAT foci (Bipindi and Campo foci), SCF, corresponding to the relative divergence between populations within the Campo focus, and STF, corresponding to the relative divergence among populations. CTF was tested by permuting populations among foci, SCF by permuting haplotypes among populations within a focus, and STF by permuting haplotypes among populations among the foci. Moreover, pairwise STF between populations were computed and their significance was tested by 10,000 permutations using ARLEQUIN.The genetic structure among http://darwin.cirad.fr/darwin). Genetic distances between populations were obtained by computing the usual Euclidian distance matrix based on haplotype frequencies. From this matrix, a dendrogram was constructed using the neighbor joining method (NJ) from Saitou and Nei The phylogenetic relationship among populations was assessed using DARwin (DARwin software http://www.fluxus-engineering.com), equally weighting each locus, and setting the epsilon parameter to 20 to obtain a full median network. The median-joining network algorithm STN and pairwise STN between populations. The STN statistic takes into account genetic distances between haplotypes STN is expected to be larger than STG. STN were compared to STG by permuting haplotypes in the genetic distance matrix between haplotypes, using the procedure implemented in the SPAGEDI program . The genome of these 244 S. glossinidius samples carried the four loci investigated in full length. The four microsatellite loci were polymorphic, and a total of 19 alleles were detected, ranging from three (ADNg 12/13) to six  alleles per locus (HE) (HE) varied from 0.35 to 0.41, corresponding to the villages Ebimimbang and Campo Beach/Ipono, respectively.The complete dataset included multilocus genotypes for the 244 er locus . Over aler locus . The meaus (HE)  was quitThe combination of the microsatellite alleles yielded a total of 35 haplotypes . The fouS. glossinidius was explored at different hierarchical levels using AMOVA  and a slight but not significant differentiation at the foci level . The genetic differentiation among the four populations was low (STF = 0.022) but significant (P = 0.006). Pairwise population comparisons of genetic differentiation (STF) are shown in STF = −0.005) nor between Campo Beach/Ipono and Mabiogo (STF = −0.001). A positive but not significant differentiation (STF = 0.009) was recorded between Mabiogo and Akak. Even though STF values were relatively low, significant differences were shown between Ebimimbang and Mabiogo, Akak and Campo Beach/Ipono, with STF of 0.018 (P = 0.023), 0.022 (P = 0.027), 0.032 (P = 0.014), respectively. This indicates a significant differentiation between the Bipindi (Ebimimbang) population and those of the three villages  in the Campo focus.The population structure of ng AMOVA . On haplS. glossinidius population (Bipindi HAT focus) with the Mabiogo population (Campo focus) and (2) the Akak and Campo Beach/Ipono populations (Campo focus), but the node is mildly supported by the bootstrap resampling .The neighbor-joining (NJ) tree calculated from the haplotype frequency using the usual Euclidian distance is shown in STN was estimated at 0.015 and was not significantly different from global STG (P = 0.72). Pairwise STN between populations are shown in STG, but the STN estimated between Akak and Mabiogo was almost significantly larger than STG (P = 0.059).The four main haplotypes , showing overall frequencies above 0.05, were shared by the populations from the four sampling areas  and wereIn addition, GenGIS was used to draw a georeferenced pattern of haplotype diversity . Figure Sodalis glossinidius), and the parasite (the trypanosomes) the vector transmits to humans and animals. This study was the first to take an interest in the population genetics of S. glossinidius in the field. Its aim was to analyze the genetic diversity of S. glossinidius populations from two human African trypanosomiasis foci, Bipindi (one village) and Campo (three villages), in South Cameroon, in order to detect possible differentiation / gene flow within or between the HAT foci. The S. glossinidius analyzed were those harbored by the flies  sampled in the different areas of the two foci. Genotyping was performed using the variable-number-of-tandem-repeats (microsatellites) approach, not yet used to investigate S. glossinidius genetic diversity in the field. Four polymorphic loci were analyzed, the robustness and resolving power of this approach being maximized when large strain collections are analyzed using multiple loci The present study was conducted within our investigations on sleeping sickness and most particularly on the tripartite interactions between the vector (the tsetse fly), its secondary symbiont , whereas one locus (ADNg 12/13) was nearly nonpolymorphic. The combination of the four markers into haplotypes led to substantial overall diversity (HEh = 0.87). However, genetic diversity was lower in Mabiogo and Ebimimbang than in Akak and Campo Beach/Ipono. The lower genetic diversity of the Ebimimbang and Mabiogo S. glossinidius population may be associated with a lower effective population size in these villages. This could be due to the lower effective population size of its host, G. palpalis palpalis, or to the existence of a selective pressure exerted by the tsetse flies on the symbiont S. glossinidius in the populations concerned. It should be noted that a lower apparent fly density per trap and per day was observed in Ebimimbang in comparison with the other three villages, and particularly Akak The microsatellite markers were polymorphic and had sufficiently high resolution to estimate the genetic structure of STF with the three villages in the Campo focus, with the foci 150 km apart and located on different river basins. The differentiation analysis, based on the pairwise STF between populations and the AMOVA, revealed that the S. glossinidius populations presented a slight but significant differentiation between the Bipindi and Campo HAT foci.Within the Campo HAT focus, differentiation between populations was not significant. The village of Ebimimbang, located in the Bipindi HAT focus, showed significant The network and the georeferenced haplotype analysis showed that three frequent ancestral haplotypes were shared between the four populations and that there was not a geographic pattern of haplotypic diversity. These data suggest either that the gene exchange between populations occurred repeatedly or that the haplotypes derived from a common ancestral population.STN did not show an impact of the alleles' phylogeography on the structure of genetic diversity, except perhaps for the relation between haplotypes from the Mabiogo and Akak populations. However, the absence of information on the mutation process of microsatellite markers in S. glossinidius does not allow inferring the liability of STN/STG comparison in this species.The information provided by the S. glossinidius populations may be considered as a single population, suggesting that gene flow occurred within the Campo HAT focus. Between Campo and Bipindi HAT foci, differentiation existed but was low. This could be explained by the fact that genes flow between the Ebimimbang population and the Campo focus is ongoing or has been maintained until recently at a level preventing strong differentiation.Finally, these results tend to show that the Akak and the Campo Beach/Ipono Glossina, with mainly a vertical transmission but also perhaps a horizontal transmission among matrilines of tsetse flies S. glossinidius depends on its host. Our results suggest that gene flow exists between tsetse flies within the Campo HAT focus and that structuring may exist between the two foci, implying a limited gene flow, at least of female flies. The slight local differentiation among the S. glossinidius populations might be related to the fly migration rate between the HAT foci. The two HAT foci are located on different river basins  between the two foci located about 150 km from each other was unexpected. This means that tsetse fly migration occurs despite this rather large distance. This finding is important in the context of sustainable vector control. Accurately estimating to what extent the genetic diversity of S. glossinidius populations depends on the population genetics of its host G. palpalis palpalis deserves to be studied: the genetic diversity analysis of tsetse fly populations will have to be undertaken within the same foci. Moreover, further investigations will consist in looking for a possible association between field tsetse fly infections by a given trypanosome species and the presence of S. glossinidius-specific haplotypes. These investigations could contribute to understanding the differences in the prevalence of S. glossinidius and trypanosomes between foci. The identification of S. glossinidius haplotypes potentially associated with vector competence could be included as diversity markers in epidemiological surveys, risk mapping and management, and vector control strategies.In conclusion, these results provide new information on the genetic diversity of Table S1S. glossinidius haplotypes found in populations from the four sampling areas: Ebimimbang (Bipindi focus), Akak, Campo Beach / Ipono and Mabiogo (Campo focus).(PDF)Click here for additional data file.


