Astacus leptodactylus) was examined by means of polymerase chain reaction (PC R) restriction fragment length polymorphism (RF LP) analysis of the cytochrome oxidase subunit I (COI) of mitochondrial DNA. A total of 194 adult specimens were collected from seven sample sites including, two in the south Caspian Sea and one each in Anzali wetland and Aras reservoir and three rivers Chafrood, Masule Rudkhan and S iah Darvishan. The PCR products were digested with 19 restriction enzymes and five enzymes revealed polymorphism patterns . Twenty eight composite haplotypes were showed with the number of haplotypes in each population sample ranging from 8 to 13. Private haplotypes were found at very low frequencies. Two regional (S iah Darvishan River and Astara) groups were clearly recognized by cluster and molecular variance model (AMOVA) analyses (P<0.0001). Each of these groups revealed dominant haplotypes while these haplotypes play less important rule in population structures of the other geographic areas. Intrapop ulatio n hap lo type (h) and nucleotide (π) diversities were high for each locality, ranging h=0.7560±0.030 and π=0.00334±0.00301, respectively. Results of this study discerned two divergent populations of narrow-clawed crayfish including S iah Darvishan River and Astara. Thus, the population structure of the narrow-clawed crayfish, as inferred from mtDN A analysis, is constituted by genetica lly separate groups that nearly reflect their geographic distribution.The genetic variation and population structure of narrow-clawed crayfish ( Astacus leptodactylus is naturally and widely distributed in lakes, ponds and rivers throughout of Iran. A. leptodactylus is a widespread species and can be found throughout Europe, Eastern Russia, and the Middle East. However it is absent from some of the Northern European countries such as Norway and S weden, and the Southern European countries such as Spain and Portugal [Astacus leptodactylus, with two subspecies, A. l. leptodactylus and A. l. eichwaldi. Only A. leptodactylus is commonly distributed in Iranian water resources. A. leptodactylus was found not only in the Caspian Sea up to 5–10 km offshore [The narrow-clawed crayfish Portugal , 2. The Portugal . This spPortugal , 5. Iranoffshore , 7. The offshore .Austropotamobius pallipes and information on genetic structure of A. leptodactylus is still rare. Assessing the relative levels of genetic diversity within and divergence among narrow-clawed populations in Iran can provide the first step towards conserving narrow-clawed genetic resources here and optimizing them for future breed improvement programs. This study is the ﬁrst molecular-based study of A. leptodactylus covering a large part of its distribution range, including river catchments of the south part of the Caspian Sea, Anzali lagoon, Southwest Caspian Sea and Aras reservoir in Northwest of Iran. The purpose of this study is to appraise the genetic status of the narrow-clawed crayfish breeds, by estimating both their relative levels of genetic diversity and the scope of genetic divergence among them using clustering procedure.The advantages of mtDNA as a tool for population genetics have been extensively reviewed , as wellA. leptodactylus were collected from seven localities . Primers 5′-GGT CAA CAA ATC ATA AAG ATA TTG-3′ (forward) and 5′- TAA ACT TC A GGG TGA CCA AAA AAT CA-3′ (reverse) were designed to amplify an approximate 1535 bp of the mtDNA COI gene. For amplification, the following reagents were added to each microtube: 2 µl of template DNA; 5 µl of 10× b uffer ; 1 µl of each primer (10 pmol); 5µl of a 5 mM solution of each deoxyribonucleoside triphosphate (dNTP); 2.5 units of Taq DNA polymerase. Enough ultrapure water was added to each sample to make a solution of 50 µl. Polymerase chain reaction conditions consisted of 35 cycles of denaturation at 94°C for 1 min, annealing at 56°C for 1 min and extension at 72°C for 1 min (5 min for the last extension only). The amplicons were exposed to endonuclease digestion using the four-base recognitio n enzymes Rsa I, Hinf I and MboІ and the five- base DdeI and TaqІ. All restriction digestions followed the standard procedure provided by the manufacturer. Restriction digestion was carried out in a 10 µl volume containing 2 µl of PCR product, 2 units of restriction enzyme, 1 µl of the approp riate b uffer and 7µl of ultrapure water. RFLP digestion was performed in an incubator at 37°C for at least 16 h. Restriction fragments were segregated on a 6% acryl amid gel, stained with silver staining technique and photographed.The mtDN A cytochrome oxidase subunit I was amplified using the polymerase chain reaction (PCR). Primers were designed manually specifically for h ) in a population were computed according to known equation [A composite mtDN A haplotype, consisting of five letters that represent the fragment pattern generated by each of the restriction endonucleases, was compiled for each individual. The nucleotide d iversity (π) and haplotype diversity . Q uantequation , 49. F-sequation .COI gene. Twenty eight different composite haplotypes were revealed with the number of haplotypes in each population sample ranging from 8 to 13 and 13 of them were private, that is, present in only one population sample (P<0.005).Variation within samples in all 194 individuals amplified successfully in the mtDN A n sample . PrivateThe geographic distributions of all haplotypes in throughout of sampling areas are given in h ) and nucleotid e diversity (π) were 0.7560±0.030 and 0.00334±0.00301, respectively. The samples with the highest haplotype diversity were from S iah Darvishan River (0.829±0.046) and Chafroud River (0.816±0.032). On the other hand, the population samples with the lowest variability were from the southern part of the Caspian Sea in K iashahr with haplotype diversity0.687±0.025 as well as the Aras River (0.702±0.038) (In the present study the mean haplotype diversity (2±0.038) . Table 3A.leptodactylus populations test showed significant genetic structures in 8 of the 21 pairwise comparisons, most involving the S iah Darvishan River and Astara samples (P<0.0001). It was shown that the main contribution to the heterogeneity of the population set was made by the A. leptodactylus samples from the S iah Darvishan River and Astara populations. Thus, three distinct genetic groups were identified . The cluster analysis also supported the existence of these three groups.The lowest Nucleotide divergence was seen between K iashahr and Anzali samples. The results of the chi square test of  samples . The χ2 A. leptodactylus populations from K iashahr and Anzali lagoon. Thus, the UP GMA tree suggested threedistinct groups; S iah darvishan River, Astara, Aras - Anzali lagoon- Chafrood River - Masuleh River . Therefore heterogeneity test of the narrow-clawed crayfish populations for haplotype frequencies and Monte-Carlo with 1000 replicates in RFLP analysis of COI gene showed significant differences (P<0.0001) and these results showed that haplotype distribution in different location were significant and populations of S iah darvishan River and Astara statistically were significant (P<0.0001).The degree of genetic differentiation among all sample pairs was tested with analysis of molecular variance (AMOVA). The results of AMOVA demonstrated that collections between Astara in the southwest Caspian Sea and also Siah Darvishan River are significant . However, according to the data acquired, the hierarchical levels of geographically isolated subdivisions were statistically significant (P<0.05). Thus concluded that the most part of the mtDNA intraspecific variation is determined by the differences among the haplotypes within a single population.variance of haplo type frequencies was subdivided into three hierarchical levels . An anal 56.85%, . The AMOC OI gene) showed the high haplotype diversity. Detecting genetic variation in mtDNA between organisms depends chiefly on the evolutionary rate of genes and the number of nucleotide bases surveyed [A. leptodactylus [Austropotamobius pallipes pallipes [Astacus astacus [A. leptodactylus corresponding to the southern part of Caspian Sea and S iah Darvishan River. This result suppo rts the hypothesis that each of these clusters include genetically differentiated populations. Most of the variation is distributed within populations, which may be explained by the existence of many shared haplotypes between populations. Analyses of the genetic structure of crustacean species with Caspian Sea tributaries distribution are relatively few and have mostly dealt with ﬁsh species [e.g. Acipenser persicus [A. nudiventris, [A. gueldenstaedtii [Results obtained from the analysis of mtDNA variation proved that most narrow- clawed crayfish samples display a wealth of diversity, presenting values of haplotype diversity. We found that the large mtDN A gene region  F ive of 7 populations showed no genetic diversity, while the most sample sites contained moderate level of genetic variability, especially because of the presence of private haplotypes. These levels of genetic variability are in accordance with the data of Grandjean and Souty-Grosset 2000) . Accordi000 . Acc F ive ofulations , 60. Add


