Orchestia montagui using mitochondrial (cytochrome oxidase subunit I), microsatellite (eight loci) and allozymic data. The species typically forms large populations within heaps of dead seagrass leaves stranded on beaches at the waterfront. We adopted a hierarchical geographic sampling to unravel population structure in a species living at the sea-land transition and, hence, likely subjected to dramatically contrasting forces.We examined patterns of genetic divergence in 26 Mediterranean populations of the semi-terrestrial beachflea Mitochondrial DNA showed historical phylogeographic breaks among Adriatic, Ionian and the remaining basins  likely caused by the geological and climatic changes of the Pleistocene. Microsatellites  detected a further subdivision between and within the Western Mediterranean and the Tyrrhenian Sea due to present-day processes. A pattern of isolation by distance was not detected in any of the analyzed data set.O. montagui is the result of the interplay of two contrasting forces that act on the species population genetic structure. On one hand, the species semi-terrestrial life style would tend to determine the onset of local differences. On the other hand, these differences are partially counter-balanced by passive movements of migrants via rafting on heaps of dead seagrass leaves across sites by sea surface currents. Approximate Bayesian Computations support dispersal at sea as prevalent over terrestrial regionalism.We conclude that the population structure of  A high number of polymorphic markers, large sample sizes and tructure . On the tructure . Phylogetructure -6. Howevtructure .Talitrid amphipods are often the dominant animal group in the supralittoral zone, where they are confined to a narrow area avoiding both the arid environment inland and submersion in seawater. Active dispersal is restricted to short, mostly nocturnal, foraging movements along the beach ,9; maximOrchestia montagui, O. mediterranea, Platorchestia platensis) that live within beached decaying seagrass, wrack or gravel, showed a shallow genetic structuring even on a wide geographical scale. Species  that are exclusively associated to sandy beaches or stranded rotting logs and have the tendency to burrow into the substrate were invariably highly structured genetically [The semi-enclosed Mediterranean Sea is surrounded by thousands of kilometers of coasts. Previous genetic studies focused on multiple Mediterranean populations of seven different species of talitrids ,13-18 haetically ,18. De Metically  interpreetically .Orchestia montagui Audouin 1826 and we expand the data available on the species both in terms of geographic sampling and kind of molecular markers used. The species is abundant along the coasts of the Mediterranean Sea and reaches the Black Sea on the East , where O. montagui is the most common talitrid, and a few populations from the Eastern Mediterranean and Aegean Sea [O. montagui[O. montagui populations from five Mediterranean basins  [O. montagui. These populations were sampled in six different Mediterranean basins  . In thatEastern)  and to pO. montagui in the central portion of the Mediterranean Sea. The species is tightly linked to stranded heaps of the seagrass P. oceanica and hence passive dispersal via rafting is a plausible hypothesis. Here we specifically ask whether such passive dispersal in O. montagui is a strong enough factor to promote genetic homogeneity over long distances. P. oceanica wrack may provide food and shelter; given the wide distribution of this seagrass in the Mediterranean Sea we suspect events of transports to be quite frequent. On the other hand, being strictly associated to a potential vector of dispersal does not necessarily imply that gene flow is realized. We indeed found a remarkably deep geographic partition in the population genetic structuring of the talitrid M. remyi, a species strictly associated to stranded rotting logs that should theoretically aid passive dispersal [The aim of this study is thus to determine the genetic structure of the beachflea ispersal . Here weispersal -27). We ispersal . FinallyThe study complies with the current laws of Italy and Germany regarding proper treatment of animals for research and was approved by the Ethical Committee of University of Rome "Sapienza", Italy.O. montagui specimens were collected by hand or using an aspirator on P. oceanica decaying leaves. A total of 26 populations were sampled for this study on rocky and sandy beaches along shores of the following Mediterranean basins: Tyrrhenian Sea (10 populations), Western Mediterranean basin (12 populations), Eastern Mediterranean basin (one population), Ionian Sea (one population), Adriatic Sea (two populations). All 26 populations were analyzed at the mtDNA level and 21 (out of 26) were genotyped for microsatellites.Additionally, we re-analyzed the dataset based on 21 allozymic loci of De Matthaeis et al. . That stIncluding the 153 samples analyzed in Pavesi et al. , we extrπn) and the mean number of pairwise differences between all pairs of haplotypes (π). The same software was used to assess the population genetic structure by hierarchical analysis of molecular variance . Geographic distances were measured as the shortest marine distances between each pair of populations, according to the circulation of surface currents [O. montagui genetic variation (populations grouped according to the SAMOVA results). Mismatch distributions were used to fit the models of sudden demographic and geographic expansion and to estimate the τ parameter. In both models τ is a mutation-scaled measure of time since expansion (demographic or geographic) and can be used to estimate time in generations (T) since expansion, using T = τ/2 μ, where μ is the crustacean COI mutation rate per site per year of Knowlton et al. [D[Fs[P-values were obtained as the proportion of simulated values smaller than or equal to the observed values (α = 0.05).Folmer et al.’s  universa . Populale tests . We alsole tests  with Arlcurrents ,37. We ucurrents . Arlequicurrents  and geogcurrents  expansion et al.  already n et al. . Goodneset al. [D and Fu’sal. [D[Fs statistiIn order to estimate genealogical relationships among all haplotypes, we used Tcs v.1.13  to carryFST values with FreeNA software. These values were computed, as described in Chapuis and Estoup [E) and observed (HO) heterozygosity, deviations from Hardy–Weinberg equilibrium and tests of linkage disequilibrium among loci. We corrected P-values using the sequential Bonferroni correction for multiple comparisons [R) for each population, and GenAlex v.6.3 [A) and their relative frequencies in each population. With Arlequin v.3.11 we calculated pairwise FST values and performed two AMOVA analyses  and Mantel test as described for the mtDNA dataset in the previous paragraph. For the AMOVA we grouped populations based on the results of Structure v.2.3.2 [We genotyped all 544 individuals (7 to 30 per population) at eight microsatellite loci . The software uses a Monte Carlo Markov Chain (MCMC) Bayesian clustering model that assumes that different loci are at Hardy-Weinberg and linkage equilibrium with one another within populations. We performed 10 independent runs for each value of K (K = 1–21) assuming an admixture model and using 100,000 replicates of the MCMC after a burn-in of 10,000 iterations. The number of clusters was calculated from the ΔK estimates using Evanno et al.’s method . AssignmWe compared the results obtained from Structure with those from Tess v.2.3 . The sofFST values, AMOVA and IBD using Arlequin. We performed the AMOVA on three groups combining populations according to their geographical position and patterns of relationships as detailed in De Matthaeis et al. [We re-analyzed the dataset of De Matthaeis et al.  consistis et al.   evaluating the relative proportions of each scenario found in the selected closest data sets  and through ii) a polychotomous logistic regression on the 1% of the simulated data sets closest to the observed data sets (default settings in DIYABC). Before estimating the posterior probabilities of scenarios, we checked whether the combination of scenarios and prior distributions of their parameters have produced data sets that are similar enough to the observed ones by performing a Principal Component Analysis (PCA) of the first 100,000 simulated data sets to evaluate the position of the observed data compared to the simulated.We used Approximate Bayesian Computation in DIYABC 1.0.4.39  to evaluThe 295 COI sequences identified a total of 82 unique haplotypes  and 9.571 (PSF) whereas πn ranged between 0.001  and 0.017 (PSF). The AMOVA detected significant population structure when we tested for three and five groups .Values of H, ps Table : in bothFCT = 0.45; P < 0.05), finding two barriers isolating the Adriatic from the Ionian populations and then these two basins from the rest. The analysis of mismatch distributions supported demographic expansion in the Adriatic group (PSSD = 0.283) only; geographic expansion was supported in all groups (PSSD ≥ 0.184). Tajima’s D and Fu’s Fs were not significant