Penaeus monodon in hatchery operations depends heavily on the evaluation of genetic diversity and population structure of brood stocks. Mitochondrial DNA (mtDNA) sequences have been widely used to study genetic variability and relationships in many crustacean groups, and these same markers may be incorporated into evaluation studies of shrimp broods and populations. For this purpose we looked at variation in mitochondrial D-loop sequences as an indicator of genetic diversity in shrimp populations from a region of India that represents the main sources of new material for brood stocks. In our study of these populations the overall mean genetic diversity was 0.191. The highest level of genetic diversity (0.357) was observed in the Kakinada population, whereas the lowest diversity (0.0171) was observed in the Nellore population. The results also indicate that overall, the populations along the Andhra Pradesh coast are genetically diverse despite the fact that there is considerable gene flow between them. From the results, it is evident that east cost of India shows high genetic diversity among P. monodon broods and no evidence of loss of diversity due to excessive inbreeding. The fact that the genetic variability of these populations has been maintained, despite ten years of dependence on these broods, shows that at the present time there is no indication of over exploitation.Quality production of the shrimp The online version of this article (doi:10.1186/2193-1801-2-671) contains supplementary material, which is available to authorized users. Penaeus represents an economically important group of shrimps and prawns  sequences are widely used to study genetic variability in aquaculture species including crustaceans, and these sequences have proved extremely useful in elucidating genetic variability and phylogenetic relationships among many crustacean groups . These produce 7882 million larvae per year, and this represents approximately 63% of total seed production in India. Keeping in mind the importance of shrimp culture to the economy and the degree to which success is primarily dependent on the health of the seed and the brood stock, the present work has been conducted to study the genetic structure and diversity of brood stocks from the Andhra Pradesh (A.P.) area in India to ensure the ability to maintain genetically diverse brood stocks for improved production.Along the coast line of the area known as Andhra Pradesh (974 Km), the dominant shrimp culture area in India, shrimp hatcheries are clustered in three areas: Vizag (North Andhra), Kakinada  and Nellore (South Andhra). Out of 280 P. monodon brooders were collected from the three regions along the A.P. coast  -5′ AAGAACCAGCTAGGATAAAACTTT 3′ and 1R (R) 5′-GATCAAAGAACATTCTTTAACTAC-3′. These were derived from Chu et al.  and Yang2, 2.5 mM dNTPs and 1.5 μM each primer. The thermal profile for hot-start PCR included initial denaturation at 95°C for 5 min, followed by 35 cycles of 20 s at 94°C, 30 s at 48°C, 60 s at 68°C and a final extension of 10 min at 68°C. The PCR product was purified by treatment with exonuclease and shrimp alkaline phosphatase at 37°C for 30 min, and the enzyme inactivation was carried out at 85°C for 15 min. Products were cleaned by ethanol precipitation and sequenced using an ABI Prism DNA analyzer 3730  and the Big dye cycle sequencing kit.The mtDNA control region was amplified in a 25 μl reaction volume with a final concentration of 1X Taq polymerase buffer and 0.6 U of Taq polymerase, 1.5 mM MgClpi) values  from the mtDNA control region, were obtained for analysis. Nine of the original samples with incomplete sequence reads were not included in analysis. The usable sequences were aligned using Bio-edit sequence editor package Hall , and datlues Nei  were callues Nei . Supportpi) for all of the samples analyzed here is 0.452 ± 0.1415, with a mean value for the populations of 0.150 ± 0.141. The average number of differences 18.8443 and the total number of segregating sites is 417  sequences of 562 bp in length (average) from 81 individuals (NCBI accession nos. JQ863127 to JQ863216) analyzed here show 43 haplotypes with a value of 14.33 for mean haplotype diversity. The overall Jukes-Cantor estimate of nucleotide diversity (The Kakinada population contained the largest number of haplotypes (21) and the highest value overall for haplotype diversity (0.931). This was closely followed by the Vizag population where 14 haplotypes produced a diversity value of 0.9195 and finally the Nellore population which had 8 haplotypes and a value for haplotype diversity of 0.864. Most of the haplotypes identified here (42 out of 43) were unique to one of these populations. The one shared haplotype was found in all three of the populations studied here.Other parameters measuring variation among the three populations are shown in Table For the three populations a total of 417 segregating sites were observed. Also for these populations overall mean numbers of 38 (transition) and 43 (transversion) type substitution mutations were observed. The Kakinada population had the highest numbers of both transitions (74) and transversions (96) whereas lower numbers  were seen in the Nellore population. An overall average of 81 substitutions were noted with the least number occuring in the Nellore population (30), followed by 43 in Vizag and the highest in the Kakinada region (170). Indels were absent in all three populations (Table k1 = 24.707 (purines) and k2 = 31.233 (pyrimidines). The overall transition/transversion bias is R = 5.623, where R = [A*G*k1 + T*C*k2]/[(A + G)*(T + C)]. All positions containing gaps and missing data were eliminated.The overall proportions of nucleotides in this dataset are 0.395 (A), 0.409 (T/U), 0.108 (C), and 0.088 (G) based on a total of 286 positions. The transition/transversion rate ratios are An Analysis of Molecular Variance (AMOVA) was performed in MEGA5.0 for 81 sequences to test for geographic variations/divisions among populations. Results from this analysis showed a percentage of variation attributable to among-population differences of 11.04% whereas most of the variation (88.96%) was attributed to variation within populations Table .Table 3The pairwise Fst comparisons in Table A Neighbour Joining (NJ) tree was constructed which depicts the overall relationships of the populations studied here Figure . This trP. monodon, a commercially important shrimp species  detected here is found within populations. Our results also suggest that overall, high levels of gene flow  are occuring between these populations. Nevertheless, as indicated by the pairwise Fst values, the mixing of lineages in ems Dale . Similarems Dale  and Mandems Dale . Also inems Dale , low levP. monodon females can enhance levels of lineage mixing because they migrate offshore when they grow and mature Authors’ original file for figure 1Authors’ original file for figure 2Below are the links to the authors’ original submitted files for images.


Pocillopora damicornis is a minor reef-builder elsewhere in the Indo-West Pacific, but is the dominant reef-building coral in the TEP, where it forms large, mono-specific stands, covering many hectares of reef. Moreover, TEP P. damicornis reproduces by broadcast spawning, while it broods mostly parthenogenetic larvae throughout the rest of the Indo-West Pacific. Population genetic surveys for P. damicornis from across its Indo-Pacific range indicate that gene flow  is generally limited over hundreds of kilometers or less. Little is known about the population genetic structure and the dispersal potential of P. damicornis in the TEP.Coral reefs in the Tropical Eastern Pacific (TEP) are amongst the most peripheral and geographically isolated in the world. This isolation has shaped the biology of TEP organisms and lead to the formation of numerous endemic species. For example, the coral P. damicornis among and within nine reefs and test for significant genetic structure across three geographically and ecologically distinct regions in Panama.Using multilocus microsatellite data, we analyzed the population structure of TEP ST = 0.162), indicating restricted gene flow , both among the three regions (RRT = 0.081) as well as within regions (RSR = 0.089). Limited gene flow across a distinct environmental cline, like the regional upwelling gradient in Panama, indicates a significant potential for differential adaptation and population differentiation. Individual reefs were characterized by unexpectedly high genet diversity (avg. 94%), relatively high inbreeding coefficients , and localized spatial genetic structure among individuals (i.e. unique genets) over 10 m intervals. These findings suggest that gene flow is limited in TEP P. damicornis populations, particularly among regions, but even over meter scales within populations.We detected significant levels of population genetic structure (global R Populations at the periphery of species' distribution ranges often exist in suboptimal and/or unstable conditions at the organism's physiological or ecological limits One of the most isolated and peripheral biogeographic regions of the world's oceans is the Tropical Eastern Pacific (TEP). It is separated from the Indo-West Pacific by 5000 km of open-ocean that Darwin Pocillopora damicornis is a minor reef-building coral in the Indo-West Pacific, but the primary reef-builder in the TEP Pocillopora damicornis in the TEP forms large, mono-specific carpets measuring tens to hundreds of hectares P. damicornis populations brood mostly asexual (parthenogenetic) larvae