Frangula alnus) is one of Ireland’s rarest tree species, and in Northern Ireland the species is now restricted to a single population in Peatlands Park, Co. Armagh numbering ca. 140 mature trees. Genotyping of 95% of the trees at nine nuclear microsatellite loci revealed that levels of genetic diversity within this population were generally lower than those reported from larger populations in Spain. Analysis of six chloroplast microsatellite loci revealed no variation. The level of FIS was significantly higher than that in the Spanish populations, as well as in other populations across Europe, potentially indicating inbreeding. Spatial autocorrelation analysis indicated some evidence of fine-scale genetic structuring, most likely due to limited seed dispersal, but the overall level of differentiation between subpopulations was low, indicating high levels of gene flow, probably due to cross-pollination by bees. Our results are consistent with a gradual population expansion from a limited number of individuals. We suggest that more immediate conservation efforts might be best focused on ensuring suitable habitat for the continued recovery of this isolated population.Alder buckthorn ( This can be further compounded if such populations have been founded by a limited number of individuals3, since genetically depauperate populations tend to have reduced evolutionary potential, which can increase the risk of extinction5. Where populations are fragmented, as is often the case in threatened taxa, reduced levels of gene flow between fragments can also aggravate the problems associated with limited genetic diversity, as there is less scope for immigration of alleles to counter the effects of drift7. Consequently, knowledge of the levels and patterns of genetic diversity in populations of threatened species are vital to the formation of well-informed, effective conservation plans9.Populations of endangered or threatened species tend to be small and/or isolated and are thus particularly vulnerable to stochastic processes. These problems are further exacerbated at the genetic level, where the increased effects of genetic drift and potential for inbreeding can lead to low levels of genetic variationFrangula alnus  is one of Ireland’s rarest tree species. Although widespread in temperate Europe, the species has a very limited and fragmented distribution in Ireland, where it has been in serious decline over the last few decades as a result of drainage of its preferred bogland habitat for alternative land use11  Order (1985) and is a Priority Species for Conservation Action. The history of this population, which currently numbers ca. 140 individuals  whether there is any evidence for a genetic bottleneck, (2) if the establishment from a relatively limited number of individuals has been accompanied by a degree of inbreeding, and (3) whether there is any significant genetic substructuring within the population. In addition, as F. alnus is considered an invasive pest species in many countries20, our findings could also shed light on the genetic demography of this recently expanded population with respect to similar invasive populations.F. alnus had been recorded previously found that the species is now restricted to a single location; Peatlands Park, Co. Armagh. The sole remaining population exists as five discrete clusters of plants numbering between 3–98 individuals, each separated by between 100–1,300 m  and from 0.331 (Subpopulation C) to 0.423 (Subpopulation D) for expected heterozygosity (HE mean = 0.387). Mean inbreeding coefficients (FIS) across loci . Allele frequencies by locus and subpopulation are given in Table HE = 0.411 vs. HE = 0.608 for Ajibe ; HE = 0.603 for Medio ; HE = 0.470 for Puerto Oscuro ; Fig. FIS for the Peatlands Park population (0.251) was significantly higher than those from the three Spanish populations  = 0.034]; FIS = −0.027 for Medio ; FIS = −0.070 for Puerto Oscuro ; Fig. The Peatlands Park population exhibited significantly lower levels of genetic diversity than two of the three Spanish populations based on the loci analyzed in the present study . The BAPS analysis indicated that the first four subpopulations belonged to the same genetic cluster, with the fifth (Subpopulation E) being genetically distinct 25. Interestingly, though, a further study on populations from Italy, France, Belgium and Sweden using 186 single nucleotide polymorphisms (SNPs) showed no decrease in genetic diversity with latitude26.The genetic analysis of the sole remaining population of 15. Although there are no records predating 1934, evidence suggests that the population remained relatively stable from this time until the end of the 1980s, after which numbers increased to the current census of ca. 140 individuals13. It may be unwise to take this at face value, though, since the power of the test is affected by several parameters, including number of loci used, number of generations since the bottleneck, the length of the bottleneck itself, and the magnitude of reduction in effective population size. Immigration, another potentially confounding factor, can most likely be ruled out, since the closest known F. alnus populations to that in Peatlands Park are found ca. 125 km away in Co. Westmeath, Ireland. Likewise, the mutation model is unlikely to have an effect, since the results were broadly consistent across the three models analyzed, and changing the parameters of the two-phase model from 90% single-stepwise mutations to 70% also had no effect. The generation time of F. alnus has been estimated to be between 5 years in Central Europe to around 20 years in the South27, so the maximum number of generations of growth is likely to be much less than 10, although the period of apparent population stability preceding this could be upwards of 10 generations. These time scales may limit the power of the heterozygote excess test to detect any possible bottleneck, but this may be balanced to some extent by the fact that, based on historical records, the effective population size (Ne) after the bottleneck is unlikely to be any higher than ca. 2015.Despite the low levels of genetic variation, the Wilcoxon test for heterozygote excess did not indicate the occurrence of a bottleneck. Seven of the nine loci indicated a heterozygote deficiency, which could be indicative of population stability or constant growthF. alnus , but this has apparently been accompanied by a degree of inbreeding, based on FIS values. Such inbreeding is most likely a result of the relatively limited genetic base of the population, particularly compared to the other populations of F. alnus. The mean value of FIS for the Peatlands Park population is significantly higher than those from the three Spanish populations studied previously21. It is also far higher than the range of values reported in F. alnus populations across Europe based on SNPs 26. Reproductive dominance by a relatively low number of highly fecund individuals within a population could give rise to biparental inbreeding29, but since F. alnus possesses a self-incompatibility mechanism, as indicated by crossing studies30, this would be dependent on the diversity of S alleles in the population. Previous studies have indicated a breakdown in self-incompatibility following population bottlenecks32, but such a possibility would have to be tested via controlled pollination experiments.Removal of rhododendron from areas of the park since the designation of the Annagarriff Nature Reserve may have facilitated the increase in numbers of F. alnus indicates a general lack of barriers to dispersal, which is unsurprising given the small spatial scale relative to the potential dispersal distances of pollinators. The overall level of population differentiation (ΦST = 0.032) was lower than the average values for outcrossing species with seeds dispersed by gravity (0.152) or ingestion (0.200) quoted by Hamrick & Godt33, as well as the average value for biparentally inherited markers in angiosperms (0.184) quoted by Petit et al.34. The spatial autocorrelation analysis suggested some fine-scale genetic structure in Subpopulation D, probably due to limited seed dispersal. Seeds of F. alnus can be dispersed by birds35, but in the closely related (congeneric in some classifications)36Rhamnus cathartica, it has been shown that 90% of fruits fall beneath the mother tree37. This scenario could at least in part be responsible for the high FIS observed in Subpopulation D, by way of a Wahlund effect.The absence of high levels of spatial structuring of genetic variation in the Peatlands Park population of F. alnus population suggests substantial levels of cross-pollination. Bees have large foraging ranges, even across sub-optimal habitats, and are likely to be important in maintaining the connectivity observed between the fragments in the present study39. In contrast to the levels of fruiting found in Peatlands Park, where almost all trees had multiple fruits, a previous study on reproduction in populations of F. alnus from Cádiz, Spain found that only 2.8% of open-pollinated flowers set fruit30. Another study on southern range edge populations also indicated that the majority of ovule losses were due to cross-pollen limitation and extensive geitonogamy, and that seed output in the populations was limited to a few large trees40. The high percentage of fruiting trees observed in Peatlands Park suggests that this population is more similar to those found in Central European populations, which have a shorter generation time and higher levels of fruit production compared to Southern Iberian populations27. If the observed high FIS values reflect some degree of inbreeding, there has been no apparent impact on fitness, at least in terms of fruit production.The area surrounding Peatlands Park has a number of apple orchards with large numbers of wild bees in the vicinity, and the high incidence of fruits in the Peatlands Park 41. Finally, only a single private allele is present in Subpopulation E, with the majority of genetic differentiation being due to the aforementioned differences in allele frequencies at several of the loci studied.Although the BAPS analysis assigned Subpopulation E to a separate genetic cluster from the other four subpopulations, this should be taken with some degree of caution for several reasons. Firstly, the subpopulation numbers only three individuals, and thus allelic frequencies will be skewed. Secondly, it has been shown previously that the BAPS algorithm tends to over-estimate the true number of genetic clusters present in the data42, in the case of F. alnus in Northern Ireland it would appear that edaphic and ecological factors are of greater importance. The land classes that are correlated with occurrence of alder buckthorn include bog, conifer and pastures, and this is consistent with the literature that claims this species likes open lowland areas with moist, fertile soils44. Nevertheless, there are large areas of apparently suitable habitat in Peatlands Park which have not been colonized during the recent expansion of the species in the nature reserve. This would suggest that although