for the Ionic  and Adriatic  groups while they were negative and highly significant for the remaining populations lumped in a single group . In light of the relatively poor performance of mismatch distribution statistics in detecting population demographic expansion compared to D and FS statistics [SAMOVA suggested the existence of three groups .AMOVA indicated a significant genetic structure and most of the genetic variability, when populations were grouped either in three 83.01%) or five groups (83.15%), was apportioned within populations Table . Populat.01% or fResults from Structure and Tess indicated respectively K = 3 (LnP(K) = −13309.7) and K = 5 (DIC = 18998.5) as the best fitting our data  Northern Sardinian populations (Western Mediterranean basin); 2) Southern Sardinian populations (Western Mediterranean basin), 3) Tyrrhenian basin, 4) GOZ , 5) Ionian plus Adriatic basins. When Structure was forced to use K = 5, the software also split the Western Mediterranean populations in two groups (corresponding to Northern and Southern Sardinian locations). Additional groups were formed by the Ionian and the Adriatic populations together and by two Tyrrhenian clusters. The Tyrrhenian groups corresponded to North Eastern Sardinian islets  and Italian peninsular sampling sites .Intriguingly, individuals carrying the four deeply divergent COI haplotypes Figure  do not pFST values ranged between 0.03 (FRA-SNA) and 0.49 (STA-SAP) (Table P = 0.0001) of the genetic variance was apportioned within populations. A Mantel test revealed a lack of significant IBD when the Greek population (GRE) was excluded from the analysis , whereas IBD was detected when GRE was included in it .We re-analyzed a dataset of 16 populations  Table . STA wasBoth Structure and Tess analyses performed on this dataset revealed K = 3 ((LnP(K) = −1767.52 and DIC = 2887.23 respectively) as the best describing the species population structuring Figure a, b. NevThe PCA analyses conducted to check whether the combination of scenarios and prior distributions of their parameters have produced data sets similar enough to the observed ones revealed in all circumstances that observed data were surrounded by many simulated data (not shown). Figure In the last two decades we have been accumulating genetic data on Mediterranean talitrids with the aim to comparatively describe the genetic structure of different species at the scale of the whole Mediterranean Basin . The marine scenario is favored regardless the marker system analyzed . It is worth noting that, in spite of this robust evidence, patterns of relationships yielded by mtDNA and microsatellites  are not mirroring one another but major differences emerged that can be explained considering the intrinsically different evolutionary properties of these markers.Present genetic data suggest that the beachflea FST values and the AMOVA results are statistically significant regardless of the marker considered. This evidence points to an overall scenario of complex genetic fragmentation that needs to be carefully evaluated to correctly resolve population structure in the species.Populations east and west of the theoretical STS boundary are significantly differentiated at the mtDNA level (SAMOVA results) while microsatellites are ambivalent on the issue . The population sampled on Gozo Is.  shares both mtDNA haplotypes and microsatellite alleles with Western Mediterranean, Tyrrhenian, Ionian and Adriatic populations. The lack of isolation by distance in the mtDNA and both nuclear datasets excludes geographic distance as the main determinant of genetic divergence. On the other hand, the vast majority of pairwise O. montagui is a small enough organism to hypothesize that it could cling, find shelter and feed on P. oceanica wrack while at sea for as long as the wrack would persist and/or strand in a new location. The reproductive biology of amphipods may enhance dispersal and recruitment; by incubating the embryos and developing juveniles in the brood pouch females provide extended parental care [T. saltator[As pointed out by Thiel and Haye , populattal care . Migrati saltator.In spite of this convincing evidence, we suggest caution in interpreting the previous and following findings in light of our incomplete sampling coverage in the Eastern Mediterranean Sea and the small sample size for one Adriatic population.The SAMOVA analysis conducted on mtDNA data detected barriers to gene flow between Adriatic and Ionian populations and between these two and the remaining basins. Patterns of mtDNA relationships within each of the above groups are not similar. Adriatic and Ionian haplotypes are scattered in the network of Figure Western Mediterranean/Tyrrhenian, Ionian and Adriatic mtDNA clusters  perfectly overlap with the corresponding biogeographic provinces identified within the Mediterranean Sea ; this evO. montagui based on a subset of six populations, we invoked male-biased dispersal to explain why the mtDNA fragmentation was not apparent at the microsatellite level [O. montagui, generally have a strongly female-biased sex ratio  cluster individuals preferentially concordant to their geographic origin, while many mtDNA haplotypes from geographically far away locations are recurrently gathered together in the haplotype network and differ by one or few substitutions. The geographically close Ionian and Adriatic populations have quite similar microsatellite profiles Figure  whereas te level . In the x ratio  and all the others analyzed in [When we compare our data within those available on a similar geographic scale for other Mediterranean species an interesting result emerges. Several marine species are genetically more homogeneous in the Western Mediterranean basin than they are in the Eastern Mediterranean, Ionic or Adriatic Sea. This was recurrently observed in fishes  or dig into rotting logs (M. remyi) [Orchestia, including O. montagui, associated with heaps of decaying wrack were thought to be genetically homogeneous, even across vast geographical ranges [M. remyi[O. montagui  ,15,18,60M. remyi lives in strong association with rotting logs stranded on sandy beaches; T. saltator burrows deeply into the sand to escape dehydration. In both circumstances, the chances for individuals to be dragged away by waves are far lower than those of O. montagui, which colonizes a temporally ephemeral habitat close to the water line. This would explain why our focus species was thought to be genetically poorly structured [O. montagui can no longer be considered a species with a shallow genetic structure, as De Matthaeis et al. [P. oceanica wrack) at sea surface due to its negative buoyancy [The intrinsic active mobility of organisms is only one determinant of the amount of the realized dispersal . Active ructured -15. On ts et al. -15 conclbuoyancy .O. montagui was believed to bear has in fact converted to an appearance of intermediate gene flow under more stringent assays.By comparing patterns yielded by microsatellites and allozymes for the same set of populations, we obtained an identical number of clusters (3) but membership of individuals was consistently higher in the microsatellite analyses, pointing – as expected – to a much better resolving power of these markers. The molecular footprint of high gene flow under low-resolution molecular assays that O. montagui within the Mediterranean Sea at a fine geographical scale. By combining molecular markers with different evolutionary properties we distinguished historical processes from present-day forces. Pleistocene climatic changes caused the isolation and divergence of mtDNA lineages. Current dynamics – namely surface marine currents jointly with rafting via seagrass wrack – produced new and different patterns of structuring, which were revealed by microsatellites. ABC gave strong support to a scenario of genetic fragmentation molded predominantly by marine forces.We were able to unveil the genetic structuring of FST and AMOVA results) suggests that the species has not yet approached equilibrium between gene flow and drift [The lack of a statistically significant pattern of IBD at both classes of markers under a scenario of genetic fragmentation  in floating wracks to ultimately link genetic and oceanographic data in a unified framework [Our results also pinpoint the difficulties in describing the population genetic structure in an ecotonal species, because of its simultaneous exposure to forces typical of two ecosystems (land and sea). The semi-terrestrial life style acquired by ramework .The authors declare that they have no competing interests.LP collected the samples, designed the project, carried out the experiments and the statistical analyses, interpreted the data, drafted and critically revised the manuscript. VK collected the samples, designed the project, carried out part of the statistical analyses, interpreted the data and critically revised the manuscript. RT provided funding for all experiments and critically revised the manuscript. EDM provided the allozymic data and critically revised the manuscript. All authors read and approved the final manuscript.