The isolation of the TEP from the Indo-West Pacific and major differences in environmental factors and species compositions between these regions enabled significant ecological and life-history shifts in many TEP species. For example, the scleractinian coral P. damicornis populations across the Indo-West Pacific indicate that larval dispersal (i.e. gene flow) is generally limited over hundreds of kilometers  and in some cases even among neighboring reefs . Some of the highest levels of genetic structure have been described at the margins of P. damicornis Indo-Pacific range P. damicornis in the Indo-West Pacific also showed significant levels of local inbreeding and contained some level of clonality Population genetic surveys among larvae-brooding P. damicornis populations in the TEP. Combosch et al. P. damicornis, using ribosomal DNA sequence data . Using multilocus genetic data, Pinzon & LaJeunesse Pocillopora species, including 7 well-recognized Indo-Pacific species . They identified three genetic clusters or types  in their multi-species TEP sample of Pocillopora. All three of these types were found in P. damicornis from across the TEP. Type I was found throughout the TEP, including Panama, Type II was found only in Clipperton Atoll and Type III was found in Panama and Galapagos. Yet, no genetic structure was detected within the broadly distributed Type I group over 3500 km of the TEP.Little is known about the population structure of broadcast-spawning One of the best-studied areas in the TEP is the Pacific coast of Panama, which is dominated by two major gulfs, the Gulf of Chiriqui in the west and the Gulf of Panama in the east. The two gulfs differ in several important ecological factors P. damicornis within and among nine Panamanian populations across the three geographically and ecologically distinct regions . We predict that high rates of vegetative fragmentation should lead to significant SGS due to clonal aggregations. In contrast, high rates of sexual reproduction by broadcast spawning should inhibit SGS due to increased outcrossing and the mixing of genotypes at the sea surface during larval development.Using multilocus microsatellite data, we examined the population genetic structure of TEP  regions . Based oPocillopora damicornis colonies from nine reef populations (A = 7). Three pairs of loci showed indications of significant linkage disequilibria, but none showed consistent significant linkage disequilibria in more than two populations. Consequently, all six loci were considered as unlinked. Significant heterozygote deficits were detected in five loci (Multilocus microsatellite data (n≥5 loci) was obtained for 207 ulations  and 2. Tive loci , but onl−5; Using the five remaining loci, 194 unique genets were identified with a high probability of identity (p<3×10IS = 0.255±0.122) and in the Gulf of Chiriqui (FIS = 0.196±0.171), but not in the Azuero Peninsula (FIS = 0.048±0.135) due to an excess of heterozygotes in Achotines. Each population contained on average 4.7 alleles per locus. Slightly more alleles were detected in the Gulf of Chiriqui (avg. NA = 5.1±0.26) than in the Azuero Peninsula (4.5±0.20) and in the Gulf of Panama (4.3±0.31). Interestingly, private alleles were only found in Gulf of Chiriqui populations, which contained the only regional-specific allele  as well.Using the genet only dataset, significant heterozygote deficits were detected in six out of the nine populations , indicatST  and global FST . For the R-statistics, population structure was equivalent among regions (RRT = 0.081) and among populations within regions (RSR = 0.089). All three regions were significantly different from each other (p<0.001), but the two gulf regions were most distinct (pairwise RST = 0.138), whereas the Azuero Peninsula, which lies between the two gulfs, showed lower pairwise genetic structure . For the F-statistics, population structure was predominantly due to differences among populations within regions  among populations, among regions, and among populations within regions . GeneticST comparisons between populations were significant after sequential Bonferroni adjustments  and the genet dataset . This indicates that corals 10 meters apart are significantly more closely related to each other than to the rest of the population. Kinship among individuals (Fij) was only slightly elevated when clones were included, reflecting the low frequency of clones in the dataset  was detected among individuals within reefs. SGS was significant over 10 m intervals in both the ramet dataset , almost equivalent to a first-cousin relatedness (FIJ = 0.125) whereas F10m-kinship in the Gulf of Chiriqui was two times lower . The genetic patch size was almost twice as large in the Gulf of Panama (70 m) than in the Gulf of Chiriqui and Azuero Peninsula (40 m). These differences in the strength and extent of SGS are reflected in the Sp statistic, which indicates that SGS is strongest in the western Gulf of Panama (0.062), intermediate in the central Azuero Peninsula (0.059) and lowest in the eastern Gulf of Chiriqui (0.049).Significant levels of SGS out to 10 m were detected in all three regions p<0.05; . AveragePocillopora damicornisP. damicornis also distinguish by its broadcast gamete-spawning reproductive strategy. Multilocus microsatellite data from nine Panamanian populations demonstrate that TEP P. damicornis populations from Panama are characterized by strong population genetic structure (RST = 0.162), within and particularly among its three major regions, the Gulf of Chiriqui, the Azuero Peninsula and the Gulf of Panama. Moreover, the large and mono-specific stands of P. damicornis that characterize TEP reefs are not dominated by a few, highly clonal genotypes as previously thought The Tropical Eastern Pacific (TEP) is one of the most geographically isolated biogeographic regions in the world's oceans P. damicornis populations (RRT = 0.081). Panama's two major gulfs – the Gulf of Chiriqui and the Gulf of Panama - which are geographically most distant, were most genetically distinct (RST = 0.138). These genetic differences correspond with pronounced ecological differences between these two regions, e.g. in species compositions and environmental factors . The most significant environmental difference is the pattern of seasonal upwelling, which is strong in the Gulf of Panama dropping temperatures down to 15°C, but is virtually absent in the Gulf of Chiriqui ST = 0.070 & 0.101).As hypothesized, we detected strong genetic differences among the three geographically and ecologically distinct regions in Panamanian Acanthaster planciP. damicornis from the non-upwelling Gulf of Chiriqui are less sensitive to higher temperatures than those from the upwelling Gulf of Panama P. damicornis populations have also been reported from the Great Barrier Reef P. damicornis indicates that even among broadcast-spawning coral populations, adaptations to different thermal regimes can occur over distances of ∼100 km.Upwellings deliver cool, nutrient-rich waters to the near-shore environment. In tropical habitats like the TEP, they have significant impacts on nutrient and temperature-sensitive reef fauna P. damicornis may also explain prolonged differences in post-disturbance reef recovery between the Gulf of Chiriqui and the Gulf of Panama. For example, the 1982/83 El Niño caused mass coral mortalities and reduced average coral cover on most Panamanian reefs by 80%, but even though mortality rates were similar in both gulfs, reefs in the Gulf of Panama had recovered by 1992, whereas reefs in the Gulf of Chiriqui recovered only 3–6% of their populations by 1997 Acanthaster predation on surviving colonies in the Gulf of Chiriqui potentially hampered initial recovery significantly, while corallivorous sea stars are absent in the Gulf of Panama. Another important factor for the differences in P. damicornis recovery between Gulfs may be geographic differences in Symbiodinium identity (or type). Baker et al Pocillopora corals with clade C bleached severely in 1997 while colonies with clade D were unaffected by bleaching, which lead to a relative increase in colonies with clade D from 43% in 1995 to 63% in 2001. Higher levels of clonality in the Gulf of Panama (e.g. Saboga) further indicate that asexual fragmentation might have played a more prominent role in the recovery of Gulf of Panama populations compared to their Gulf of Chiriqui counterparts.Limited inter-regional gene flow in Panamanian Pocillopora are comprised of three distinct genetic types (Type I–III), independent of species identity and morphology. This is controversial because they included at least seven well-recognized Indo-Pacific species that must then either be reclassified or synonymized by their genetic types. Their dataset included a limited number of P. damicornis samples from the Gulf of Panama (n = 26), which were comprised of two of the three genetic types (Type I & II). Using a similar set of microsatellites, our STRUCTURE analyses do not support two distinct genetic types, but instead identified five genetic clusters Similar levels of population genetic structure over scales of hundreds of kilometers have also been observed in other brooding and broadcast-spawning coral species in the Indo-Pacific and the Caribbean  as 0.39 . Some ofP. damicornis microsatellite datasets from peripheral populations in East-African ST = 0.02 over 860 km) than among TEP populations (FST = 0.05 over 400 km). Indonesian populations show the same degree of genetic differentiation as TEP populations (FST = 