the low levels of genetic diversity and potential inbreeding revealed in the population are of some concern with respect to evolutionary potential, more immediate conservation efforts might be better focused on ensuring suitable habitat for the continued recovery of this isolated population. Additionally, consideration should be given to possible supplementation using material from the populations in the Republic of Ireland, which are larger and may harbor additional genetic diversity.Whilst it is true that knowledge of the evolutionary dynamics of natural populations with respect to demography and gene flow allows the management of threatened plant populations to go beyond simple “conservation gardening”Frangula alnus Miller (syn. Rhamnus frangula L.) is a small tree or shrub found across temperate Europe from northern Scandinavia and Russia to the Mediterranean, where it is comparatively scarce43. In Britain and Ireland, the species tends to be found in damp, boggy habitats, but can also colonize drier ground outside of its native range, particularly in North America where the species is now considered an invasive alien35. Reproduction in F. alnus is exclusively sexual30. Flowers are hermaphroditic and well-adapted to insect pollination as they have nectar, colour and odour. They are generalised entomophiles, with orders Hymenoptera, Diptera and Coleoptera providing the main pollinators, and occasional pollination by Lepidoptera40. Controlled pollination studies revealed almost no selfing or geitonogamy, indicating the existence of self-incompatibility mechanisms, although occasional selfing in the absence of insect pollinators has been reported30. Field studies suggest that limited fruit initiation is primarily due to low levels of cross-pollination40. Seeds are dispersed either by gravity or via ingestion by birds37.F. alnus had been found previously based on records from the National Biodiversity Network (NBN) Gateway (http://data.nbn.org.uk) and the Centre for Environmental Data and Recording (CEDaR: http://www.habitas.org.uk/cedar/). Frangula alnus is restricted to the southern shores of Lough Neagh, where it exists as a fragmented population from a single location in Annagarriff Nature Reserve in Peatlands Park, County Tyrone  could not be consistently amplified, and three  were monomorphic, leaving nine polymorphic loci: FaB101, FaA110, FaB7, FaA104, FaB106, FaB4, FaA7, FaA3 and FaB9. All reactions were carried out on a MWG Primus thermal cycler. PCR was carried out in a total volume of 10 μl containing 100 ng genomic DNA, 10 pmol of dye-labelled forward primer (HEX), 1 pmol of tailed forward primer, 10 pmol reverse primer, 1x PCR reaction buffer, 200 μM each dNTP, 2.5 mM MgCl2 and 0.25 U GoTaq Flexi DNA polymerase (Promega). PCR conditions were as described previously19. Genotyping was carried out on an AB3730xl capillary genotyping system. Allele sizes were scored using the GeneMapper software package  and LIZ-500 size standards, and were checked by comparison with previously sized control samples.We attempted to genotype all mature plants for sixteen previously described microsatellite loci for F. alnus chloroplast genome sequences either from GenBank or in regions amplified and sequenced de novo using universal chloroplast primers22. The trnT-trnF, atpH-atpI, atpI-rpoC2, rps18-clpp, psbC-trnS, trnS-trnfM and rpl16-rps3 (UCP6) regions were amplified and sequenced using the primers described in Grivet et al.47 and Provan et al.48. Species-specific primers were designed using the Primer3 program to amplify six chloroplast microsatellites  and expected (HE) heterozygosity, and fixation indices (FIS) were calculated using the Arlequin (V3.5.1.2)50 and Fstat (V2.9.3.2)51 software packages respectively. Significance of FIS was determined by 10,000 randomization steps. To test for the occurrence of a genetic bottleneck, the Wilcoxon test for heterozygote excess was performed under the infinite alleles model (IAM), the stepwise mutation model (SMM) and a two-phase model (TPM) incorporating 90% single-stepwise mutations using the program Bottleneck (V1.2)52. The Wilcoxon test was used as it is recommended for a relatively low number of loci.Subpopulations of the Peatlands Park population were classed as distinct groups of trees with no other trees of this species growing within 100 m between groups, resulting in five distinct subpopulations  framework54 using Arlequin. To identify possible spatial patterns of gene flow, the software package BAPS (V5)55 was used to identify clusters of genetically similar subpopulations using a Bayesian approach. Ten replicates were run for all possible values of the maximum number of clusters (K) up to K = 5, the number of subpopulations, with a burn-in period of 10 000 iterations followed by 50 000 iterations. Multiple independent runs always gave the same outcome. To further identify possible spatial patterns of gene flow, spatial autocorrelation analysis was carried out for Subpopulation D, the largest of the five subpopulations, using SPAGeDi (V1.4)56. Mean coancestry coefficients (θxy)57 between pairs of individuals were calculated at 25 m distance class intervals, and plotted as a correlogram, with 95% confidence intervals calculated from 1,000 permutations of individuals within each distance class, and for estimates of θxy using 1,000 permutations.The level of genetic differentiation between the five fragments was estimated using Supplementary Info


Poa annua on King George Island (Maritime Antarctic). The remoteness of this location, its geographic isolation, and its limited human traffic provided an opportunity to trace the history of an invasion of the species. Poa annua was recorded for the first time at H. Arctowski Polish Antarctic Station in the austral summer of 1985/6. In 2008/9, the species was observed in a new locality at the Ecology Glacier Forefield (1.5 km from “Arctowski”). We used AFLP to analyze the genetic differences among three populations of P. annua: the two mentioned above (Station and Forefield) and the putative origin of the introduction, Warsaw (Poland). There was 38% genetic variance among the populations. Pairwise ФPT was 0.498 between the Forefield and Warsaw populations and 0.283 between Warsaw and Station. There were 15 unique bands in the Warsaw population (frequency from 6% to 100%) and one in the Station/Forefield populations . The Δ(K) parameter indicated two groups of samples: Warsaw/Station and Forefield. As indicated by Fu's Fs statistics and an analysis of mismatch distribution, the Forefield population underwent a bottleneck and/or founder effect. The Forefield population was likely introduced by secondary dispersal from the Station population.We studied an invasion of  Samples from the Polish population  were collected in May 2009. The population found growing on the Forefield of the Ecology Glacier within the Antarctic Specially Protected Area 128  following the manufacturer's recommendations. To assess genetic variability, we used the amplified fragment length polymorphism (AFLP) procedure ‐labeled primers. Eight selective primer pair combinations were used  for each population from binary data assuming Hardy–Weinberg equilibrium  to quantify the amount of variation of the likelihood for each K. The range of possible Ks tested was 1–10. In order to determine the optimal number of clusters (K), an ad hoc statistic ΔK  was created  parameter revealed the presence of two groups of samples. No additional structuring was observed  for excess heterozygosity implemented in the bottleneck software produced significant el Table .4KpnI/MseI platform was highly efficient in differentiating the analyzed populations as pointed out I value  identified caryopses of P. annua among diaspores and phyto‐remains of 46 other plant species in cargo transported to Arctowski. Thus, it cannot be excluded that the Antarctic population was founded by multiple introductions from different sources, which is supported by the presence of one band exclusive to both Antarctic populations. Multiple introductions are a common feature of biological invasions , this supports the hypothesis that the Forefield population was introduced directly from Station and did not originating as a new introduction. In order to study such effects using dominant markers, the infinite allele model can be used to test the mutation–drift versus the bottleneck hypothesis  and Colobanthus quitensis (Kunth) Bartl., Caryophyllaceae , with the local population of P. annua show that, the Antarctic populations of P. annua still have much higher levels of genetic variability, even in the Forefield population (Table Poa annua in Antarctica is in the early stages of invasion, so the Antarctic populations are probably still unstable and undergoing dynamic demographic processes.Comparisons of genetic variability of the only two Antarctic angiosperm on Table . Poa annP. annua may also inflate its intrapopulation genetic variability. This species is an allotetraploid and thought to be derived from a cross between Poa infirma H.B.K. and Poa supina Schrad., both 2n = 2x = 14 (Heide, The polyploidy of 4 Heide, . Polyplo4 Heide, , with ma4 Heide, . It is c4 Heide, , which c4 Heide, . This is4 Heide, .Forefield population was very likely introduced from the vicinity of Arctowski. Due to a limited number of individuals, this population was influenced by a bottleneck or founder effect and strong selection pressure, with parallel expansion. A critical factor in the success of this species is the ability to adapt rapidly to new environments following introduction (Galera et al., Our results show that the P. annua on King George Island become an important conservation issue.Biological invasions have become regarded as “natural experiments,” offering unique insights into ecological and evolutionary processes occurring in real time Lee, . UnderstNone declared.KJC and MW conceived of this project. KJC collected plant samples and performed AFLP analysis. PTB and AP analyzed all data. MW, KJC, HG, and AZ wrote the manuscript. HG and AZ discussed ecological part of the manuscript. All authors commented on and contributed to the final manuscript.