Coffea arabica L. (arabica coffee), the only tetraploid species in the genus Coffea, represents the majority of the world's coffee production and has a significant contribution to Nicaragua's economy. The present paper was conducted to determine the genetic diversity of arabica coffee in Nicaragua for its conservation and breeding values. Twenty-six populations that represent eight varieties in Nicaragua were investigated using simple sequence repeat (SSR) markers. A total of 24 alleles were obtained from the 12 loci investigated across 260 individual plants. The total Nei's gene diversity (HT) and the within-population gene diversity (HS) were 0.35 and 0.29, respectively, which is comparable with that previously reported from other countries and regions. Among the varieties, the highest diversity was recorded in the variety Catimor. Analysis of variance (AMOVA) revealed that about 87% of the total genetic variation was found within populations and the remaining 13% differentiate the populations . The variation among the varieties was also significant. The genetic variation in Nicaraguan coffee is significant enough to be used in the breeding programs, and most of this variation can be conserved through ex situ conservation of a low number of populations from each variety. Coffea arabica L. (arabica coffee) is a self-fertile allotetraploid species that belongs to the genus Coffea in the family Rubiaceae [n = 4x = 44), the remaining species being diploid with 2n = 2x = 22 chromosomes [Coffea canephora (robusta coffee) and C. eugenioides or their ecotypes in the plateaus of Central Ethiopia [ubiaceae , 2. Out omosomes . ArabicaEthiopia .Coffee production is an important economic activity in Central America and accounts for about 10% of the world coffee production . In NicaSeveral studies have shown that the genetic diversity of arabica coffee is low when compared to that of robusta coffee , 12–19 dDetecting and quantifying genetic variation in crop species is important for successful conservation of genetic resources and plant breeding. Molecular marker techniques, such as random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), and simple sequence repeats  have been used for genetic diversity analysis in wild and cultivated coffee , 29–31. Coffee seeds from twenty-six populations representing eight arabica coffee varieties were used in this study. Each population was represented by ten individual coffee trees. Fresh coffee berries were collected between December 2009 and February 2010 from the main coffee growing provinces of Nicaragua . The berGCTTCT” to avoid a nontemplated addition of a single nucleotide by Taq DNA polymerase to the PCR product, as described in Ballard et al. [Twenty-five SSR primer-pairs were initially screened for good amplification, polymorphism, specificity to their target loci, and suitability of the allele size for multiplexing. This led to the selection of twelve primer-pairs for final analysis . The ford et al. .μL containing 25 ng genomic DNA, 0.3 μM forward and reverse primers, 2 mM MgCl2, 0.3 mM dNTPs, 1 U Taq DNA polymerase , and 1 × PCR buffer . The reactions were performed using the GeneAMP PCR system 9700 thermocycler using the following temperature profiles: initial denaturation at 95°C for 3 min, followed by six touchdown cycles of denaturation at 94°C for 30 sec, annealing at X-Y°C (−1°C/cycle) for 30 sec and extension at 72°C for 45 sec, and then 32 cycles of denaturation at 94°C for 30 sec, annealing at Y°C for 30 sec, and extension at 72°C for 45 sec, and a 20 min final extension step at 72°C. The annealing temperature (Ta) was changed based on the melting temperature (Tm) of each primer-pair  at Genomics Core Facility of the University of Gothenburg, Sweden.For each locus, amplification was confirmed by running 5 The allele peaks were visually inspected and then analyzed using PEAK SCANNER V1.0 software (Applied Biosystems) based on the internal Genescan-500 LIZ size standard. Each peak was considered as an allele at a codominant locus and the genotype of each individual at each locus was recorded. The Free Tree-Freeware program  was used838, DCM06, Sat235, and SSR06 (Cam35) to 0.55 (SSR09). In addition to SSR09, the other loci with a relatively high level of gene diversity were CM5, Sat207, and Cam03 with HT of 0.50, 0.50 and 0.54, respectively  and the within-populations gene diversity (HS), estimated based on Nei's gene diversity [HLoci), which is the average gene diversity across the eight polymorphic loci, and the percent polymorphic loci (PPL) were also analyzed. HLoci ranged from 0.23 to 0.47, whereas %PL ranged from 0.33 to 0.58  and Cam35 (frequency = 0.025), respectively. Population-specific rare alleles, with frequencies ranging from 0.025 to 0.1, were detected in five of the 26 populations. An 89 bp allele unique to population B3 was detected at locus Sat207  at a freGST; [FST; [GST and FST were 0.23 and 0.13, respectively (Tables GST varied from 0.00 (CM5) to 0.62 (Cam16) when calculated for each locus. The values of both GST and FST at locus CM5 were zero although each population has a gene diversity of 0.5. This is due to the fact that all individuals were heterozygous for the two alleles at this locus. The same is true for locus Sat207 except that few individuals in three of the 26 populations had additional rare alleles. At these two loci, both the total and within-population gene diversity was high but with no differentiation between the populations.The genetic differentiation of populations was estimated based on gene diversity  and AMO; [FST; ). The ovy Tables . The estP < 0.0001; FCT = 0.08; P = 0.023) contributing 7.9% to the total genetic variation. The presence of rare alleles in four of the eight varieties contributed to the significant differentiation obtained. On the other hand, AMOVA revealed no significant variation among the two coffee growing regions and among the eight provinces  to 0.392 (B3 versus CM2) with the overall mean of 0.060 . The genCAM35 is a hexanucleotide repeat SSR [CM5, a trinucleotide repeat SSR locus [Sat207 appeared to be due to a combination of differences in the repeat motif and other types of variation, such as indels in the flanking sequences, as the difference in length between the alleles varied from three to four nucleotides  with Sat235 more closely linked to the gene than Sat207 [Colletotrichum kahawae that may cause severe damage in arabica coffee. In addition to their application for analysis of genetic diversity, SSR markers have several other applications that include their use as markers for desirable traits. Among the SSRs used in the present study,  Sat235 was monomorphic across the 26 populations, and it is less likely that it can be a useful marker for genetic linkage analysis of Ck-1 in Nicaraguan coffee. On the other hand, Sat207 was polymorphic with two major alleles (ca 82 bp and 93 bp) and two rare alleles (ca 89 bp and 97 bp). Taking into consideration the amphidiploid nature of arabica coffee, it is most likely that the 82 bp allele on one hand and the other three alleles on the other hand originated from different progenitor genomes of arabica coffee , re, reCk-1 e to CBD  with thee to CBD , as thisCM5. Two alleles were detected at this locus and all the 260 individual plants studied were heterozygous for the two alleles. Baruah et al. [Coffea , 41CM5. a , 1918, 1 McCouch  reportedHT) and within-population (HS) gene diversity were estimated to be 0.35 and 0.29, respectively. The estimates for these parameters were 0.22 and 0.07, in that order, for the RAPD-based study of the Ethiopian arabica coffee by Anthony et al. [HT of 0.37 using ISSR markers in Ethiopian forest coffee. Thus, the level of genetic variation in Nicaraguan arabica coffee is comparable to that previously reported from several countries and regions. The presence of the major SSR alleles across all the populations in very high frequencies in the present study suggests a narrow gene pool of arabica coffee in Nicaragua in line with previous reports. This suggests some difficulties in finding genotypes bearing desirable traits, such as resistance to diseases and pests within the domesticated arabica coffee gene pool.In the present study, the mean Nei's total . A significant differentiation between these two varieties may not be expected due to the relatively short time elapsed since the development of the variety Bourbon, especially at selectively neutral loci that include most of the SSRs used in the present study. Similarly, Catuai was the result of a cross between Mundo Novo and Caturra and thus there may not be a clear genetic differentiation between the Caturra and Catuai varieties at this stage. A relatively close relationship between the Bourbon, Caturra, and Catuai varieties can be observed from The cluster analysis of the SSR data for the 26 populations revealed that, in most cases, the clustering pattern of the populations was not in line with their varietal classification. The principal coordinate analysis (PCoA) of the 260 individual plants (data not shown) revealed the presence of divergent genotypes in populations B2, B3, CM3, CT2, CT5, and PA, which partly explains the poor clustering of populations according to their variety of origin. Given that arabica coffee is an autogamous species; such a poor clustering pattern of populations according to variety of origin is somewhat unexpected. However, the processes through which these varieties were developed may partly explain the lack of a clear differentiation between the populations of the different varieties. For example, the variety Caturra was developed from mutant genotypes of the variety Bourbon . This variety is interesting not only because of its high genetic diversity but also because two of its populations (CM2 and CM3) were significantly differentiated from all the other populations . At locua coffee , 23. Roba coffee , 19). ThMeloidogyne cause major damage in coffee worldwide, and coffee breeding for durable resistance to RKN is now a major goal in coffee producing countries [Meloidogyne exigua and M. incognita are known RKN attacking arabica coffee in Nicaragua [M. arabicida in Costa Rica, which suggests that identification of resistant genotypes to M. exigua and M. incognita from arabica coffee in Nicaragua may be possible. In addition, the resistance gene Mex-1 identified in robusta coffee and successfully introgressed into arabica coffee by Noir et al. [Coffea species, such as the variety Catimor for pest and disease resistance.Root-knot Nematodes (RKN) of the genus ountries . Meloidoicaragua . The besicaragua  reportedr et al.  suggestsOverall, the level of genetic diversity of arabica coffee in Nicaragua is generally low and is comparable to that previously reported for arabica coffee from other countries and regions. Therefore, it should be promoted through crossing with other closely related species such as robusta coffee. In addition, the presence of rare alleles in some populations suggests the need to explore such populations in order to identify mutants bearing desirable traits. The significant differentiation between most Nicaraguan arabica coffee varieties suggests that varieties grown in the country should be analyzed for resistance/tolerance to major biotic and abiotic stresses. On the other hand, the absence of a significant differentiation between the coffee populations based on regions of origin suggests that germplasm collecting missions should prioritize the representation of coffee varieties over coffee growing regions in the country.