0.05) over a 10-fold wider geographic distance (3000 km vs. 400 km). We observed a much lower allelic diversity in the TEP (NA/locus = 7) compared to P. damicornis in Indonesia and East-Africa . Lower allelic diversities are expected for peripheral populations and the lower diversity in TEP populations, compared to the similarly peripheral East-African populations, is likely due to the extreme isolation of the TEP and very limited transpacific gene flow . As expected, inbreeding was found to be highly significant in both peripheral populations, in the TEP and in Eastern Africa P. damicornis allozyme studies confirm all three population genetic trends in peripheral populations: 1) higher genetic structure [e.g. 62 vs. 56], 2) decreased allelic diversity Direct comparisons of population genetic data are possible with two Our results indicate lower genetic diversity and higher levels of population structure among broadcast-spawning populations (TEP) than among larvae brooding populations (Indo-West Pacific), which may reflect intrinsic differences between the two reproductive strategies. In Indo-West Pacific populations, parthenogenetic larvae brooding preserves genotypes, which reduces the rate of genetic drift and allelic loss, while allelic diversity in TEP populations is likely reduced due to reproductive sweepstakes, promoted by its broadcast-spawning reproductive strategy IS = 0.18) indicate that gene dispersal is limited in Panamanian P. damicornis, not only between reefs, but within reefs as well. Fine-scale SGS arises from a variety of historic, demographic, and evolutionary processes P. damicornis was predominantly driven by significant non-random relatedness among individual genets out to 10 meters, which is unusual for free-spawning benthic marine populations.The observed fine-scale spatial genetic structure (SGS) and the high inbreeding coefficients  or proximity recruitment (i.e. SGS among parents and recruits). In brooding coral species, proximity recruitment is facilitated by the internal (i.e. stationary) larval development and the frequent settlement of larvae within meters of the maternal colony P. damicornis do not release brooded larvae P. damicornis, larval development occurs externally and larval settlement after gamete release takes at least 1–3 days The two most likely explanations for the observed SGS among genets in TEP Pocillopora  and several other coral species  are known to be negatively buoyant P. damicornis gametes or larvae are positively or negatively buoyant is currently unknown, since gamete spawning has not yet been observed directly.One potential scenario that could significantly reduce passive dispersal would be if gametes and/or larvae sink down and escape the flow of the water column. Although coral gametes typically float on the sea surface during fertilization and development P. damicornis might facilitate inbreeding in several ways. Firstly, the external fertilization of broadcast-spawners lacks the potential parental control of internal fertilization in brooders IS = 0.26), and significant inbreeding is common in P. damicornis populations throughout the Indo-Pacific, regardless of reproductive strategy, local environment, population history, and analyzed genetic marker  and a roughly comparable genetic patch size (80 m). Among broadcast-spawning corals, Stoddart Acropora digitifera and Goniastrea favulus, respectively. However, Miller and Ayre Platygyra daedalea, including significantly positive kinship in the smallest distance interval (F1 = 5 m) and a Genetic Patch Size of only 17 m. This is the only other documented broadcast-spawning coral with genet SGS. To account for this pattern, Miller and Ayre While SGS is common in plants , it has only been documented in four other coral species. Underwood Antipathes fiordensisStyela plicataA. fiordensis) or eggs (S. plicata).Small-scale SGS over tens of meters has been documented for at least eight other sessile marine invertebrates, including two sponges P. damicornis reefs were long thought to be highly clonal, persisting locally through vegetative asexual fragmentation P. damicornis. Simulation models by Neigel & Avise Acropora cervicornis, which depends heavily on vegetative fragmentation, 5% sexual recruitment  is enough to generate high genotypic diversities (∼60%), independent of initial genet diversity.Because of its large mono-specific stands and little to no detectable larvae recruitment P. damicornis studies, levels of clonality in the TEP (6%) seem low. However, direct comparisons among studies are biased by different sampling strategies and genetic markers. Lower levels of clonality tend to be detected with microsatellites compared to allozyme markers  across a well-documented upwelling gradient [e.g. 73] along 1400 km of coastline in Panama  DNA buffer for genetic analysis On each reef, 18 to 36 nubbins (2 cm) of et al.et al.Ten microsatellite loci were amplified using primers developed by Magalon Population genetic statistics, including allele frequencies, clonality, probability of identity, F-statistics and Analyses of Molecular Variance (AMOVA) were calculated using GenAlEx 6.1 STSTST and FST is based on 999 permutations. RST recovered more genetic structure, especially among regions, whereas FST had a lower resolution and higher intra-regional variation (FRT = 15% of FST compared to RRT = 50% of RST). As a result, we preferentially focus on RST , genetic patch size and Sp. F(1) is the average kinship among individuals within the smallest distance interval (10 m). Its statistical significance (p) was obtained by comparing the slope b(F) of F(r) on ln(r) to 9999 random permutations of individuals among locations within populations using a Mantel test. The genetic patch size is the distance that corresponds to the first x-intercept of the kinship correlogram [cf. 148]. Within the genetic patch, individuals are more closely related than the population average, i.e. the association between genotypes is positive, while individuals outside of the patch are genetically independent, i.e. their correlation is negative. The Sp statistic (Vekemans and Hardy (1) and the decrease of SGS with distance (b(F)).Fine-scale Spatial Genetic Structure (SGS) within populations was analyzed using the program SPAGeDi 1.2 Figure S1Results of the Bayesian clustering approach implemented in STRUCTURE 2.3  that were used to infer the number of genetic clusters (K) in the microsatellite dataset. The graph shows the mean log likelihood L(K) for K = 1–19 (±SD) and ΔK, the second order rate of change of L(K) for K = 2–18 (EPS)Click here for additional data file.


Genetic admixture between wild and introduced populations is a rising concern for the management of endangered species. Here, we use a dual approach based on molecular analyses of samples collected before and after hatchery fish introduction in combination with a simulation study to obtain insight into the mechanisms of admixture in wild populations. Using 17 microsatellites, we genotyped pre- and post-stocking samples from four Atlantic salmon populations supplemented with non-native fish to estimate genetic admixture. We also used individual-based temporally explicit simulations based on realistic demographic and stocking data to predict the extent of admixture. We found a low admixture by hatchery stocks within prestocking samples but moderate to high values in post-stocking samples (from 12% to 60%). The simulation scenarios best fitting the real data suggested a 10–25 times lower survival of stocked fish relative to wild individuals. Simulations also suggested relatively high dispersal rates of stocked and wild fish, which may explain some high levels of admixture in weakly stocked populations and the persistence of indigenous genotypes in heavily stocked populations. This study overall demonstrates that combining genetic analyses with simulations can significantly improve the understanding of admixture mechanisms in wild populations. Genetic admixture between wild indigenous populations and introduced conspecifics from distant and/or captive stocks is a growing concern in conservation biology  populations from the Baie-du-Mont-Saint-Michel (BMS), Lower-Normandy, France, using microsatellite DNA analyses of pre- and post-stocking samples. From 1989 to 2003, about 1 052 000 young-of-the-year salmon have been introduced in the Couesnon (COU), Sélune (SEL), Sée (SEE), and Sienne (SIE) rivers. Individuals released were non-native and produced by progenitors captured in distant French populations: the Aulne River (Brittany) and to a lesser extent the Gave d'Oloron River (Aquitania). Scale samples of fish caught by anglers have been collected since the late 1960s in BMS populations. In addition, estimates of population sizes are available and the numbers and origins of fish stocked in each river are known. We used available demographic, genetic, and stocking data to run individual-based simulations to produce expected admixture rates according to various demographic scenarios. Specifically, this study aimed at (i) comparing the genetic structure and admixture levels of the study populations before and after stocking, (ii) simulating realistic expected admixture rates in each population according to stocking and demographic data, and (iii) comparing the observed data with simulated admixture data to determine whether lower survival and higher dispersal of stocked fish relative to wild individuals may explain the observed patterns of admixture.Here, we assessed the admixture between wild and hatchery fish in