Puma concolor) provide an excellent model for studying the genetic impacts of migrants on populations isolated by increasing human development. In densely populated southern California, USA, puma populations on the east and west side of interstate highway 15 (I-15) have become fragmented into a small inbred population on the west side (Santa Ana Mountains) and a relatively larger, more diverse population on the east side (Eastern Peninsular Range). From 146 sampled pumas, genetic analyses indicate seven pumas crossed I-15 over the last 15 years, including four males from west to east, and three males from east to west. However, only a single migrant (named M86) was detected to have produced offspring and contribute to gene flow across the I-15 barrier. Prior to the M86 migration, the Santa Ana population exhibited inbreeding and had significantly lower genetic diversity than the Eastern Peninsular Range population. After M86 emigrated, he sired 11 offspring with Santa Ana females, decreasing inbreeding measures and raising heterozygosity to levels similar to pumas in the Eastern Peninsular Range. The emigration of M86 also introduced new alleles into the Santa Ana population, although allelic richness still remained significantly lower than the Eastern Peninsular population. Our results clearly show the benefit of a single migrant to the genetics of a small, isolated population. However, ongoing development and habitat loss on both sides of I-15 will increasingly strengthen the barrier to successful migration. Further monitoring, and potential human intervention, including minimizing development effects on connectivity, adding or improving freeway crossing structures, or animal translocation, may be needed to ensure adequate gene flow and long-term persistence of the Santa Ana puma population.Migration is essential for maintaining genetic diversity among populations, and pumas ( ImmigPuma concolor) populations are becoming models for the study of genetics in small, isolated populations [P. concolor coryi) resulting in severe inbreeding is the extreme example [In the United States, fragmented puma  are the primary dispersers [Within southern California, our research team has been monitoring puma populations in the Santa Ana Mountains and Eastern Peninsular Range since the early 2000s . Pumas ispersers ,36 and aspersers . Our teaspersers ,34. HoweTo assess the effects that migration can have on small, isolated populations, we studied the impact of inter-population movements on inbreeding, heterozygosity and allelic richness between the Santa Ana and Eastern Peninsular Range puma populations ,34. We u2.2.1.et al. [et al. [We captured, marked and monitored radio-collared pumas from 2001 to 2016 in the Santa Ana Mountains and Eastern Peninsular Range. Capture methods are detailed in Vickers et al. . We exam [et al. .2.2.et al. [et al. [We analysed genetic samples from 146 pumas among the Santa Ana Mountains, San Gabriel Mountains, San Bernardino Mountains and the Eastern Peninsular Range. Genetic sampling is detailed in Ernest et al. . Briefly [et al. .2.3.We used spatially explicit Bayesian population assignment programs GENELAND v. 4.0  and TESSIn TESS, K must be tested over a range of possible values. First, we ran 10 non-admixture models for each K from 2 to 10. For model comparisons, TESS computes a deviance information criterion (DIC). We ran 10 spatially conditional auto-regressive admixture models for each K to the DIC plateau of non-admixture models. All models included pairwise great circle geographical distances, 100 000 iterations, and a 25% burn-in period. We retained 20% of the models which contained the lowest DIC scores and used CLUMPP v. 1.1.2  to perfo2.4.We used program CERVUS v. 3.0.7  to const2.5.ĤE) in GenAlEx 6.502 [ĤE is a robust genetic diversity estimator for small sample sizes [FST) was also calculated in GenAlEx; significance testing was based on 999 permutations [To assess inbreeding, we calculated internal relatedness using package Rhh 1.0.2 in program R v. 3.3.0 ,45. InteEx 6.502 ,47. Unlile sizes ,48. To mle sizes ,50. PopuT-test to determine if the internal relatedness of offspring was higher than the average internal relatedness of their consanguineous parents. Temporal and population differences in heterozygosity and allelic richness were tested using a repeated-measures (RM) ANOVA with locus treated as the repeated measure. We used Tukey's honest significant differences post hoc tests. Statistics were computed in program R using base software for linear models  and packages lme4 1.1–12 [Internal relatedness, heterozygosity and allelic richness data met the assumptions of linear models. Population differences in internal relatedness were calculated using analyses of variance (ANOVAs). To assess the validity of internal relatedness as a measure of inbreeding, we used a 4 1.1–12  and lsme4 1.1–12  for mixe3.3.1.N = 6), temporal measures of internal relatedness, heterozygosity and allelic richness could not be calculated and further analyses were restricted to SA and EP. Pedigree analyses indicated the additional substructure within SA was composed of a male migrant  and his 11 offspring . Substructure within EP primarily corresponded to a female puma (F20) and her offspring, but several substructure-assigned individuals were not first-order relatives  of F20 .Based on our Bayesian population assignment analyses, we identified three geographically structured populations  with additional substructure within SA and EP figures  and 2. Gffspring b,c. Subs) of F20 b,c.FiguWith our pedigree analyses, we identified frequent mating within, but not among, populations. There was only one observed instance of mating among populations, which occurred when M86 migrated from EP into the Santa Ana Mountains in 2010 and mated with F61, F89, F133 and mated with his offspring F92 . NotablyT137 = 5.4, p < 0.001) higher in SA (mean ± s.e.: 0.13 ± 0.02) than EP .Internal relatedness was significantly (3.2.c). M86 and his offspring had significantly lower internal relatedness compared with other SA pumas , indicating M86 decreased internal relatedness of his SA-offspring to comparable levels of EP pumas  and allelic richness (Ar) than EP in all time periods (RM-ANOVA: Tukey HSD p < 0.0001). After the M86 migration, ĤE increased in SA and was no longer significantly different from EP . Although Ar in SA significantly increased after the M86 immigration, SA had significantly lower Ar than EP in all time periods (p < 0.001). SA–EP population divergence (FST) decreased after the M86 immigration; however, populations remained significantly diverged at all time periods  population is inbred and genetically depauperate compared to the Eastern Peninsular Range (EP) population . Our anaIt is possible that the emigration of M86 has resulted in genetic rescue . However, genetic rescue is difficult to confirm given the natural variation in individual survival/reproduction rates, population dynamics, and environmental conditions which affect individuals and populations . Pumas iN = 11) prior to his death. However, his contribution to gene flow was foreshortened because he was killed by a vehicle strike, and over half of his offspring are either deceased or in captivity. Another migrant from EP to SA (M151) was apparently killed prior to successfully siring offspring in the SA. This puma had the lowest internal relatedness of any migrant pumas we sampled, but he was unable to enhance genetic diversity in the SA because he was legally shot on a depredation permit after repeatedly preying on domestic animals. Further monitoring of the only remaining migrant (M119) in the Santa Ana Mountains, as well as M86's surviving offspring, will be important for tracking population viability, and assessing the long-term impacts of migration.The significant increase in heterozygosity and allelic richness caused by the emigration of M86 suggests that little or no gene flow occurred between the SA and EP populations in the period immediately prior to this study. Although low sample sizes through 2000–2006 in SA could have biased genetic diversity estimates, the low sample size estimates (i.e. 2000–2003 and 2004–2006) were consistent with each other and with the more-intensively sampled time period prior to M86 (i.e. 2007–2009). Additionally, our estimates of genetic diversity are robust to differences in sample size. Given the high mortality rates in the SA, it is remarkable that M86 produced a large number of offspring  and are also isolated by a major roadway ,27. Over4.2.The loss of rare alleles and accumulation of deleterious alleles can occur in small populations through genetic drift and inbreeding ,55,56. WHeterozygosity is a classic measure of genetic diversity and has been correlated to disease resistance ,64, resi4.3.We identified three puma populations in this region, including the SA, SGB and EP populations. Although population assignment models GENELAND ,71 and TWe identified a total of seven migrants, all males. Four moved east and three moved west across I-15. With the exception of M56 who was GPS-collared when he crossed I-15 in 2010, it is not known exactly when these migrants crossed the I-15 barrier. If they migrated at their dispersal ages, which is most likely, then based on their estimated ages when sampled their crossings would have all occurred between 2008 and 2014. TESS underestimated the number of migrants by including M86 into a SA subpopulation, whereas GENELAND overestimated the number of migrants by incorrectly assigning an inbred offspring (M124) of M86 to the EP population. The addition of our pedigree analysis allowed us to more accurately identify migrants and family groups, and showed that only one of three migrants into the SA population successfully established a territory and mated. Interstates, including I-15, have been reported to be major barriers to puma movement ,28,34,76FST), and SA pumas still face threats from disease, human development, and stochastic demographic, genetic and environmental events [Despite the increases in heterozygosity and allelic richness in the SA population, and despite being only separated by a single interstate highway, the SA and EP populations remain significantly diverged (i.e. significant pairwise l events ,33,34,77l events ,53,78,79l events ,62,80–82l events ,13, multThe ability of pumas to cross I-15 is very limited, and will only decrease. Several pumas have been killed on I-15 in the past 30 years, but road mortalities are only one aspect of barrier effects. Other barrier effects include the combination of noise, light, human presence, adjacent development and other anthropogenic factors. It is essential that the ability of pumas to cross over or under I-15 not be reduced further, and mitigation measures to reduce road barrier effects should be pursued. As of this writing, large residential developments are proposed to be constructed within the two primary puma travel corridors between the Eastern Peninsular and Santa Ana Ranges, and immediately adjacent to the existing crossing structures –86. ConsThese results, in combination with the challenges of preserving and improving the state of genetic connectivity for pumas in this region, emphasize that despite M86's success in improving some genetic parameters of the SA population, the population remains at risk of further genetic decline. The political and conservation barriers are large, and positive change will depend on a great deal of political-will, and both public and private investments. In the case of the Florida panther and the Santa Monica Mountains pumas, large amounts of funding were mobilized once these subpopulations were threatened with extirpation. The SA population, and possibly other puma populations in California in the future, may also need human intervention to persist.