Pinus strobus is a highly invasive tree species in Central Europe. Using ten polymorphic microsatellite loci we compared various aspects of the large-scale genetic diversity of individuals from 30 sites in the native distribution range with those from 30 sites in the European adventive distribution range. To investigate the ascertained pattern of genetic diversity of this intercontinental comparison further, we surveyed fine-scale genetic diversity patterns and changes over time within four highly invasive populations in the adventive range.North American Our data show that at the large scale the genetic diversity found within the relatively small adventive range in Central Europe, surprisingly, equals the diversity found within the sampled area in the native range, which is about thirty times larger. Bayesian assignment grouped individuals into two genetic clusters separating North American native populations from the European, non-native populations, without any strong genetic structure shown over either range. In the case of the fine scale, our comparison of genetic diversity parameters among the localities and age classes yielded no evidence of genetic diversity increase over time. We found that SGS differed across age classes within the populations under study. Old trees in general completely lacked any SGS, which increased over time and reached its maximum in the sapling stage.Based on (1) the absence of difference in genetic diversity between the native and adventive ranges, together with the lack of structure in the native range, and (2) the lack of any evidence of any temporal increase in genetic diversity at four highly invasive populations in the adventive range, we conclude that population amalgamation probably first happened in the native range, prior to introduction. In such case, there would have been no need for multiple introductions from previously isolated populations, but only several introductions from genetically diverse populations. Many studies of plant invasions have focused on comparative aspects of ecology, with emphases on understanding either the properties of species that determine their invasive potential Even if introduced species encounter physical environmental (e.g. climatic or edaphic) conditions in their adventive ranges that are similar to those in their native ranges, they face new biotic environments Several mechanisms by which invasive populations can retain sufficient genetic diversity to enable adaptation have been suggested. First, rapid population expansion can occur immediately after an introduction, allowing retention of genetic diversity The time taken for introduced populations to acquire sufficiently high frequencies of adaptive genotypes might explain the substantial time lags (of years or decades) between initial establishment and the manifestation of invasiveness. These time lags have been well documented Although many studies have reported multiple introductions of invasive species, the impacts of admixture on the fitness of invasive species have rarely been studied. Those studies that have been done have all shown a strong influence of the resulting genetic diversity on colonization success per se but also its distribution in space is an important aspect of population dynamics  and North America to test theoretical predictions concerning genetic diversity of an invasive species in its native and adventive ranges, and evolution in its adventive range on large as well as fine scales. In studying the pattern of large-scale genetic diversity, we specifically asked (1) whether there are any differences in population genetic diversity and structure between the native and the adventive ranges and between invasive and naturalized populations within the adventive range; (2) whether there is any indication of bottleneck in introduced populations in the adventive range, and (3) whether populations of P. strobus are derived from independent introduction events and from which areas. In a subsequent study we investigated the pattern of fine-scale genetic diversity and SGS and their changes over time within four highly invasive populations in the species’ adventive range. Such a fine-scale study might help clarify the species’ population dynamics at different localities over the course of its invasion history. In particular we: (1) investigated the pattern of genetic diversity within and among different age classes and in different highly invasive populations; and (2) asked if there is any SGS that is maintained within and among different age classes in different invasive populations.We used The study did not involve endangered or protected species. To perform our study in the Bohemian Switzerland National Park, a collaboration agreement was made between the Institute of Botany of the Academy of Sciences of the Czech Republic and the National Park authorities. Other forests on the Czech side are managed by the Forests of the Czech Republic, State Enterprise, i.e. forests managed under public ownership, for which no special permission is required to collect small samples of needles from an alien invasive species. We did not procure special permissions for collecting in any of the US localities, as we collected just several needles from a very common tree species. We could confirm that none of the collection sites were privately owned.Pinus strobus L.  is a conifer native to northeast North America P. strobus stands were deforested by the late 19th century Pinus strobus is highly invasive in some sandstone areas of the Czech Republic Pinus strobus was introduced into mixed conifer forests to increase community diversity and inhibit pest infestation in species-poor forests on nutrient poor sandy soils and sandy loams Dicrano-Pinetum) and oak-pine (Vaccinio vitis-idaeae-Quercetum) forests Pinus strobus is an outcrossing, wind-pollinated species whose winged seeds are dispersed by wind. In the adventive area, individual trees can be fertile at the age of twenty years, with the substantial production of seeds at the age of 50 years and older. Münzbergová et al.Pinus strobus possesses high seed and pollen dispersal capacity. Wind-dispersed seeds released from the upper canopy can travel several hundreds meters from the source tree, with the maximum recorded distance of a self-sown tree from the potential seed source found to be 757 m. However, the observed median distance for 50% of self-sown trees was only 52 m and only 1% of self-sown trees reached 502 m In both large- and fine-scale studies, we aimed at obtaining samples from different phases of the invasion process. We thus sampled trees of several consecutive age classes which we define, and hereafter use, as follows: (1) “old trees” – trees 50 years old and older. While these trees are canopy trees (in the adventive range mostly planted); (2) “mature trees” – between 21 and 49 years old trees; (3) “saplings” – trees of age between 6 and 20 years; and (4) “seedlings” – trees not older than 5 years. Individual age classes were determined on the basis of a combination of mortality rate and fecundity estimated in a related study  along the eastern edge of its native range. Because the distribution of P. strobus in North America is quite broad We sampled 60 localities, 30 in the native and 30 in the adventive range. Based on a comparative morphological study of seedlings, Musil P. strobus populations in Central Europe, we sampled plants at the same interval in the adventive range to ensure sampling consistency. Within a population, we would locate individuals to be sampled by creating a 60 by 45 m rectangular grid with the gridlines spaced at 15 m intervals, and then collecting from those trees located at the intersections of the gridlines. If no P. strobus tree was located at one of these points, we would use the plant nearest to the point but not further than 1 m away in cases of trees not older than 10 years, 2.5 m away in cases of trees not older than 50 years, and 5 m away in cases of trees older than 50 years. If the resulting area surrounding a point did not contain any usable tree, we skipped that point and extended our grid by 15 m to generate a replacement point. Where possible, we would continue sampling until we collected samples from twenty trees. In total we collected 1194 samples.We collected several needles from each of 20 plants at each locality wherever possible. Some localities, however, had such small numbers of trees that we could only sample fewer individuals there . At eachet al.http://geoportal.gov.cz).In the Czech populations, we later used the number of points in our grid not occupied by trees for discrimination between invasive and naturalized populations  Bynwere see . To obtahttp://www.cuzk.cz).In each of the four populations, we intended to collect 50 samples of each age class from regularly distributed plots, if possible. To ensure sampling consistency, we used the same sampling strategy at all localities. At each locality, we sampled all old trees because their number was always limited. Afterwards, in the core of each stand, we created a 40 by 100 m grid with gridlines spaced at 10 m intervals. We then took one sample of each age class within each square (except for old trees), i.e., 50 samples from each age class were taken. Saplings were originally divided into old (50 samples) and young saplings (50 samples) that were later merged due to our inability to precisely define these groups on the basis of mortality and fecundity parameters. In most cases, we were unable to collect all 250 samples at each locality, in particular due to the lack of old trees. In total, we collected 954 samples and recorded the spatial coordinates of each. The positions of all trees were recorded using a post-processing DGPS technique (GPS Trimble Pathfinder Pro XRS). All GPS positions were corrected to achieve sub-meter accuracy using data from two-reference base stations located in distance of up to 50 km  under the following conditions: an initial denaturation step of 15 min at 95°C followed by 40 cycles of 30 s at 94°C, 90 s at 60°C, 60 s at 72°C and a final extension of 10 min at 72°C.We analysed genetic variation at ten nuclear microsatellite loci in both the large-scale genetic diversity study  and the fine-scale genetic diversity study . These loci were RPS1b, RPS2, RPS12, RPS25b, RPS34b, RPS39, RPS50, RPS84, RPS118b and RPS127 PCR products were electrophoresed in an ABI PRISM 3130 sequencer . One microlitre of PCR product was mixed with 0.2 µl of GeneScan-500 LIZ (Applied Biosystems) and 12 µl of formamide (Applied Biosystems). Allele sizes were determined using GeneMapper version 4.0 software (Applied Biosystems). The raw data are available by request from the authors. An individual was declared null (nonamplifying at a locus) and treated as missing data after at least two amplification failures. The diagnostic results using MICRO-CHECKER A), mean gene diversity overall loci (HS), mean allelic richness (RS)  statHO) and expected (HE) heterozygosities were calculated using the program Arlequin stat. Sequential Bonferroni corrections were applied to adjust P value according to Rice Summary data for SSR loci, including the average number of alleles per locus  was estimated by alleles permutated across the