four Atlantic salmon  is presented in Institut National de Recherche Agronomique) and ONEMA . The age of each individual was determined from its scales. As post-stocking samples, we focused on cohorts 2002–2003  buffer, and chelex, at 55°C 2 h and then at 100°C for 10 min  were also conducted with Fstat. Expected heterozygosity, He,  genetic distances .We used M2 Goudet . Tests f He, Nei  and obsetructure software . We ran Structure from 1 to 6 genetic clusters (k) with 15 replicates for each k. Each run started with a burn-in period of 50 000 steps followed by 300 000 Markov Chain Monte Carlo replicates and estimating 90% credible intervals. We selected the k with the highest likelihood  and sex was set randomly . F-statistics and multilocus genotypes were recorded every generation. There was no dispersal between AUL, GAV, and the four other BMS populations before stocking events.We implemented the following semelparous life cycle: (i) breed; (ii) dispersal; (iii) random regulation of local populations size, which reduced the pool of competing individuals to the local carrying capacity ; (iv) reproduction during which females were assigned a fecundity value drawn from a Poisson distribution with a mean value of 40 offspring and mated with one randomly chosen male (monogamy). We also investigated the potential effect of multiple mating by testing 20% and 50% of polyandry for one of the best scenarios. Adults died after reproduction and the cycle started again. This assumption of semelparity is realistic as the estimated proportion of multispawners in BMS salmon populations is extremely low: from 0.9% to 2.1% for the 1992–2002 and 1972–1982 periods, respectively  with the same settings used for molecular data. To estimate population admixtures, we averaged admixture values over the three Structure runs and then averaged values of the three Nemo runs. To identify the simulated scenario(s) that best fitted the observed data, we used two approaches. First, we used chi-square tests to test whether mean-simulated admixture values for each scenario significantly differed from observed values. Second, we used a least-squares approach: we summed the squared differences between observed and simulated individual admixture values for each scenario in each population and defined the best scenario as the one with the smallest sum of squared differences. The best scenario for the situation where admixture rates were averaged over the four populations was defined as the smallest of the four sums of squares, which were divided by the corresponding number of individuals in each population. We also used factorial correspondence analyses computed with Genetix 4.05.2 to display the genetic structure among samples and compare observed and simulated data.Three SFST) was found between successive cohorts in SIE, SEE, and SEL, indicating temporal stability of allele frequencies within populations. We thus combined genotypes from successive cohorts for each population in our analyses. We found some evidence of null alleles or large allele dropouts for six of 238 tests. Considering the overall data set, there was no evidence of departure from HWE . In contrast, FSTs between COU03 and the other BMS samples were significant (0.01–0.03). FSTs among temporal samples were relatively low but significant for SIE86-SIE03, SEL86-SEL03, and AUL69-AUL03  than among contemporary samples  corresponding to AUL, GAV, and BMS samples . Similarly, the differentiation among prestocking samples from BMS was low in all scenarios (FST < 0.01). The factorial correspondence analysis highlighted the existence of three genetic groups among simulated populations before stocking . The differentiation between post-stocking BMS samples and GAV and AUL was comparable to the one observed in real data (average stocking . In the tructure program identified three genetic clusters (k) corresponding to AUL, GAV, and BMS samples . 90% credible intervals were relatively narrow and of the same magnitude as for molecular data  produced similar number of scenarios not differing from the observed data when considering global levels of admixture . At the Overall, admixture proportions for the scenario C7 fitted well with the observed data . This scThis study showed moderate to high admixture levels between wild and stocked salmon in post-stocking BMS samples. Importantly, a moderate level of admixture by Gave d'Oloron was observed in the SIE population while fish from the former had not been stocked in the latter but in other BMS populations. This demonstrates that stocked fish (or their wild-born descendants) can disperse into unstocked rivers and modify their genetic makeup. By comparing molecular data with realistic temporally explicit simulations, we showed that stocked salmon may have a 10–25 times lower survival than wild individuals. Simulations did not support the hypothesis of a higher dispersal of stocked fish relative to wild individuals, but this aspect requires further investigations.We found significant admixture rates with Aulne and Gave in post-stocking samples from BMS (from 12% to 60%) while prestocking samples were weakly admixed (from 2% to 5%). Furthermore, our results showed a decrease in genetic differentiation among donor and stocked populations. Some of these BMS populations have been intensively stocked during the last decades and were the only ones in France to be still stocked with non-native fish in the 2000s. Therefore, while the weak admixture observed in prestocking samples most probably results from low levels of ‘natural’ gene flow, high contributions of donor populations within post-stocking samples are consistent with stocking data and corroborate previous results on these populations . This suggests that our simulation approach cannot be used to infer relative survival rates of stocked fish when admixture is very low (<10%). The overall admixture rate by Aulne and Gave d'Oloron in BMS populations was 25% (range 12–60%), which allowed meaningful comparisons with simulation scenarios. For instance, the overall observed admixture was always significantly different from simulated scenarios with an equal survival of wild and stocked fish, which predicted 46–90% of admixture. Scenarios compatible with overall observed admixture suggested a 10–25 times lower survival of stocked fish relative to wild individuals. At the population level, our simulations suggest that only 2–9% of the native gene pool may remain in COU if the survival of stocked and wild fish was equal, whereas we observed that 40% of the native gene pool remains. The level of admixture in the observed data could result from a low survival of hatchery fish in the wild  must have dispersed into the Sienne River. Several studies have reported high straying rates of stocked salmonids Quinn , which mEmpirical data suggested a high dispersal of stocked fish leading to relatively high admixture rates in weakly stocked SIE and SEE populations. Accordingly, the high admixture with the local cluster observed in the previously almost extinct COU population suggests significant levels of dispersal of wild BMS individuals. Our simulation study suggested that both 6% and 15% dispersal rates were compatible with overall observed admixture data. However at the population level, 15% dispersal rates produced more scenarios compatible with the observed data in COU, SEL, and SIE populations, suggesting that high dispersal was an important factor in the establishment of high admixture rates in these populations. Nevertheless, simulated scenarios implementing a higher dispersal rate of stocked fish relative to wild individuals did not better fit the observed data than scenarios with equal dispersal rates for the two groups of individuals. Straying rates were chosen according to Jonsson et al. , but higtructure software for computational reasons. Our aim was mainly to demonstrate that a combined approach using simulated and empirical data can provide some insights into the process of genetic admixture beyond the mere description of admixture patterns.Despite the use of a restricted set of simulation parameters, we detected some combinations of parameters that fitted remarkably well with the real data when admixture was not too low (above 10%). Other approaches  would probably allow a more thorough investigation of the parameters' space. However, the millions of data sets generated by an ABC approach could anyway not be analyzed by the Sin situ observations made by INRA and fisheries organizations. Other sets of parameters could have been tested including various values of population sizes or straying rates (Jonsson et al. The simulation parameters were chosen according to the literature and from To conclude, this study confirms that stocking practices can greatly alter the genetic integrity of managed populations but also the genetic structure among donor and recipient populations. This implies that BMS rivers should be managed independently from other genetic groups to maintain the local genetic diversity and putative local adaptation. From a management perspective, this study allowed estimating the relative performances of stocked and wild individuals and highlighted the role of dispersal as a ‘spreading agent’ of non-native genes into wild salmon populations. This study further demonstrates that the combination of simulation tools and analyses of recent and old archived samples could greatly contribute to the understanding of genetic admixture following the introduction of non-native and/or domestic individuals in wild populations.