Camellia reticulata is an arbor tree that has been cultivated in southwestern China by various sociolinguistic groups for esthetic purposes as well as to derive an edible seed oil. This study examined the influence of management, socio‐economic factors, and religion on the genetic diversity patterns of Camellia reticulata utilizing a combination of ethnobotanical and molecular genetic approaches. Semi‐structured interviews and key informant interviews were carried out with local communities in China's Yunnan Province. We collected plant material  from five populations at study sites using single‐dose AFLP markers in order to access the genetic diversity within and between populations. A total of 387 DNA fragments were produced by four AFLP primer sets. All DNA fragments were found to be polymorphic (100%). A relatively high level of genetic diversity was revealed in C. reticulata samples at both the species  and population  levels. Findings further revealed a relatively high degree of genetic diversity within C. reticulata populations . The higher genetic diversity within populations than among populations of C. reticulata from different geographies is likely due to the cultural and social influences associated with its long cultivation history for esthetic and culinary purposes by diverse sociolinguistic groups. This study highlights the influence of human management, socio‐economic factors, and other cultural variables on the genetic and morphological diversity of C. reticulata at a regional level. Findings emphasize the important role of traditional culture on the conservation and utilization of plant genetic diversity. This species has been widely described in classical literature including poems and inscriptions  Ming, . The culpe Ming, . By the C. reticulata have bred this resource with other Camellia species to produce colorful cultivars with different colors and blossom periods , and Lijiang  at the study sites who were willing to participate in surveys. A total of 120 informants participated in this study including 38 from Kunming, 20 from Chuxiong, 25 from Dali, 26 from Tengchong, and 11 from Lijiang. In addition, we examined relevant literature regarding beliefs, traditional knowledge, and customs on C. reticulata at the study sites.Research was carried out in the main distribution and production areas of g Figure . EthnoboA total of 190 individuals from five populations were sampled from the five study sites. Their local name and the morphological characteristics including flower color, flower type, and blooming period were recorded according to ethnobotanical survey as well as plant database research . PCR reactions were performed in a GeneAmp PCR system 9700 . The preamplification program consisted of 25 cycles of 30 s at 94°C, 1 min at 56°C, and 1 min at 72°C. Evaluation of the restriction digest, adapter ligation, and the preamplification occurred on a 1.5% TAE buffered agarose gel, using λ Pst as size reference (Tables S2 and S3). Selective amplification was performed with four EcoRI/MseI primer combinations with six selective bases (fluorescent labeling of the EcoRI primers): EcoRI‐ACT/MseI‐CAC (PC1), EcoRI‐ACC/MseI‐CAA (PC2), EcoRI‐ACC/MseI‐CTC (PC3), and EcoRI‐ACG/MseI‐CTC (PC4). The amplification program consisted of 2 min at 94°C, 30 s at 65°C, 2 min at 72°C, followed by 8 cycles of 1 s at 94°C, 30 s at 64°C, and 2 min at 72°C  and finally 23 cycles of 1 s at 94°C, 30 s at 56°C and 2 min at 72°C (Table S4). AFLP fragments were separated on a 3130xl Genetic Analyzer (Applied Biosystems). GeneMapper v4.0 (Applied Biosystems) was used to record the signal peak height, the signal area, and the fragment size. The data were exported to access, and a scoring table was generated as a starting point for the data analysis. The bands were scored as either present (1) or absent (0) across all loci , Nei's Gene Diversity Index  was calculated at two levels: the average diversity within populations (Hpop) and the total diversity (Ht). The proportion of diversity among populations was estimated using the following equation: (Ht−Hpop)/Ht. An analysis of molecular variance (AMOVA) was performed using Arlequin  from 1 to 14. The optimum number of clusters (K) was processed and identified by STRUCTURE HARVESTER through comparing log probabilities of data for each value of K , Nei's Gene Diversity (H), Shannon's Information Index (I), total genetic diversity (Ht), genetic diversity within populations (Hs), and gene differentiation coefficient (GST) is shown in Table Ne = 1.6041, H = 0.3578, Ht = 0.3483, and Hs = 0.3285), whereas PC4 showed the lowest level of genetic diversity .A total of 387 AFLP unambiguous bands 100%) with a gene size ranging from 50 bp to 455 bp were obtained from the 190 plant samples collected for this study with the four 00% with C. reticulata populations at our five study sites in Yunnan, the total percentage of polymorphic loci (PPL), Ne, H, and I were 85.63%, 1.4917, 0.2937, and 0.4421, respectively, shown in Table H and I varied from 0.3509 to 0.1199 and from 0.5227 to 0.1750, separately. The highest value of PPL (100%) was detected in the Kunming population with values of H = 0.3402 and I = 0.5145. The lowest value of PPL (28.94%) was found in Chuxiong population with the lowest values of Ne = 1.2046, H = 0.1199, and I = 0.1750. Compared to Kunming, Lijiang possessed a lower percentage of polymorphism loci, but had the highest values of Ne = 1.5895, H = 0.3509, and I = 0.5227.For the five 3.3Ht = 0.3483) was primarily distributed within populations (Hs = 0.3285) on the basis of the genetic variation detected by PC2 as shown in Table GST (0.0536), findings indicate that relatively low level genetic differentiation exists between the five populations; rather, the majority of genetic variation is found within populations. Based on the GST value, the estimated number of migrants (Nm) was 15.2925 and indicates that the rate of gene flow is high enough for transferring genetic diversity among populations.Total gene diversity (C. reticulata was conducted and is shown in Table p < .001), which was notably and significantly higher than that among populations .Analysis of molecular variance (AMOVA) on genetic differentiation among and within populations of 3.4C. reticulata were calculated using TFPGA Software v1.3 and are shown in Table The genetic distances among all five populations  of C. reticulata . However, an R2 value of 0.037 indicated that about 97% of the variation is not explained by the distance between population pairs, but may be caused by other factors such as hybridization by human cultivation practices.The correlation analysis between genetic distances and geographic distances among the five populations is presented in Fig. S1. Findings indicate that there was significant correlation between genetic distances and geographic distances among the five populations of C. reticulata individuals.The UPGMA dendrogram based on Nei's genetic distances that was constructed through POPGENE32 v1.32 , pink (4.74%), red (8.95%), white (3.16%), and multicolor (3.68%). The flower type of individuals primarily contained double , semi‐double (13.68%), and single flower type (13.16%). The blooming period of individuals was found to be either early , middle (49.47%), and late (8.42%) . The optimum number of clusters (K) was processed and identified by STRUCTURE, which was K = 7 . About 88.60% of flower data of all samples were covered by these 12 flower characteristics as shown in Table C. reticulata, followed by semi‐double‐pink‐early (17.37%) and double red‐middle (14.21%) . Group B mainly consists of C. reticulata cultivars that have pink petals, double type flowers, and early maturity. C. reticulata samples in group A had a closer relationship to C. japonica, compared to C. reticulata samples in group B. This pattern is potentially due to the inclusion of many other Camellia species in the formation of C. reticulata's polyploidization that have also contributed to its high genetic diversity. C. japonica as an outgroup in the phylogenetic tree was used to further discriminate the relationship of different cultivars of C. reticulata.Based on the genetic and morphological data, an UPGMA dendrogram was constructed using NTSYSpc‐2.11F software  in A and two subgroups (V and VI) in B Figure . Cultiva3.7C. reticulata during our ethnobotanical survey and named these cultivars based on their morphological features, local customs, people's interests, and values. The population of C. reticulata in Kunming, Chuxiong, Dali, and Tengchong all has rich cultivar diversity. However, the populations from Kunming, Dali, and Tengchong have higher genetic diversity than the population of Chuxiong   Table . TherefoC. reticulata with genetic data collected from populations of close congeners, C. taliensis    by this study, relatively low genetic diversity existed within populations. This genetic trend is likely because of overexploitation, frequent human activities, and insufficient conservation management  and AMOVA (3.69%) values indicated no significant genetic differentiation among the studied populations. The low genetic differentiation exhibited by C. reticulata between populations can be explained by ancient economic and cultural exchange including the trade of natural resources such as tree and flower germplasm that has resulted in massive gene flow across ecosystems and populations in Yunnan. For example, the ancient Silk Road fostered economic and cultural exchange between Yunnan Province and its surrounding regions (Wang & Zhao, C. reticulata in these areas. Except for Chuxiong (PPL = 28.94%; Table C. reticulata also has rich cultivar diversity in these four populations (Figure C. reticulata