entire dataset, f(FIS) was estimated by the permutation of alleles within populations, and θ(FST) was estimated by the permutation of alleles among populations. The allele frequency-based FST was used rather than the allele size-based RST of Slatkin RST and FST values are not expected to differ greatly for short-term differentiation of populations within species FST-based estimates of differentiation are considered more reliable when fewer than 20 loci are used Weir & Cockerham’s stat with 10,000 permutations.Comparisons of genetic diversity parameters between groups  were performed with Fstat). To test differences in allele frequencies, we used the exact test for population differentiation http://www.marksgeneticsoftware.net).We used the Mantel test to assess the model of isolation-by-distance using genetic distance for pairs of populations We tested for an excess of heterozygosity, which reveals the loss of rare alleles in a bottlenecked population relative to that expected under the mutation-drift equilibrium  for an observed level of heterozygosity K) and to fractionally assign individuals sampled in North America and Europe to the inferred groups. We applied the model which allows population admixture with prior population information K) was set at each value from one through ten, and the simulation was run ten times at each K value to confirm the repeatability of the results. Each run comprised a burn-in period of 150,000, followed by 300,000 Markov chain Monte Carlo (MCMC) steps. Of the various ways to estimate the ‘true’ number of genetic clusters using Bayesian assignment techniques K is employed as an ad hoc estimator of the second order rate of change of the lnP(K|X) to furnish an initial estimate of K .The effects of ecological factors  on stat with 10,000 permutations. Pairwise FST values were used to compare differences in gene frequencies among different age classes within each locality with Fstat, and a Bonferroni correction was used to adjust observed P values for multiple comparisons.Genetic diversity parameters were calculated as described in the large-scale study (see above). Comparisons of genetic diversity parameters among populations were performed with FGenetic variation at the level of populations and age classes was investigated with a nested analysis of molecular variance  was estimated between all pairs of individuals within each age class following the methods of Loiselle et al.et al.ijf) coefficient, we calculated ijf for 5 m distance intervals up to 100 m. Mean values of ijf were obtained for distance intervals of 5 m by averaging over all pairs of individuals located within that interval. Spatial autocorrelation analysis was also computed for all individuals within each population irrespectively of age class and among age classes within each population to test whether the spatial structure of individual age classes were dependent on each other. When ij  = f0, there is no significant correlation among individuals at the spatial scale of interest; when ijf >0, individuals in a given distance class are more closely related than expected by chance; and when ijf <0, individuals within a given distance class are less related than expected by chance with respect to the local population. Co-ancestry coefficients were computed using SPAGeDi software Fine-scale genetic structure was assessed by spatial autocorrelation analysis of genetic relatedness between pairs of individuals as described in Sp statistics, calculated as –b/(1–F(1)), where F(1) is the ijf for the first distance class. F(1) can be considered an approximation of the kinship between pairs of neighbours, provided the first distance class contains enough pair of individuals to obtain a reasonably precise F(1) value We also evaluated the strength of isolation by distance using A  = 5.1 vs. 5.4) (RS), where the difference was marginally significant (f(FIS) was lowest in native population A47 (0.033), and highest in native populations A33 and A35 (0.326), which indicates the existence both of populations in Hardy-Weinberg equilibrium and of populations showing excesses of homozygotes, suggesting some level of inbreeding  . No genenificant , suggestnificant . Only 13breeding .f(FIS) was quite high and significantly different from zero in most cases within both ranges (θ(FST) values reached by populations in the adventive range (f(FIS) nor θ(FST) differed significantly between the native and adventive ranges .To investigate the pattern of inbreeding and population differentiation further, we analysed genetic variation and structure for individual microsatellite loci and over all loci separately for native and non-native populations. The mean value of h ranges , with a ve range . Howevere ranges . When wer = –0.003, R2 = 0.001, P  = 0.947 and r = –0.081, R2 = 0.66, P  = 0.088, respectively).There was no consistent association between genetic and geographic distances in both the native and the adventive range, as indicated by the Mantel tests . We note, however, that the heterozygote excess/deficit test has been known to miss bottlenecks, as it was accurate in only 50–75% of test studies K indicated that two clusters best explained the genetic structuring of P. strobus populations  of the studied Czech populations was not affected by elevation above sea level, precipitation, temperature, or soil types, as indicated by Logit models (data not shown).HS), although the differences were very weak (RS), observed heterozygosity (HO), inbreeding f(FIS) or fixation θ(FST) coefficients .Invasive and naturalized populations differed in gene diversity  showed similarities for allelic richness , gene diversity  and fixation indices , there was a significant difference in observed heterozygosity (HO) and inbreeding coefficient . Especially the locality Hrad showed a lower level of heterozygosity and consequently higher inbreeding , observed heterozygosity (HO), gene diversity (SH) and inbreeding coefficient (FIS) over time, i.e. from old trees to seedlings, with the dependent factor being the genetic diversity parameter and the independent one being time, were not statistically significant (data not shown).Regression analyses of parameters of genetic diversity, i.e. allelic richness (–6). Only 19.25% (P<10–6) was partitioned among individuals within age classes and 0.007% (P  = 0.446) was partitioned among age classes within localities. The remaining 0.97% (P<10–6) of the variation was due to differences among localities.A hierarchical analysis of molecular variance (AMOVA) revealed that a majority of the genetic diversity was partitioned within individuals . Allele frequencies at the locality Hrad significantly differed between seedlings and mature trees (P  = 0.01), seedlings and old trees (P  = 0.05) and between saplings and mature trees (P  = 0.05) (data not shown).Pairwise b) between the kinship coefficient (ijf) and the log of the physical distance between individuals as well as Sp statistics corroborate these results  and fine-scale  genetic diversity in invasive tree a comparably high level of genetic diversity present in the native and the adventive ranges, with an absence of strong large-scale genetic structure in both distribution ranges . In otheThe genetic diversity of trees introduced to the adventive localities approximately one hundred years ago was high enough to ensure the maintenance of high genetic diversity. On the fine scale we found no evidence for any increase in genetic diversity parameters over time; however, we observed the development of fine-scale genetic clustering over time.The finding of comparably high levels of genetic diversity has two important implications. First, the amount of genetic diversity present in the adventive range is so high that it is difficult to envisage a severe demographic bottleneck having occurred either during or after the introduction to Europe. This finding is consistent with the supply of large amounts of seeds from diverse sources since the beginning of the introduction process .Pinus strobus populations in the native range show a pattern of genetic diversity distribution that is less structured than that of many invasive plant species studied so far  within the four localities in the adventive range shows how genetic diversity changed through the invasion process. We did not find any increase over time in the genetic diversity parameters measured, further supporting our explanation that the presence of a high genetic diversity in the adventive range is due to massive introduction of genetically highly diverse material since the beginning of the invasion process. Based on the absence of a difference in genetic diversity between the native and the adventive ranges, together with the lack of structure in the native range, we propose that in this species, genetic structure in the native range did not play a significant role for invasion success in the adventive range as in invasive species having highly genetically structured populations Second, even though theory predicts high genetic diversity in the adventive range to be a consequence of multiple introductions, the pattern of genetic diversity in the native range can provide clearer insight into the invasion history.  but see . Hence, Our Bayesian assignment test identified two genetic clusters . They wePinus strobus, as do some other conifers, shows low population differentiation and considerably high pollen-mediated gene flow P. strobus in the timber industry, resulting in disruption of the genetic integrity of original populations due to admixture of populations from different sources. Historically, by the early 1600s, the first colonists were quick to make use of P. strobus forest resources in the eastern part of North America, and by the early 1900s the area of destruction of the primeval white pine forests reached from the Atlantic seaboard through the Great Lake states Interestingly, we did not detect any strong structure even within the native range. There are basically two non-exclusive explanations that can clarify the pattern. (1) The very low genetic structure of the native populations is worth considering in the light of Taylor and Keller’s study P. strobus to the Czech Republic, there is historical evidence of large-scale importations of seeds, which were likely sufficient to largely maintain the genetic diversity of the mixed populations from which they came. For example, there is a historical record that in 1784 10 kg  of P. strobus seeds were bought and sown in forests in the north-western part of the Czech Republic. These seeds came from Germany with a declared origin in England. Another example is the historical record concerning nearly 32 kg  of white pine seeds imported directly from North America in 1800 and planted in the south-eastern part of the Czech Republic In the case of the introduction of P. strobus may have been also affected by recent human activities, mainly by the transport of propagules.Moreover, we did not see any significant correlation between genetic and geographical distance in either Europe or North America. This pattern is in contrast to studies of other species in which isolation by distance was found in their native ranges and was attributed to a long history of genetic isolation P. strobus