Iris brevicaulis and Iris fulva. Specifically, we asked how populations are structured and if population structure coincides with potential barriers to gene flow. We also asked whether there is evidence of hybridization between these two species outside Louisiana hybrid zones. We used a genotyping-by-sequencing approach and sampled a large number of single nucleotide polymorphisms across these species' genomes. Two different population assignment methods were used to resolve population structure in I. brevicaulis; however, there was considerably less population structure in I. fulva. We used a species tree approach to infer phylogenies both within and between populations and species. For I. brevicaulis, the geography of the collection locality was reflected in the phylogeny. The I. fulva phylogeny reflected much less structure than detected for I. brevicaulis. Lastly, combining both species into a phylogenetic analysis resolved two of six populations of I. brevicaulis that shared alleles with I. fulva. Taken together, our results suggest major differences in the level and pattern of connectivity among populations of these two Louisiana Iris species.Identifying processes that promote or limit gene flow can help define the ecological and evolutionary history of a species. Furthermore, defining those factors that make up “species boundaries” can provide a definition of the independent evolutionary trajectories of related taxa. For many species, the historic processes that account for their distribution of genetic variation remain unresolved. In this study, we examine the geographic distribution of genetic diversity for two species of Louisiana Irises,  Molecular Ecology Special Issue: Genotyping By Sequencing in Ecological and Conservation Genomics) , which stretches from Illinois to the Gulf of Mexico, is the historic floodplain of the lower Mississippi  How is genetic diversity partitioned and how are populations structured? (2) Does population structure coincide with potential geographic barriers to gene flow, such as the Mississippi River? (3) Is there evidence of hybridization between these species beyond the hybrid zones defined in the state of Louisiana?lis Fig. . Iris brIris fulva is found at lower elevations in intermittently flooded, forested wetlands. Iris brevicaulis occurs at slightly higher elevation in mixed hardwoods  for sequencing. DNA from each individual was separately digested using EcoT221, a six base cutter, and the fragmented DNA was then ligated to a barcoded adaptor and a common adaptor. Within a 96-well plate, each well contained DNA from a different individual and a barcode adaptor unique to that well. Individuals were barcoded to allow discovery of genetic variation both within and between species. The resulting libraries were sent for sequencing using single-end 100-bp reads on the Illumina HiSeq 2000 with 48 samples sequenced per lane.DNA was extracted from eight individuals for six populations of each species throughout the species' range Fig. . Extracthttp://www.maizegenetics.net/gbs-bioinformatics), was used for SNP discovery and genotyping both between and within species. UNEAK takes raw Illumina sequence files and converts them into individual genotypes. Reads are retained, and trimmed to 64 bp, when they possess a barcode, cut site, and no ‘N's in the first 64 bp of sequence after the barcode. Identical reads are clustered into tags and counts of these tags present in each barcoded individual are stored. Pairwise alignment, then, identifies tag pairs having a single base pair mismatch, and these single base pair mismatches are considered candidate SNPs. Any tag pair that contains more than one mismatch is discarded to minimize SNPs resulting from alignment of paralogous sequences. Error tolerance rate was set at 0.03, so that only reciprocal pairs of tags are retained for SNP calling according to standard protocols of Cornell Institute for Genomic Diversity.The nonreference pipeline, Universal Network-Enables Analysis Kit , which is analogous to FIS by Wright  for K = 1–7, where K = number of populations. Optimal number of populations was identified as the one in which the BIC was the lowest value and after which the BIC either increased or decreased by the least amount.Discriminant analysis of principle components (DAPCs) . All parameters had effective sample size (ESS) values > 250. Convergence onto posterior topology was assessed using AWTY  with the default settings. SNAPP, part of BEAST 2.0, is a package for inferring species trees and species demographics from independent (unlinked) biallelic markers  were blasted against the nonredundant, nucleotide database collection using BLASTN. Almost all tag pairs examined resulted in E-value scores which were not lower than 10−4; however, four tag pairs had an E-value of below 10−4 that matched sequences from chloroplast DNA. One tag pair with a significant E-value was associated with an I. brevicaulis IRRE transposon marker.A total of 151,189 SNPs were identified from individuals from all populations of I. brevicaulis, within-population observed heterozygosity (Ho) ranged from 0.101 to 0.259 and expected frequency of heterozygotes over all populations (He) ranged from 0.144 to 0.217. The inbreeding coefficient (GIS) ranged from 0 to 0.3, with three population GIS values significantly different than zero  (Table S1a). For I. fulva, within-population observed heterozygosity (Ho) ranged from 0.314 to 0.561 and expected frequency of heterozygotes over all populations (He) ranged from 0.357 to 0.403. The inbreeding coefficient (GIS), for I. fulva, was approximately zero for all populations (Table S1b). Genetic differentiation (pairwise population FST) for I. brevicaulis populations was moderately high .No isolation-by-distance was detected (data not shown) in either species. The slope of the regression line in the Mantel test (correlation of geographic and genetic distance) was not significantly different than zero , although with only a slight difference between the two. At K = 2, we do not see two distinct clusters, but a proportion of each individual's genome is shared with both clusters. Because the proportion of the individuals assigned to each of the two populations is roughly symmetric, we conclude that there is basically no population structure. When individuals from both species were combined and analyzed using STRUCTURE, the optimal number of clusters is K = 2  for within a species with 1–7 clusters and between species with 1–13 clusters. ΔK, the statistic used to detect the most likely number of clusters, I. brevicaulis DAPC analysis, the BIC gave the most support to three genetic clusters , which differs from STRUCTURE . However, there is subtle genetic structure where the first principle component separated populations 1, 4, and 6 from populations 2, 3, and 5. The second component separated the first group into populations 1 and 4 (Arkansas and Louisiana) from population 6 (Mississippi). For the second group , the second function separated 2 and 3 (Illinois and Kentucky) from 5 (Missouri) , which was sister to all other samples . The remainder of the genotypes demonstrated a geographic partitioning with populations becoming more nested with increasing latitude. When species were combined, individuals from Alabama and Louisiana formed one clade (bootstrap support = 98%) that was more closely related to I. fulva genotypes than to other I. brevicaulis individuals or populations .Nodes with <50% support were collapsed for RAxML trees. Most nodes in the I. brevicaulis  the Louisiana sample being sister to all other populations and (2) the Arkansas sample being sister to the four other populations  approach to genotype individuals. Advantages of using a GBS approach include genotyping nonmodel, large genome, organisms using a large panel of SNPs at a cost that is comparable to traditional Sanger sequencing for a handful of molecular markers. Library preparation is much simpler than traditional RAD approaches and can be performed relatively quickly and is amenable to a high level of multiplexing. Essentially, GBS does marker discovery and genotyping simultaneously compared with independent marker discovery, assay designs, and genotyping following the classical approach. Lastly, by generating fragments of DNA spread throughout the genome, one can potentially sequence regions inaccessible to other sequencing approaches. However, GBS suffers the same bioinformatics hurdles as all next-generation sequence methods such as generating and analyzing massive amounts of data, dealing with complex genomes, and the presence of missing data.I. brevicaulis and I. fulva. Our results are in line with those previously reported for two species of frog, Rana catesbiana and Pseudacris crucifer  does show differentiation between populations located on either side of the Mississippi River, consistent with the river being a barrier to gene flow  compared with the remaining populations. Additionally, both the RAxML and SNAPP trees failed to detect genetic differentiation due to the Mississippi River. Specifically, we did not resolve two reciprocally monophyletic groups of populations distributed on opposite sides of the Mississippi River. Instead, the phylogeographic pattern for I. brevicaulis is one of higher latitude populations being more derived relative to more southerly distributed samples.In contrast to the Loblolly pine, I. brevicaulis, it is possible to test whether this species conforms to the central–marginal hypothesis  would be expected to exhibit characteristics typical of central populations, such as having high levels of genetic diversity as measured by expected heterozygosity  into one single locus. We were, however, able to resolve the population phylogeny using a species tree approach. We found that the Louisiana population was a sister lineage to all other populations. Future analyses estimating migration rates between collection localities should provide further definition of patterns of gene flow among I. fulva populations.In contrast to ons Fig. A. FurtheI. brevicaulis is experiencing limited gene flow between populations, while I. fulva is not. Our results are not consistent with a geographic barrier (i.e. the Mississippi River) to gene flow and there are other processes that could lead to the observed patterns of genetic differentiation. Within plants, selection on various floral morphologies has been well documented and shown to be influenced by both abiotic and biotic factors  are well known for spring migratory flights that cover great distances encompassing most of eastern North America .Outcomes from natural hybridization are varied and often have significant