in these four populations is not notably differentiated. This lack of differentiation may be due to economic exchange along the Silk Road that likely fostered the genetic diversity and cultivar diversity of C. reticulata. Traders exchanged the cultivars or other Camellia species from different places following their trade routes and play an important role in the high levels of genetic diversity within populations.In our study, both Nei's genetic differentiation index among population (C. reticulata is dispersed by birds or insects naturally (Kunitake, Hasegawa, Miyashita, & Higuchi, C. reticulata populations, but rather artificial propagation by humans that has been facilitated by cultural exchange and other cultural factors between different geographies.Although pollen of 5Camellia reticulata resources have relatively high genetic diversity in Yunnan Province of southwestern China and that cultural factors may be a notable driving influence on fostering this diversity compared to geographic distance. AFLP was validated to be useful for examining the genetic evolution of C. reticulata as well as for elucidating genetic relationships of different C. reticulata cultivars by cluster analysis. This is the first study to provide evidence on the genetic diversity, structure, and differentiation within and among populations of C. reticulata. Traditional cultural practices and beliefs of different sociolinguistic groups in the study area of China have likely served an important role in the conservation and enhancement of C. reticulata diversity. We expect this study will be helpful for supporting biodiversity conservation, efforts for cultivar introduction, and further studies of C. reticulata and related species that are valued by different cultural groups.This study highlights that The authors declare that they have no conflict of interest.T.X. and W.J.H. wrote the manuscript as co‐first authors. C.L.L., J.D.R, and X.T. devised this study. T.X. collected the samples and conducted experiments. T.X. and W.J.H. analyzed the data. S.Z. and J.V.H were involved in the sampling and analyzing data. C.L.L., S.A., and W.J.H revised the manuscript.Camellia are furthermore provided within the Appendix The AFLP data used to determine the genetic diversity of  Click here for additional data file. Click here for additional data file. Click here for additional data file.


Geographic isolation is an important factor that limit species dispersal and thereby affects genetic diversity. Because islands are often small and surrounded by a natural water barrier to dispersal, they generally form discrete isolated habitats. Therefore, islands may play a key role in the distribution of the genetic diversity of insects, including flies.Bactrocera dorsalis, we analyzed a dataset containing both microsatellite and mtDNA loci of B. dorsalis samples collected from six offshore islands in Southern China. The microsatellite data revealed a high level of genetic diversity among these six island populations based on observed heterozygosity (Ho), expected heterozygosity (HE), Nei’s standard genetic distance (D), genetic identity (I) and the percentage of polymorphic loci (PIC). These island populations had low FST values (FST = 0.04161), and only 4.16 % of the total genetic variation in the species was found on these islands, as determined by an analysis of molecular variance. Based on the mtDNA COI data, high nucleotide diversity (0.9655) and haplotype diversity (0.00680) were observed in all six island populations. F-statistics showed that the six island populations exhibited low or medium levels of genetic differentiation among some island populations. To investigate the population differentiation between the sampled locations, a factorial correspondence analysis and both the unweighted pair-group method with arithmetic mean and Bayesian clustering methods were used to analyze the microsatellite data. The results showed that Hebao Island, Weizhou Island and Dong’ao Island were grouped together in one clade. Another clade consisted of Shangchuan Island and Naozhou Island, and a final, separate clade contained only the Wailingding Island population. Phylogenetic analysis of the mtDNA COI sequences revealed that the populations on each of these six islands were closely related to different populations on mainland China.To characterize the genetic structure of island populations of B. dorsalis. Such knowledge is helpful for a better understanding of evolutionary processes of the species of island populations.Our study suggests that these island populations have high genetic diversity, experience frequent gene flow and exhibit low or medium levels of genetic differentiation among some island populations. Therefore, the geographic isolation of the six islands does not appear to be a major dispersal barrier to The online version of this article (doi:10.1186/s12898-016-0101-0) contains supplementary material, which is available to authorized users. Genetic diversity is a critical component of biodiversity and affects the survival and evolution of species , 2. GeogBactrocera dorsalis (Hendel) (Diptera: Tephritidae), the oriental fruit fly, is a quarantined pest that is found worldwide, is highly fecund and highly adaptable. This fly species causes serious economic losses to fruit production in tropical and subtropical areas [B. dorsalis populations has been reported to be from tropical or subtropical Asia [B. dorsalis has spread over Asia and to many regions around the Pacific, including Hawaii [Bactrocera dorsalis was first recorded in Taiwan in 1911 but has since spread to many other provinces in China [B. dorsalis in the Chongqing region in China. Their results indicated that the height of these mountains in this region was insufficient to prevent the long-distance dispersal of B. dorsalis and suggested that there would be a high frequency of gene flow among fly populations. However, Li et al. [B. dorsalis and has resulted in genetic differentiation between these regions. To date, no studies have explored the genetic diversity of B. dorsalis on islands. In this study, we hypothesized that six island populations of B. dorsalis in South China would have lower levels of genetic diversity due to their isolation from the mainland. Furthermore, the isolation of islands may also result in high levels of genetic differentiation between different island populations. In this study, we generated and analyzed a dataset of both microsatellite and mtDNA loci from B. dorsalis samples collected from six offshore island populations in South China to estimate the genetic divergence and dispersal ability of these flies. Our results may also suggest possible strategies for the control of this species based on their dispersal patterns.al areas –16. The cal Asia . To dateg Hawaii . Bactrocin China . Wan et in China  examinedBactrocera dorsalis were sampled from six offshore islands in South China , 2 µL of a dNTP mixture (10 mmol/L), 1.5 µL of each primer (10 mmol/L), 0.2 µL of Taq DNA polymerase (5 U/µL) and 15.3 µL of ddH2O. Cycling conditions for amplification were as follows: 3 min at 94 °C followed by 35 cycles of 30 s at 94 °C, 30 s at 48 °C, 45 s at 72 °C, and an extension of 5 min at 72 °C.DNA from individual specimens was extracted using the TIANamp Genomic DNA Kit . In total, DNA was extracted from 20 flies from each island. Eight polymorphic microsatellite loci  were analyzed, all of which were previously developed , 21–23 . Alleles were scored using PROSize 2.0 .A 759-bp fragment of the cytochrome oxidase subunit I (COI) gene was amplified using primers ) and following the method described by Tang et al. , 25. Thehttp://www.wbiomed.curtin.edu.au/Genepop/Genepop_op1.html) [http://www.montepllier.inra.fr/URLB) [NE), Shannon’s information index (I), observed heterozygosity (Ho), expected heterozygosity (HE), the percentage of polymorphic loci (P), Nei’s genetic diversity (Nei’s), gene flow (Nem), Nei’s original measures of genetic identity (I) and genetic distance (D) for paired populations. These parameters were then analyzed using Popgene Version 1.3.1 [http://www.taylor0.biology.ucla.edu/structureHarvester/#). Structure Harvester was used to calculate six iterations per K-value and obtained the best K-value using the ΔK method of Evanno. [Nei’s genetic distance (D) for the six island populations was reconstructed for the population phylogenetic tree using the unweighted pair-group method with arithmetic mean (UPGMA) in the tools for population genetic analyses (TFPGA) program [Deviations from Hardy–Weinberg proportions and linkage disequilibrium were calculated using Genepop version 5.0 (p1.html) . We estifr/URLB) . Populaton 1.3.1 . The popon 1.3.1 , which u Evanno. . Nei’s g program . We used program .Isolation by distance was measured using the TFPGA software. Matrix correlations using pairwise genetic distance versus geographic distance were estimated and significance was determined using the Mantel test.FST) and the sum of squared size differences [Analysis of molecular variance (AMOVA) was performed with ARLEQUIN ver. 3.5 to partition the genetic variance within and among populations based on the numbers of different alleles  were downloaded from NCBI [Hd), the average number of differences (k), and nucleotide diversity (π) were calculated for each population using DnaSP 5.0 [Published COI sequences of rom NCBI , 34 to crom NCBI . A populnaSP 5.0 , 36.FST) and gene flow (Nem) were estimated using ARLEQUIN ver. 3.5 [Nem > 4 [FST < 0.05); a medium degree of genetic differentiation (0.05 ≤ FST < 0.15); a high degree of genetic differentiation (0.15 ≤ FST ≤ 0.25); and a very high degree of genetic differentiation (FST > 0.25) [The fixation index  , 39.FIS for each locus in each population ranged from 0.0365 to 0.8353, which indicated that there were deficiencies in heterozygotes. Over all populations and loci, the locus MS12A for the DAD population had the highest null allele frequency (Na = 0.41419). The null allele frequency of the other loci and populations ranged from 0 to 0.37780. The presence of null alleles was the primary contributor to the deficiencies in heterozygotes and the departure from the Hardy–Weinberg equilibrium. There was no significant linkage disequilibrium between pairs of loci for all populations (P > 0.05). Therefore, the eight loci were inherited independently.All loci and samples significantly deviated from Hardy–Weinberg proportions (P < 0.01). The average values of HE) of each locus ranged from 0.5397 to 0.8806, and the observed heterozygosity (HO) ranged from 0.1008 to 0.6441. The observed number of alleles per island population ranged from 5.75 (Dong’ao Island) to 8 (Wailingding Island)  to 19 (Locus 6.8A), with an average of 13.375  and 47.9 %  of the total variation, respectively. In addition, within individual variation accounted for 47.23 %  and 46.75902 %  of the total variation for the one group and three groups analyses, respectively. Most of the total genetic variation was explained by the variation among individuals within populations.Hierarchical ls Table . The genNei’s standard genetic distance (D)  among six island populations ranged from 0.0853 to 0.4021 and Nei’s genetic identity (I)  varied from 0.6689 to 0.9182. The population genetic identity (I) between the Wailingding island population and the Weizhou island population was 0.6689, which was the minimum among all paired island populations. Accordingly, there was high genetic distance, which was 0.4021 between these paired island populations .The relationship between genetic distance and geographic distance was determined among island ranges. The result showed that there was no relationship between genetic distance and geographic distance  for each population ranged from 0.889 to 0.995. The average number of differences (k) ranged from 3.895 (Wailingding Island) to 6.553 (Naozhou Island). Nucleotide diversity (π) for each population ranged from 0.513 % (Wailingding Island) to 0.863 % (Naozhou Island)  and haplotype diversity (0.9665). Haplotype diversity (d) Table .Table 4HFST significance tests. The FST values between paired groups ranged from −0.03194 to 0.05413. The Dong’ao Island population had a low degree of differentiation with the Wailingding Island, Hebao Island and Shangchuan Island populations (0.01024 ≤ FST ≤ 0.01895). However, Dong’ao differentiated from the Weizhou and Naozhou Island populations  (Table FST < 0) (Table Nem) between each pair of populations was over four, which suggested that there was a full exchange of genes between the six island populations. The isolation of the islands did not prevent gene flow between the populations. These results were consistent with the microsatellite data.There was genetic divergence between the six island populations, as estimated by population pairwise B. dorsalis population maximum likelihood tree suggested that there were no independent groups in the island populations. In the phylogenetic tree, these six island populations of Southern China were clustered in one branch with different mainland populations of China , which showed that among populations there was a high levels of gene flow and a low and medium level of differentiation among some paired island populations. These results were consistent with the results from our microsatellite data that shows a low Nei’s standard genetic distance (D) and a high genetic identity (I). Pairwise FST values indicated that there was a medium degree of population differentiation between Weizhou Island and Naozhou Island. The microsatellite data indicated that population genetic variation was mostly partitioned among populations. We divided the six island populations into three groups, which were supported by the FCA. Our results were also similar to the levels of population differentiation between mainland China populations. Wan et al. [B. dorsalis from six populations in Qiongqing using eight microsatellite loci. The results showed that the Qiongqing populations had low levels of population differentiation. Yao et al. [FST was 0.25 between South China and Southeast Asia populations, which suggested that these populations had genetic divergence due to geographic isolation. Our study suggested that island B. dorsalis populations had high levels of genetic diversity and a low or medium level of differentiation among some paired island populations and the genetic distance of pairing populations had no correlation with geographic isolation . There are several possible explanations for this pattern. First, Tephritid fruit flies are capable fliers that can fly more than 25 km under windless conditions [Bactrocera dorsalis can migrate to and from islands on its own or with the aid of typhoons [B. dorsalis, such as banana, pawpaw and others. Many tropical fruits, such as guava, carambola, and mango, are imported and exported from the mainland to the islands. Bactrocera dorsalis can lay eggs on these fruits, and the infested fruits can subsequently be transported to other areas. Therefore, gene flow is likely frequent between islands. The low levels of genetic diversity are also likely caused by the short distance between the islands and the mainland.For COI data, the value of n et al.  analyzedo et al.  also exao et al. . Li et ao et al.  reportedo et al.  also repnditions , 53. Thetyphoons . The isoB. dorsalis have relatively high levels of genetic diversity, whereas populations from offshore islands exhibited low genetic differentiation.This study showed that island isolation may not significantly influence the genetic diversity of tephritid fruit flies. We have found that offshore island populations of


Crassostrea gigas, is one of the major aquacultural shellfish species that has been introduced to Europe and America from its native source in the West Pacific. In Taiwan, the cultivated cupped oysters along the west coast have been identified as C. gigas for over centuries; however, several molecular phylogenetic studies have cast doubt upon the existence of this species in Taiwan and adjacent waters. Indeed, our analyses of mitochondrial cytochrome oxidase I (COI) sequences from 313 Crassostrea collected from 12 locations along Taiwanese and southern Chinese coastlines confirm that all samples were the Portuguese oyster, C. angulata, rather than C. gigas. Multiple lines of evidence, including haplotypic and nucleotide diversity of the COI gene, demographic history, and population genetics, suggest that Taiwanese C. angulata is unique, probably experienced a sudden population expansion after the Last Glacial Maxima around 20,000 years ago, and has a significantly limited genetic connectivity across the Taiwan Strait. Our study applies an extended sampling and DNA barcoding to confirm the absence of C. gigas in natural and cultivated populations in Taiwan and southern China, where we only found C. angulata. We highlight the importance of conserving the gene pool of the C. angulata population in Taiwan, particularly considering the current threats by large-scale environmental disturbances such as marine pollution, habitat destruction, and climate change.The Pacific cupped oyster,  Crassostrea are among the most important cultivated shellfish species in the worldCrassostrea listed by the FAO, the Pacific cupped oyster, C. gigas, contributed an estimated 555k tons to global aquaculture production in 2013C. gigas for over a century and has produced 27,793 tons in 2013 having an economic value of over US$20 millionCrassostrea species have raised concern over the taxonomic status of C. gigas in Taiwan345678C. angulata, using small numbers of Taiwanese cupped oysters as the Asian reference, unexpectedly showed that those from Taiwan were grouped with C. angulata from the Atlantic3C. gigas from Japan. C. angulata is a species assumed to be native to the northeastern Atlantic but is morphologically and enzymatically indistinguishable from C. gigas561213Crassostrea oysters confirmed that C. gigas and C. angulata are closely-related and probably diverged about 2.7 million years ago (mya)15C. angulata has a wide distribution in southern China, with the Yangtze River separating it from C. gigas in northern China817C. angulata and C. gigas might prove to coexist along the Taiwan coast if intensively sampled. In addition, geological events such as the last glacial maximum (LGM~20 ka BP) are speculated to have affected the phylogeography of marine biota in the western Pacific1920Crassostrea species in Taiwan.Cupped oysters belonging to the genus Crassostrea species from eight populations in Taiwan and four along the southern China coast  were retrieved from GenBankDNA barcoding based on the mitochondrial cytochrome oxidase I (COI) fragment has been a standard way to discriminate between closely related species, identify new, cryptic, or invasive species, and assess species assemblages of communities across many animal phyla. The general rule in DNA barcoding is that intraspecific COI variation is <1%, whereas interspecific divergence is normally >2%na coast . In addiCrassostrea samples collected from 12 locations along the Taiwanese and Chinese coasts. In a total of 43 haplotypes with 41 variable sites, 14 phylogenetically informative sites were identified for Crassostrea. The COI phylogenies constructed using ML and Bayesian methods produced identical topologies , with high bootstrap support (99) and Bayesian posterior probability (100). ITS-1 phylogeny showed that