in the Czech Republic can be classified as invasive. Some populations are represented only by old trees and almost completely lack sapling and seedling stages. The test of whether this behavior is influenced by environmental conditions did not reveal any significant relationships. Invasive and naturalized populations only differed in gene diversity (HS)  see . AlthougPinus strobus from different parts of the Czech Republic and explored them in detail to answer this question. By comparing the different age classes in four populations, we found that the initial population of old trees did not suffer from low genetic diversity due to the introduction of a low number of individuals. Conversely, our populations of old trees did not differ from younger age classes, with the exception of the locality Hrad, where we detected lower heterozygosity and a consequently higher inbreeding coefficient (FIS). This means that foresters sowed large amounts of seeds that were genetically diverse enough that no admixture was necessary in this case. Even in highly invasive seedlings, which completely fill the space available in the forest, we did not detect any increase in genetic diversity in comparison with other age classes. These results are concordant with studies performed on a large scale study conclusions presented above.When a species is introduced to a new range from several genetically distinct populations, further recombination within the adventive range may dramatically increase genetic diversity outside the native range Pinus strobus populations has been intensively studied in the native range et al. P. strobus to the longer dispersal distance of seeds and a historical overlap of seed shadows from adults outside of the plot coupled with an overlap of seed shadows from younger, more recently established reproductive adults. They also discuss the possible influence of disturbance and colonization history, mating system and ecological factors on SGS and stress the role of thinning processes, which can weaken the initial structure present in seedlings within the parental population.Our results concerning SGS corroborate those presented above. Old trees completely lacked any SGS. SGS increased over time and reached its maximum in the sapling stage, but on different distance classes in different populations with the lowest SGS in Bynovec locality , Table 6et al.Pinus halepensis population founded by five reproductive individuals. They found no SGS in the early stages of invasion and suggested the random distribution of genotypes could be the result of density-dependent grazing. In our study on invasive Pinus strobus, we also did not detect a SGS in early stages, i.e. in old trees. These trees were planted by foresters, and individual gene combinations were introduced randomly. Old populations therefore cannot have any SGS, and the spatial tree distribution is not an outcome of a biological process but of a human activity. However, many studies of conifer species demonstrate a weak SGS in old-age classes in wind-dispersed species as an outcome of biological processes such as consanguineous mating, low level of pollen flow and short-distance seed dispersal without human influence Troupin Pinus halepensisPinus strobus. We have observed very weak spatial structuring in the seedling stage at all localities, which should be the result of two processes that are not mutually exclusive, i.e. long-distance seed dispersal and spatial-temporal overlap in seed shadows. Based on these processes, as the population of an invasive tree rapidly increases, propagule pressure increases as well. In combination with the high dispersal capacity of Pinus strobus , seedlings do not form strongly genetically structured populations, as different genotypes are widely mixed due to pollen and seed dispersal over distances longer than 100 m. Hadincová et al. et al. P. strobus is able to spread very effectively. They estimated that P. strobus can disperse up to 750 m away from the parental source in different localities. As a result, there is great seed shadow overlap from adults within the population as well as adults adjacent to the plot . This has the effect of reducing both the level and spatial scale of relatedness in studied populations and is especially pronounced in the seedling stage. On the other hand, 750 m is the maximal detected dispersal distance, a majority of seeds is dispersed up to 100 m apart and that approximately 80% of them are distributed up to 20 m apart. This dispersal pattern may generate fine-scale genetic clustering up to 15 m among age classes.Epperson P. strobus reveals a surprisingly complicated story. The amount of the genetic diversity in the adventive range is attributable to either multiple introductions, similar to findings on other species th century and later. In such a case, there was no need for multiple introductions from previously isolated populations but only several introductions from genetically diverse populations. Moreover, we also found evidence of differentiation between invasive and naturalized populations in the adventive range. Highly invasive populations were clearly distinguished by different allele frequencies. The fine-scale genetic diversity study further supports these results. The genetic diversity of trees introduced to the adventive localities approximately one hundred years ago was high enough to ensure the maintenance of high genetic diversity. At the fine scale within the invasive populations, we found no evidence for any increase in genetic diversity parameters over time. Furthermore, in invasive populations, we observed the development of fine-scale genetic clustering over time. This occurred at the maximum distance of 25 m, at which old trees completely lacked any spatial genetic structure that increased over time and reached its maximum in the sapling stage. These results support the hypothesis that rather than admixture, a single introduction from a genetically diverse source or multiple introductions from different sources of similar genetic diversity pattern, or a combination of such events, was likely responsible for the high genetic diversity of P. strobus populations in the adventive range.In summary, the comparative population genetics of native and adventive


Tor tambroides, an important freshwater fish species in Malaysia, using fifteen polymorphic microsatellite loci and sequencing of 464 base pairs of the mitochondrial cytochrome c oxidase I (COI) gene. A total of 152 mahseer samples were collected from eight populations throughout the Malaysia river system. Microsatellites results found high levels of intrapopulation variations, but mitochondrial COI results found high levels of interpopulations differentiation. The possible reasons for their discrepancies might be the varying influence of genetic drift on each marker or the small sample sizes used in most of the populations. The Kelantan population showed very low levels of genetic variations using both mitochondrial and microsatellite analyses. Phylogenetic analysis of the COI gene found a unique haplotype (ER8∗), possibly representing a cryptic lineage of T. douronensis, from the Endau-Rompin population. Nevertheless, the inclusion of nuclear microsatellite analyses could not fully resolve the genetic identity of haplotype ER8∗ in the present study. Overall, the findings showed a serious need for more comprehensive and larger scale samplings, especially in remote river systems, in combination with molecular analyses using multiple markers, in order to discover more cryptic lineages or undescribed “genetic species” of mahseer.This study examines the population genetic structure of  Tor tambroides, commonly known as mahseer, Kelah, or Empurau, is a highly priced cyprinid fish in Malaysia due to its delicious flesh and has a great potential for the freshwater aquaculture industry [T. tambroides which require clean running water with gravels and rocks are rapidly being degraded due to anthropogenic disturbances  and uncontrolled fish harvesting [T. tambroides and given its fragmented distributions and population size, studies on the population structure and level of genetic variations throughout its distribution range are required for the effective management and conservation strategies of this important freshwater resource.industry , 2. It iindustry . Unforturvesting . TherefoT. tambroides and its congeneric species T. douronensis have been conducted by several authors such as [T. tambroides from 5 populations in Malaysia using the mitochondrial COI sequences and found low mitochondrial variations at the intrapopulations level but slightly higher variations at the interpopulation level. Although mitochondrial DNA has proven to be a powerful marker for detecting genetic subdivisions in many organisms [The phylogenetic relationship and taxonomic status between  such as –7 who ge such as  subsequerganisms , informarganisms .T. tambroides using microsatellites, a biparentally inherited nuclear marker. The highly variable microsatellites can provide a finer resolution of population-level dynamics, suitable for evolutionary and conservation genetics studies [T. tambroides.In this study, we investigated the population structure of  studies . The mic studies  using miT. tambroides used in this microsatellite study were similar to those described in [T. tambroides, T. douronensis, and Neolissochilus stracheyi haplotypes found in [Samples of ribed in  with thefound in  were alsfound in –16.cytochrome c oxidase I (COI) gene was amplified with the oligonucleotide primers COIf  and COIe  [μL reaction mixture containing 50 mM 10X buffer, 2 mM MgCl2, 0.2 μM of each dNTP (Promega), 0.1 μM of each primer, and 0.5 units of Taq DNA polymerase (Promega). The cycle parameters consisted of 35 cycles of denaturation , annealing , and extension . The amplified products were visualized on 2% agarose gel containing ethidium bromide, ran for approximately 30 min at 90 V, and photographed under UV light. The purified PCR products were directly sequenced using the BigDye Terminator v3.0 Cycle Sequencing kit (ACGT) on an ABI 377 automated sequencer (PE Applied Biosystem) using only the forward primer (COIf). A sequencing reaction using the reverse primer (COIe) was subsequently carried out on some of the samples (haplotypes) to verify the polymorphism in the DNA sequence initially detected using the forward primer.Total DNA was extracted using the CTAB method  in the preverse) . ApproxiT. tambroides using primers from other cyprinids [Cyprinus carpio , Barbusd Barb62 ), and Ba (Bgon13 ).