evolutionary consequences Arnold . In the I. fulva, or hybrid plants, into a third species of the Louisiana iris species complex, I. hexagona , which apparently underlie adaptations to ecological settings within these iris species (Martin et al. In a previous study of g et al.  found thn et al. , 2006 doHelianthus debilis var cucumerifolious and Helianthus annuus ssp annuus (Kim and Rieseberg Helianthus annuus into novel environments and in producing its unique phenotype. These hypotheses were tested using a variety of experimental hybrid and parental genotypes/phenotypes, in both greenhouse and field experiments. A pattern of overrepresentation of H. debilis alleles suggested that these genomic components might confer a fitness advantage to the introgressed H. annuus. Additionally, introgression apparently altered multiple traits of the H. annuus phenotype causing adaptive changes in components interacting with the biotic and abiotic environments (Whitney et al. Adaptive trait introgression has been inferred in a number of organisms other than iris. For example in I. brevicaulis populations appear much more isolated from intraspecific gene flow than do I. fulva populations. However, neither species demonstrates phylogeographic breaks indicative of the Mississippi River acting as a barrier to gene flow. In contrast to isolation among I. brevicaulis populations, we detected asymmetric introgressive hybridization of alleles largely from I. fulva into I. brevicaulis, with some of the allele transfer possibly being adaptive. For example, the introgressed, Alabama population of I. brevicaulis was found in an I. fulva-like habitat, that is, in standing water. Martin et al. (Martin et al. I. fulva into I. brevicaulis that allowed the latter to survive an extreme flooding event. Performing a transplant study, in order to evaluate whether I. fulva parents or hybrid individuals survive as well or better in the I. brevicaulis collection site in Alabama, should provide a further test of the hypothesis of adaptive trait introgression within this species complex.The present study demonstrates species-level differences in connectivity among populations of Louisiana irises. In particular,


Origanum vulgare in restored semi-natural grassland patches. We compared the genetic diversity and differentiation of fourteen recent populations with that of thirteen old, putative source populations, and we evaluated the effects of spatial configuration of the populations on colonization patterns. We did not observe decreased genetic diversity in recent populations, or inflated genetic differentiation among them. Nevertheless, a significantly higher inbreeding coefficient was observed in recent populations, although this was not associated with negative fitness effects. Overall population genetic differentiation was low (FST = 0.040). Individuals of restored populations were assigned to on average 6.1 different source populations (likely following the ‘migrant pool’ model). Gene flow was, however, affected by the spatial configuration of the grasslands, with gene flow into the recent populations mainly originating from nearby source populations. This study demonstrates how spontaneous colonization after habitat restoration can lead to viable populations in a relatively short time, overcoming pronounced founder effects, when several source populations are nearby. Restored populations can therefore rapidly act as stepping stones and sources of genetic diversity, likely increasing overall metapopulation viability of the study species.In most landscapes the success of habitat restoration is largely dependent on spontaneous colonization of plant species. This colonization process, and the outcome of restoration practices, can only be considered successful if the genetic makeup of founding populations is not eroded through founder effects and subsequent genetic drift. Here we used 10 microsatellite markers to investigate the genetic effects of recent colonization of the long-lived gynodioecious species  In many landscapes, large scale habitat restoration has proven to be the only way to establish self-sustaining ecosystems that are resilient to future perturbation Metapopulation genetic theory can help to understand the potentially complex genetic consequences of early colonization following habitat restoration φ) that two colonizing individuals originate from the same source population (with φ = 1 for the propagule pool model and φ = 0 for the migrant pool model) is therefore a good indication of the extent of founder effects k the number of colonists and Nm the effective number of migrants To what extent founder events reduce population genetic diversity and increase the magnitude of genetic differentiation among populations is dependent on (i) the number of colonizing individuals that arrive in the new habitat, relative to the number of migrating individuals between extant populations, and (ii) the degree of common source population origin of colonizing propagules As the extent of genetic founder effects is strongly mediated by the amount and the direction of gene flow between populations, it can be expected to depend strongly on the spatial configuration and the connectivity of the restored populations and the source populations Origanum vulgare (Lamiaceae) in newly restored semi-natural grassland patches. This species is a diploid (2n = 30), aromatic perennial herb, mainly occurring in grasslands on relatively dry, nutrient-poor to moderately nutrient-rich calcareous soils Origanum vulgare exhibits gynodioecy, in which both hermaphroditic and functionally female individuals co-occur O. vulgare bare exclusively male-sterile flowers, which contain one receptive stigma and four aborted (sterile) anthers and are considerably smaller than those of hermaphroditic plants O. vulgare in Western Europe has been found to vary between 1–62% of male sterility sensuO. vulgare was classified as forming a persistent seed bank in 41% of all performed seed bank studies including this species O. vulgare, seeds were found to survive longer than 5 years in the soil In this study we investigated the genetic consequences of recent colonization of the long-lived plant species Origanum vulgare, we compared genetic diversity, linkage disequilibrium and genetic differentiation between fourteen recently colonized populations and thirteen old established populations, using 10 highly polymorphic microsatellite markers. Since we sampled a large number of possible source populations, we were able to investigate whether the spatial configuration of source populations, relative to the colonizing populations, mediated patterns of gene flow between old and recent populations, and influenced the degree of genetic differentiation among recent populations. Because our study system consisted of a large set of recently restored grassland patches, all founder populations were of relatively recent origin (<10 years old). This allowed us to examine the impact of source population genetic diversity on the genetic makeup of the recent populations, after only very limited effects of possible drift and inbreeding effects, which are expected to become more likely with increasing number of generations and thus with aging To investigate the genetic consequences of the recent colonization of The sex ratio in natural populations of gynodioecious plant species can be highly variable, particularly in recently established populations, due to random sampling effects during colonization Origanum vulgare structure genetic colonization patterns?Does the spatial configuration of old relative to recent populations of O. vulgare characterized by impoverished genetic diversity, increased linkage disequilibrium and inflated genetic differentiation, caused by founder events?Are recent populations of Do our results concur with either the ‘propagule pool’ or the ‘migrant pool’ model of Slatkin 2 . These mature grasslands occur on stony hills ranging in altitude from 150 to 250 m and are surrounded by forests and cultivated land th century, most grasslands had become small and highly fragmented due to plantation of Pinus sylvestris and P. nigra and the abandonment of grazing, often leading to Buxus sempervirens encroachment. Recent restoration practices between 2001 and 2007 have led to the creation of ca. 100 ha restored historical calcareous grassland through the removal of trees and shrubs, which reduced the spatial isolation of the remaining grasslands O. vulgare is mainly restricted to remnant and restored calcareous grasslands. After tree and shrub removal, spontaneous colonization of typical grassland species was allowed. Colonization of grassland species was only facilitated through the movement of sheep across grassland patches for grazing, with no deliberate introduction of seeds or seedlings. Origanum vulgare responded very quickly to restoration practices and was successful at establishing large populations on several recently restored grasslands.The study area comprises several calcareous grasslands in the valley of the river Viroin (Southern Belgium) and covers c. 80 kmIn total, we randomly selected 27 populations: 14 populations were located in recently restored calcareous grasslands, and 13 populations in mature calcareous grasslands . In the In the laboratory mean seed weight per population was obtained using a high precision balance (accuracy = 0.001 g). For each population, three replicates of 20 seeds were placed on moist Whatman paper in separate 10-mm Petri dishes. Seeds were incubated at 5°C for 2 weeks, followed by 4 weeks at 20°C with a 12h photoperiod. The number of germinated seeds was counted daily. Reproductive success per population was quantified using two estimates: mean seed weight and the percentage of germinated seeds (germination rate) at the end of the experiment.http://dx.doi.org/10.5061/dryad.08gc2.We extracted DNA from collected leaf samples using the Nucleospin DNA-extraction kit . DNA quality and concentration were estimated using a NanoDrop ND-2000 spectrophotometer . For this study we used ten microsatellites developed by Novak et al. E) and observed heterozygosity (HO) were calculated for each population using GenAlEx 6.5 IS) was estimated based on Wright`s F-statistics with GenAlEx 6.5 After checking the microsatellite data for scoring errors due to stutter bands, null alleles and large allele dropout with MICRO-CHECKER O, HE, FIS and LD as dependent variables. The models contained population age (recent vs. old) as a factor, and population size and the percentage of female plants as covariates and all first order interaction terms. All non-significant terms were removed using stepwise model reduction to obtain