all oyster samples collected in this study were of C. angulata, not C. gigas , and variations consisted predominantly of transition substitutions (Ti: Tv = 11.78). The number of haplotypes (nh) ranged from 4 in Beihai to 14 in Chiayi. The number of endemic haplotypes (neh) was significantly higher in populations along the Taiwanese coast than from the Chinese coast . Haplotypic diversity (h) was high in all populations, with a mean value of 0.882 ± 0.012. In contrast, nucleotide diversity (π) was low, with a mean value of 0.00396 ± 0.00017  showed that the two C. angulata populations along the Taiwanese and Chinese coasts experienced different demographic histories (s (−27.30487) values along the Taiwanese coast were significantly negative (p < 0.05), indicating these populations had a sudden population expansion, although most individuals were not statically significant in these two parameters. In contrast, Tajima’s D (0.01183) was positive and Fu’s Fs (−1.11287) along the Chinese coast was not significantly negative, suggesting that these populations have suffered a recent bottleneck or expansion. The different demographic history across the two sides of the Taiwan Strait is also illustrated by mismatched distributions . The MJN analysis showed a complex star-like network of six haplotypes accounted for 79.87% of the COI DNA sequences collected from angulata . In contaplotype . The MJNistories . The Tajibutions . The comibutions . In contibutions . Tau valears ago . When esears ago . In contears ago . In contears ago .st values between C. angulata populations along the Chinese coast and 20 of 28 pairwise Fst between populations along the Taiwanese coast were not significant . The overall population subdivision between Chinese and Taiwanese coasts was highly significantly different , suggesting a limited gene flow across the Taiwan Strait. An analysis of molecular variance (AMOVA) supported this tendency with a significant among-group ΦCT (0.09787) between Taiwanese and Chinese coast populations, although 88.26% of the variance existed within populations  that was adapted to the warmer SSTs along the West Pacific coast in the late Pliocene. When glacial-interglacial cycles emerged during the Pleistocene, dramatic changes in the area and configuration of the coastline along the West PacificC. gigas. Haplotypic diversity, nucleotide diversity, and demographic analyses of the COI gene also supported C. angulata and C. gigas being distinct species and that C. angulata is an older lineage than C. gigas. C. angulata had twice as much haplotypic diversity (h: 0.882) and four times the nucleotide diversity (π: 0.00396) of C. gigas (h: 0.457 and π: 0.00097)C. angulata was complex and star-like, being complicated by multiple connections and high-frequency internal haplotypes, the MJN of C. gigas from the northwestern Pacific was simple and star-like, which is the most common and widespread haplotype and the one that is typically assumed to be the most ancestralC. angulata in the warm, subtropical southwestern Pacific and C. gigas in the cool, temperate northwestern Pacific, with the Yangtze River as a natural biogeographic barrier to separate not only these two cupped oyster species but also other coastal marine species along the Asian continent919By intensively sampling cupped oyster populations island-wide, we confirmed that the ity h: 0.82 and foC. angulata along the coast of Taiwan and southern China based on different lines of evidence including network, mismatch distribution analysis (MDA), and neutral tests. For example, populations in Taiwan and southern China experienced different expansion models, with the former having a sudden population expansion and unimodal frequency distribution and the latter with a recent bottleneck or expansion and a negative binomial frequency distribution. The different expansion history of C. angulata, as indicated by MDA, is that the Chinese population expanded around 118 Kya and the Taiwanese population around 64 Kya. Beginning 140 Kya during the last interglacial period, Earth’s air temperature rose to approximately 2 °C above the present level and sea level rose to 5–6 meters or more above present ocean levels by 130–127 KyaC. angulata populations might have had the chance to colonize and expand along the coast of southern China as well as western Taiwan. However, this scenario was not supported because, following this interglacial high stand and several more oscillations, sea level dropped 120–130 m below the present level during the last glacial maximal . Thus, C. angulata populations must be extinct because southern China and western Taiwan became connected when the sea level dropped during the LGM. In contrast, the Bayesian sky plot (BSP) matched the sequences of demographic histories for Taiwanese and southern Chinese populations to the effects of the LGM. That is, the BSP fits the time needed for their expansion to stable population sizes after another rapid rise in sea level (over a 12 Kya period) stabilized close to the present level 7,000 years agoC. angulata in this region because in consistent estimations of demographic expansion between MDA versus the BSP analysis have been noticed in several marine species in the marginal seas of the northwestern Pacific19e.g., microsatellites, SNPs) should be considered for confirming the population expansion of C. angulata along the Taiwanese and southern Chinese coasts.Significant population expansion was identified for mal LGM;  Kya. Thumal LGM;  Kya. Thust between Taiwanese and southern Chinese populations of C. angulata is still highly significant, suggesting that cross-strait gene flow is limited. The Taiwan Strait, situated between the north end of the South China Sea (SCS) and south side of the East China Sea (ECS), has been recognized as a corridor for the migration of marine species between the two seas, aided by complex ocean currents around Taiwan  C. gigas samples were purchased from importers and restaurants. Ten additional species of Crassostrea were selected as outgroups . Collected samples were frozen and transported to the laboratory. Adductor muscle tissue was cut into pieces and preserved in 95% alcohol. All reference samples are preserved in the Molecular Systematic Laboratory, Marine Fisheries Division, Fisheries Research Institute.In total, 313 individual oyster specimens were collected from 12 locations that were subdivided into two geographical regions (Taiwanese coast and Chinese coast) . Eight f2O was added to make a volume of 50 μL. A model 2400 thermal cycler (Perkin-Elmer) was used for PCR and conditions were set as follows: one cycle at 95 °C for 4 min, 40 cycles at 95 °C for 1 min, at 50 °C for 1 min, at 72 °C for 3 min, and a final elongation at 72 °C for 7 min. The reaction was stopped and products were preserved at 4 °C.For initial species diagnosis, COI was selected as the molecular marker. Total genomic DNA was extracted from adductor muscle tissue using a commercial DNA isolation kit . The LCO-1490  and HCO-2198  primer set that amplifies COI was usedPCR products were examined on 1% agarose gel by electrolysis to confirm that the correct fragment length had been obtained, and then eluted using the QIAquick Gel Extraction kit . Purified PCR products were sent to Mission Biotech  for DNA sequencing.C. gigas and hybrids (C. gigas × C. angulata) occur in Taiwan, we also sequenced the nuclear ITS-1 region with primers described by Hedgecock et al.Escherichia coli JM109 according the manufacturer’s protocol. Plasmids were screened for inserts and inserts were sent to Mission Biotech  for DNA sequencing.To reinforce the accuracy of species diagnosis and to determine whether 7 generations and a sampling frequency of 100 generations (standard deviation of split frequencies = 0.008835), and 3 × 106 generations for the ITS-1 analysis .COI and ITS-1 sequences were aligned by ClustalW in MEGA 6.0h), endemic haplotypes (eh), and nucleotide diversity (π) for each population using DnaSP 5.10. Pairwise genetic divergence between populations was estimated by using Fst values and significance was verified through 10,000 permutations and adjusted by sequential Bonferroni correctionGene diversity was described as haplotype diversity  ver. 2.1.3Ne) across time were inferred using Bayesian skyline analyses. To infer demographic history, coalescence methods require an initial demographic model to be specified. Where evidence of population expansion was found, the timing of expansion in generations (t) was estimated from τ = 2 μt, where τ (tau) is a parameter of the time to expansion in units of mutations and μ is the mutation rate per generation for the DNA sequence under study. The mutation rate of C. angulata was calculated as 2.785% per nucleotide per Myr since the divergence time was about 2.7 Myr for C. angulata and C. gigasTo detect any differences in genetic structure among populations using analysis of molecular variances (AMOVA), we grouped populations into three types: (a) those along Taiwanese  and Chinese coasts , (b) those along the Taiwanese  and Chinese coasts, and (c) those along Taiwan’s east  and west coasts  and the China coast . This first analysis was carried out to determine whether there were any differences in genetic structure across the Taiwan Strait. Oyster spats are regularly transported between Taiwanese coast and two islands, MT and KM, along the Chinese coast for marine cultureThe haplotype network of the COI gene was inferred using the median-joining algorithmHow to cite this article: Hsiao, S.-T. et al. DNA barcoding reveals that the common cupped oyster in Taiwan is the Portuguese oyster Crassostrea angulata , not C. gigas. Sci. Rep.6, 34057; doi: 10.1038/srep34057 (2016).