μL, containing 25–50 ng of genomic DNA, 1X PCR buffer , 2.0 mM MgCl2, 0.2 mM of each dNTP, 5 pmol of each primer, and 1.5 units of Taq DNA polymerase. Amplification conditions were 94°C for 5 min followed by 25 cycles at 94°C for 40 s, Ta  for 40 s and 72°C for 1 min, with a final extension of 72°C for 4 min.Polymerase chain reaction (PCR) amplifications were performed in a final volume of 10 μL/mL), and photographed under UV light using an Alpha Imager 2200. The alleles were designated according to PCR product size and calculated relative to a standard molecular marker .Microsatellites were run on 4% high resolution MetaPhor agarose gels for 2 h at 78 V/cm, stained with ethidium bromide  was done using a close-neighbor-interchange (CNI) option implemented in MEGA (version 4.0 ). The NJA maximum parsimony (MP) tree was estimated using heuristic searches, as implemented in PAUP* v4.0b10 . Heurist6 generations with trees being sampled at every 100 generations.Phylogenetic tree was also estimated using the maximum-likelihood (ML) approach also implemented in PAUP* v4.0b10 . BootstrHO), and expected (HE) heterozygosity [G-based test) that assumes random samples of genotypes also using GENEPOP. Linkage disequilibrium to confirm independence between loci was tested by log-likelihood ratio G-tests  implemented in FSTAT. F-statistics for each locus and over all loci [FIS) as a measure of heterozygote deficiency or excess [FIS for significant departure from zero. The probability was estimated as the proportion of permutations which gave as large or larger statistic as the observed one. Sequential Bonferroni adjustments were made for all multiple comparisons [For microsatellite analysis, the software package MICRO-CHECKER2.2.1  was usedzygosity  availablzygosity . Allele zygosity . GENEPOPzygosity  was usedall loci  and inbrr excess  were calparisons .T. tambroides populations was estimated by performing a hierarchical analysis of genetic diversity using the analysis of molecular variance (AMOVA) using ARLEQUIN version 3.01 [FST, calculated according to [The significance of the spatial variation in gene diversity of ion 3.01 . Differerding to , also usrding to . An Unwerding to .α = 0.01.To compute the probability that an individual belonged to each reference population, assignment tests were performed on the basis of multilocus genotype data using GENECLASS version 2.0 . The BayK) in the data set without prior information of the sampling locations, available in STRUCTURE version 2.0 [λ was set at 1, the default value). The software was run with the option of admixture, allowing for some mixed ancestry within individuals, and α was allowed to vary. Ten independent runs were done for each value of K (K = 1 to 8 for each species) with a burn-in period of 25,000 iterations and 25,000 replications.A Bayesian approach was used to infer the number of clusters  variable/polymorphic sites including 56 (12.1%) parsimony-informative sites while 377 (81.2%) were conserved sites. The mean total of the nucleotide composition was 25.7% A, 32.3% T, 22.6% C, and 19.4% G. In total, 24 substitutions were found among haplotypes, of which there were 21 transitions and three transversions. All substitutions occurred at the third codon position, a common characteristic of mitochondrial DNA . The pron = 47) clustered into haplotype HKE4, while haplotype HKE12 comprising a single sequence was clustered within the T. tambroides group. Interestingly, ER8* which was another unique haplotype that consisted of four sequences was positioned intermediately between the T. tambroides and T. douronensis clusters based on all methods of phylogenetic analyses  generally produced similar tree topologies with slight differences in their bootstrap support values; thus only the Bayesian tree was chosen to show the mahseer phylogenetic relationships. Accordingly, the COI sequences of the 71 samples of mahseers from the Endau-Rompin, Baleh and Ulu Limbang populations produced a total of only 4 haplotypes with two unique haplotypes (HKE12 and ER8*) were only found from the Endau-Rompin population . All seqanalyses . Thus, tT. tambroides . There were no indications of scoring errors attributable to stuttering or large allele dropout. The general excess of homozygotes for most allele size classes may hence indicate the presence of null alleles or a Wahlund effect. For the microsatellites developed by , ten outmbroides . PolymorT. tambroides. However, only two loci (Tt2.B02 and Barb62) still displayed significant deviations from Hardy-Weinberg expectations after adjustments with Bonferroni correction. In all populations, FIS values were significantly different from zero (P < 0.05) in both species except in the Endau-Rompin population, indicating a loss of heterozygosity within this population in accordance with significant deviations from Hardy-Weinberg equilibrium. Linkage disequilibrium among pairs of loci was tested for 105 combinations over 15 loci with 26 (20.5%) that were in linkage disequilibrium (only one significant deviation after Bonferroni correction). After pooling all populations, 12 comparisons were highly significant (only one after Bonferroni correction).Randomization tests showed that six (40.0%) of the 15 single locus permutation tests were inconsistent with Hardy-Weinberg equilibrium expectations in FST over all loci showed that 22 (78.5%) out of 28 pairwise estimates showed significant genetic differentiation with the Kelantan population showing the highest degree of differentiation from all other populations  . Pairwise estimates of genetic distances computed by [AMOVA results revealed that the majority of variance (83.94%) was from within individual variations, and only 13.13% of total variance resulted from inter-population difference . Pairwis < 0.05) . In all puted by  among poResults of the assignment tests using GENECLASS2 showed that on average 42.8% (66 out of 152) of the individuals were correctly assigned to their original sampling site . The PerK value identified for T. tambroides was K = 3  reported in most freshwater fish species (13 species [The mean expected heterozygosity (0.3184) at 15 microsatellite DNA loci found in  species ) but was species , 54–56.Empirical studies of natural populations have commonly reported departures from Hardy-Weinberg equilibrium in a wide range of fish species –59. ObseT. tambroides . Nevertheless, significant differentiation in 78.5% of the pairwise comparisons among populations from the eight rivers confirmed their population genetic divergence. AMOVA analyses further supported the low inter-population differentiation among the T. tambroides populations with the majority of variation resulting from inside the populations compared to only 13% among populations. The pattern of genetic structure of the T. tambroides populations based on the present microsatellite analyses is most likely to be modulated by historically high gene flow between populations and then followed by genetic isolation. The mixed pattern of population differentiation was further supported by the assignment test results where samples from the Perak population showed a high percentage of correctly assigned individuals, followed by a moderate percentage in the Pahang and the Endau-Rompin populations, and a low percentage in other populations. Accordingly, a high percentage of correctly assigned individuals may indicate strong genetic divergence among populations and, conversely, low assignment success may indicate weak divergence [The results of the microsatellite analyses showed mixed levels of population differentiation among extant populations of vergence .T. tambroides. Microsatellites results found high levels of within- (intra)population variations in the present study, but mitochondrial results found high levels of among- (inter)populations differentiation. Furthermore, microsatellites results showed low genetic differentiation among the three populations of Peninsular Malaysia , but significant differentiation was found between them and the Kelantan population, which was slightly different compared to the overall low genetic differences found among the four populations observed using a mitochondrial dataset [T. tambroides populations was evidently higher based on comparing mitochondrial and microsatellite dataset.The patterns of genetic variations differed between results generated using microsatellites in the present study and results from mitochondrial COI sequences done by  in T. ta dataset . The popT. tambroides population structure can be discussed based on two assumptions. Firstly, differences between these datasets may reflect the varying influence of genetic drift on mitochondrial and nuclear genomes. Mitochondrial genes have a 4-fold lower effective population size, relative to their diploid/biparentally inherited nuclear counterparts, due to their uniparental  inheritance and haploid genotypes [T. tambroides populations was due to the small sample sizes in most of the populations, where some of the alleles could not be detected, thus reducing their overall level of genetic variations [Thus, the discrepancies between the mitochondrial and microsatellite datasets regarding the enotypes . Given triations .T. tambroides population from Kelantan , which is consistent with a low mitochondrial variation  found by [T. tambroides. Null alleles are alleles that are not amplified during PCR because of mutation events changing the DNA sequence in one of the primer sites (mostly at the 3′ end), which causes the primer to no longer anneal to the template DNA during PCR [T. tambroides. Hence, in the present study, the possible causes for an excess of homozygosity can be speculated as habitat destruction and overexploitation in the upper reaches/headwaters of the Kelantan River, especially the northwestern part of the headwater, where the samples might have been collected. This is because mass reduction to the effective population size of T. tambroides in this area would therefore lead to large reductions in both mitochondrial and microsatellite variations. Although bottleneck analyses showed no evidence of a recent bottleneck occurring within the Kelantan population  without any information regarding their specific location of capture and detailed morphological description by a private conservation company. Therefore, we suggest that a fine scale genetic analysis using multiple markers must be done throughout the upper reaches of Kelantan River in order to confirm the actual level of genetic variation in the T. douronensis and the T. tambroides clades, instead of a grouping with the other Endau-Rompin haplotypes in the T. tambroides clade (T. douronensis (4.5%–5.9%) compared to T. tambroides (5.9%–6.6%). This finding was of particular interest because the T. douronensis lineage has never been described from rivers in Peninsular Malaysia, although their distributions commonly overlap with T. tambroides in the upper streams rivers of the Sarawak state of the Borneo Island [Phylogenetic analysis found a unique haplotype lineage (ER8*) from four fish individuals from the Endau-Rompin population. This particular haplotype showed conflicting phylogenetic positioning within both the es clade . The paio Island .T. douronensis samples from a few populations  as well as the four individuals representing the ER8* haplotype (grouped together as a single population) were included in the analyses. Microsatellite analysis showed marked differences in allelic frequencies at a few loci between T. tambroides and T. douronensis with both species harbouring private alleles, although no fully diagnostic loci could be found between them (data not shown). Accordingly, T. douronensis samples exhibited a low “120” private allele at the Tt2.D01 loci not found in the T. tambroides populations while the T. douronensis samples also lacked allele “180” at the Tt1.C10 loci which was found at high frequencies in the