the final models. Population size and the percentage of female plants (%F) were log transformed to obtain homogeneity of the variances. We tested for effects of population age and population size on the percentage of female flowers with an analogue GLM model. We also performed a Levene’s test to test for differences in variance in the percentage of female flowers between recent and old populations. Mean population size was compared between restored and old populations with a t-test. To test for founder effects at the level of reproductive success, mean seed weight and germination rate were correlated to FIS using Pearson correlations and compared between recent and old populations using a t-test.To test for the occurrence of founder effects on the different metrics of within population genetic diversity we used first-order factorial General Linear Models (GLM) in Statistica 10 ST) was calculated. Because genetic differentiation measured by FST may be underestimated for multi-allelic markers, such as microsatellites ST and Jost’s D which are not affected by marker variability. G’ST is the original GST as defined by Nei ST(max)) Pairwise genetic differentiation among populations based on Wright`s F-statistics . An analogous analysis was performed to compare pairwise geographical distances between recent and old populations. Total genetic diversity was partitioned among recent and old populations (among groups), among populations and within populations by performing a hierarchical analysis of molecular variance (AMOVA) on FST with GenAlEx 6.5 ST, G’ST and Jost’s D) distances on pairwise logarithmic spatial distances using Mantel tests in GenAlEx 6.5 Genetic differentiation among populations was compared between recent and old populations based on a 2-tailed t-test on the pairwise F4 and runs of 105 repetitions for each value of K (number of population clusters), varying from 1 to 16. We performed 20 iterations for each tested value of K. The entire model was also rerun using the LOCPRIOR option. This allows the model to use population identity of the individuals as prior information to assist the clustering. The true number of K for both models was identified based on the approach of Evanno et al. To characterize the overall genetic structure, we applied a Bayesian clustering approach implemented in STRUCTURE 2.3.3 P = 0.05. Based on the assignment analysis, we calculated pφ for each recent population, i.e. the probability that two gene flow events into a recent population originate from the same source population, by combinations of probabilities pφ for each recent population was calculated as p (with p = 1 to m), sk the number of plants in the recent population p genetically originating from source population s (with s = 1 to n) and tk the total number of plants in the recent population . The overall value of pφ for the study system was calculated as the mean pφ over all recent populations. To infer the effects of spatial configuration on gene flow, we correlated the geographical distance between each recent population and the different source populations, with the number of plant individuals assigned to each of these source populations. Since these data points are not independent, we performed a linear regression with bootstrapping (10000 bootstraps) (SPSS Statistics 19.0).Finally, to infer colonization patterns, individual plants of recent populations were assigned to old populations based on the Monte Carlo resampling procedure MICRO-CHECKER results indicated no problems with scoring errors due to stutters or allelic dropout in any of the 10 loci. We did, however, detect the occurrence of a homozygote excess in 22% of the populations for 2 loci (OR 12 and OR 75), possibly indicating the occurrence of null alleles. For this reason, all analyses were rerun excluding these loci. As the obtained results were similar to those obtained using all 10 loci, we decided to include all 10 loci in the analyses.O of 0.44 (range: 0.41–0.54) , the percentage of female flowers  or the variance in the percentage of female flowers . The percentage of female flowers was not related to population size. The mean number of alleles increased with increasing percentage of female plants. However, this pattern was influenced by population size, with a decrease in the positive correlation with decreasing population size  was not affected by any of the measured population characteristics. Linkage disequilibrium (LD) on the other hand, decreased with increasing population size, but was unaffected by population age , but not for recent populations . We found no evidence of recent genetic bottlenecks in any of the 27 populations. Reproductive success was not affected by FIS  or population age .Recent populations had a median of 708 plants (range: 150–5000), with median of 9.7% female plants (range: 3.3–25.9%). The number of alleles per population (A) varied between 3.1 and 4.1 alleles per population (average: 3.6), whereas observed heterozygosity varied between 0.37 and 0.48 (average: 0.42) for recent populations . Old pop41–0.54) . No signulations , Fig. 3.tion age . When weST = 0.040; G’ST = 0.058; Jost’s D = 0.039), but significant according to the AMOVA based on FST (P<0.001) . We observed significant isolation by distance based on FST, G’ST and Jost’s D when including all populations, a trend that was even stronger when only including old populations. However, no isolation by distance was observed when including only recent populations  at all values of udy area .β = –0.25, P = 0.018)  . We founulations .Origanum vulgare, resulting in several new populations in less than 10 years time. Contrary to our initial predictions, the percentage of female plants was not related to population age or population size, suggesting that no sampling effects had occurred in the sex structure of the founder populations after colonization Beta vulgaris ssp. maritima, which showed a higher variability in sex structure compared to older populations The restoration of large patches of calcareous grassland following tree removal led to fast colonization of Salix reinii on Mount Fuji mainly consisted of seed recruitment of nearby populations supplemented by only limited seed recruitment over long distances. The diverse genetic origin of the individuals of recent populations in our study system (pφ = 0.22) suggests that it more likely follows the ‘migrant pool’ model according to Slatkin ST = 0.040) The individuals of the newly established populations were assigned to on average 6.1 different source populations, suggesting considerable gene flow within our study area. This was confirmed by the lack of genetic differentiation between old and recent populations. Nevertheless, we observed a significant effect of the spatial configuration of the grasslands on gene flow, with gene flow into the recent populations mainly originating from nearby source populations. This was also reflected in the spatial aggregation of three of the four genetic groups defined by STRUCTURE. These results are in accordance with the findings of Myrica cerifera during range expansion on Hog Island off the coast of Virginia (USA). Similar results during primary succession were found after colonization of glacier forelands by Geum reptansSaxifraga aizoidesVaccinium membranaceum at Mount St Helens in Washington Antirhea borbonica on the Piton de la Fournaise volcano on La Réunion Nassauvia lagascae var. lanata on the Lonquimay volcano in Chile Primula veris during secondary succession in young ‘grassland’ fragments in the Oxford region (UK).Our results indeed showed that recent populations were not more genetically differentiated from each other than old populations. This is in accordance with previous research on the colonization of plant species that are characterized by high levels of gene flow and low overall genetic differentiation. Erickson et al. O. vulgare were found to be little differentiated, old populations showed a higher degree of genetic differentiation. Furthermore, we observed significant isolation by distance for old populations, but not for recent populations. This suggests that old populations are moving towards a migration-drift equilibrium. These populations likely became smaller and more isolated when the grassland area decreased in the years prior to restoration Whereas recent populations of As predicted by the ‘migrant pool’ model of Slatkin O. vulgare, resulting in a higher inbreeding coefficient in these populations. The mean FIS value of 0.083 for recent populations was low however, and together with the absence of fitness effects on germination rate or seed weight in these recent populations, this suggests that inbreeding depression is absent. We can expect that future gene flow and population expansion of the recent populations will probably lead to a decreasing FISObserved heterozygosity, on the other hand, was significantly lower in recent populations of Origanum vulgare is known to form a persistent seed bank in our study area O. vulgare were still present within the soil seed bank at the time of restoration. Recruitment from these seeds could have influenced the observed levels of gene flow, cf. Finally, the absence of pronounced founder effects in this study may also partly be explained by the occurrence of seed dispersal through time O. vulgare metapopulation and also to mitigate the consequences of the genetic drift observed in the original source populations.This study demonstrated that spontaneous plant colonization after habitat restoration can lead to new and viable populations, overcoming potentially important genetic founder effects, when several source populations are nearby. It has been suggested that the severity of founder effects is largely dependent upon the characteristics of the study area and the traits of the focal species, through mediating gene flow and population growth rate after colonization Table S1Pairwise genetic differentiation among Origanum vulgare populations. Lower left triange: FST estimates; Upper right triange: G’ST estimates. Significant values are shaded in grey.(XLS)Click here for additional data file.Table S2Pairwise genetic differentiation among Origanum vulgare populations. Lower left triangle: Jost’s D estimates Significant values are shaded in grey.(XLS)Click here for additional data file.Table S3Inferred ancestry of individual plants. Analysis based on the Bayesian clustering of STRUCTURE 2.3.3 for K = 4, using the admixture with correlated alleles model with LOCPIOR option. qg =  the probability of individual ancestry from group g. Each individual was assigned to the group with the highest qg value (assigned group).(XLS)Click here for additional data file.