T. tambroides populations. In addition, all the T. douronensis populations also exhibited high frequencies of allele “140” to allele “160” at the Barb62 loci compared with a lower allelic range  commonly found in the T. tambroides populations.Thus, analysis of hypervariable microsatellite loci was conducted in order to provide further information regarding the genetic identity of haplotype ER8*. The T. tambroides and T. douronensis at different frequencies but also exhibited a few private alleles themselves . Results from Bayesian cluster analysis  the clustering of haplotype ER8* within the T. douronensis clade based on phylogenetic analyses of COI sequence as well as UPGMA analysis of microsatellite data sets and (ii) its closer genetic relationship with other T. douronensis haplotypes compared with T. tambroides haplotypes.Therefore, several possibilities could be made regarding the genetic identity of haplotype ER8* based on both the microsatellite and mitochondrial results of the current study. First, haplotype ER8* could represent a cryptic T. douronensis lineage in the Endau-Rompin River can be explained in relation to the biogeographical history of South East Asia . Geological evidence suggests that the river systems of the southern parts of Sarawak were historically interconnected with most major river systems of Peninsular Malaysia during the Tertiary and Quaternary periods (10–5 Ma) via the North Sunda River [T. douronensis of the Endau-Rompin populations from their Borneo population counterparts. Similar evidence of a close genetic relationship between freshwater fishes of Borneo and mainland Asia in relation to their biogeographical history was discussed by several other authors including [Hemibagrus nemurus and Hampala macrolepidota, respectively.Furthermore, the possible occurrence of a da River , thus alda River . The risncluding , 66 in rT. tambroides itself in the Endau-Rompin population but with a high genetic divergence compared with the other T. tambroides haplotypes which exhibited slight differences in their allelic frequencies as well. The high proportional membership of microsatellites data into the T. tambroides cluster (92.8%) identified by STRUCTURE further supported this hypothesis. Thus, the higher number of samples analyzed from the Endau-Rompin population has also increased the chances of detecting new or cryptic lineage compared with smaller samples sizes analyzed from other populations.Secondly, haplotype ER8* might represent a cryptic lineage of T. tambroides and T. douronensis since freshwater fishes, such as cyprinids, commonly hybridize in nature [T. tambroides and T. douronensis could indicate that they were hybrids, possibly from backcross generations. However, we lack a fully diagnostic microsatellite loci and, along with an unresolved phylogenetic relationship of ER8* haplotype, restricted us from confirming this hybridization issue. On the other hand, it is worthy to note that no hybrid individuals were found based on our genetic analysis from the other sympatric locations of the two mahseer in Sarawak such as in the Batang Ai and the Ulu Limbang populations. This appears to suggest that a hybridization event between T. tambroides and T. douronensis might be absent (but is not impossible) due to undetermined reproductive isolation factors. Nevertheless, further studies are needed using more microsatellite loci and larger sample sizes from their sympatric locations in order to reconcile and confirm the hybridization issue.Thirdly, ER8* presumably represent a hybrid population between n nature . The shaT. tambroides populations in Malaysia. The utilization of both the mitochondrial and microsatellites markers showed different degrees of genetic differentiation and levels of resolution within and among populations, but their combination proved to be a better choice for future population genetic studies of mahseer as well as other freshwater species. The preliminary findings of a potential cryptic lineage of T. douronensis from the Endau-Rompin population showed a need for more comprehensive and larger scale surveys, especially in remote river systems in order to find more cryptic lineages or undescribed “genetic species” of mahseer.In summary, this study has managed to provide further insights into the population structure and levels of genetic diversity among


One of the most significant biological disturbances on a tropical coral reef is a population outbreak of the fecund, corallivorous crown-of-thorns sea star, Acanthaster planci. Although the factors that trigger an initial outbreak may vary, successive outbreaks within and across regions are assumed to spread via the planktonic larvae released from a primary outbreak. This secondary outbreak hypothesis is predominantly based on the high dispersal potential of A. planci and the assertion that outbreak populations (a rogue subset of the larger population) are genetically more similar to each other than they are to low-density non-outbreak populations. Here we use molecular techniques to evaluate the spatial scale at which A. planci outbreaks can propagate via larval dispersal in the central Pacific Ocean by inferring the location and severity of gene flow restrictions from the analysis of mtDNA control region sequence . Substantial regional, archipelagic, and subarchipelagic-scale genetic structuring of A. planci populations indicate that larvae rarely realize their dispersal potential and outbreaks in the central Pacific do not spread across the expanses of open ocean. On a finer scale, genetic partitioning was detected within two of three islands with multiple sampling sites. The finest spatial structure was detected at Pearl & Hermes Atoll, between the lagoon and forereef habitats (<10 km). Despite using a genetic marker capable of revealing subtle partitioning, we found no evidence that outbreaks were a rogue genetic subset of a greater population. Overall, outbreaks that occur at similar times across population partitions are genetically independent and likely due to nutrient inputs and similar climatic and ecological conditions that conspire to fuel plankton blooms. Acanthaster planci, are widely recognized as a major threat to coral reef ecosystems. Ecologically, outbreaks severely impact reef systems A. planci reduce the aesthetic value of coral reefs, thereby negatively impacting economies that depend on tourism. To reduce the impact of these corallivores, costly control and eradication programs have been established in several countries Outbreaks of the crown-of-thorns sea star, Outbreaks may arise from a single mass recruitment event or from the progressive accumulation of sea stars from multiple cohorts A. planci larvae disperse widely, en mass on oceanic currents. The pan-tropical Pacific range  of A. planci is a potential indicator of broad dispersal, and available genetic evidence using allozyme, mitochondrial COI DNA, and nuclear microsatellites supports high dispersal ability, with few detected barriers to gene flow A. planciThe foremost assumption of the secondary outbreak hypothesis is that On the GBR, primary outbreaks exhibit a subset of the genetic diversity in the total population A. planci's range , and is based upon dated allozyme assays. Nevertheless, this hypothesis has become an accepted theory to explain outbreaks that occur consecutively among the discontinuous coastlines of islands, archipelagos, and regions throughout the tropical Pacific Ocean A. planciOverall, there have been very few direct tests of the secondary outbreak hypothesis. It has only been supported with genetic data in a limited portion of Acanthaster planci across the Pacific Ocean, from Yap in the western Pacific to Hawai'i and Mo'orea in the central Pacific, testing the extent to which the larvae of A. planci readily disperse, thereby defining boundaries to secondary outbreak propagation via larval dispersal. We specifically test the spatial scale of genetic partitioning  and the level of genetic differentiation between outbreak and non-outbreak populations. Additionally, we test for differences in the genetic diversity within and among outbreak and non-outbreak populations. The results of this study advance our understanding of the propagation of outbreaks via larval dispersal, highlight the genetic complexity of such a widespread planktonic species, and contribute to improving the efficiency and focus of current management strategies for this destructive corallivore.Here we examine the genetic structure of the highly variable mitochondrial control region  A. planci (n = 656 sea stars) were collected between 2005 and 2008 from 23 sites across the Pacific Ocean: the northwestern Pacific (NW) region, the north central Pacific (NC) region, and the south central Pacific (SC) region , and three habitat types were sampled at Pearl & Hermes Atoll .Adult ) region , Table 1in situ, while either free diving or scuba diving Live sea stars were sampled non-lethally by snipping off an arm tip A. planci is considered to be an outbreak varies depending upon the survey's method and spatial scale. Due to opportunistic sampling, three different methods were used to diagnose populations as outbreaks: towed-diver surveys (a similar procedure to the established manta-tow technique), belt transects, and swim surveys (−2 (15 ha−1) are considered to be undergoing a population outbreak −2 at NW2*, 11,050 sea stars km−2 at SC1*, and 50,500 sea stars km−2 at NC8*. For belt transect surveys, where sea stars are more thoroughly quantified, densities <10,000 km−2 (100 ha−1) are generally considered to be low density populations (non-outbreak), while values greater than this are considered to be high density populations (outbreak) −2 (4500 ha−1) −2 (3500 ha−1), and 660,000 sea stars km−2 (6600 ha−1) respectively. In timed swimming surveys, >40 sea stars per 20 minute swim characterizes an outbreak population The density threshold at which a population of  surveys . For manSamples from these outbreak populations were collected between 2005 and 2008: NC8* 2005, SC1* 2006, NW5* 2007, NW2* 2007, NC2* 2008, and SC3* 2008 . Based oTwo different procedures were used for DNA extraction and amplification, based on tissue type and age of samples. DNA was extracted from tube feet, as described in Jessop 5′-CAAAAGCTGACGGGTAAGCAA-3′ primer and the COTS-ctrl-rvs 5′-TAAGGAAGTTTGCGACCTCGAT-3′ primer 2O, 10 µL of undiluted template DNA, 10 µL of each primer (5 µM), and 50 µL of Promega MasterMix. Thermocycling was performed with an initial denaturation at 94°C for 5 min, 34 cycles , and a final extension for 10 min at 72°C. PCR products were prepared for cycle sequencing with the UltraClean PCR Clean-Up Kit .Approximately 530 base pairs of the noncoding mitochondrial DNA control region (mtDNA) were amplified with polymerase chain reaction (PCR), using the COTS-ctrl-fwd The PCR for historical samples utilized 25-µL reactions with 2.5 µL of 10X buffer, 5 µL of each primer (0.2 µM), 0.5 µL of undiluted template DNA, and 1.5 U of Immolase Taq polymerase (Bioline USA). Thermocycling for all samples was performed with an initial denaturation at 94°C for 5 min, 34 cycles of , and a final extension for 10 min at 72°C. PCR products were treated with 1.5 unit of exonuclease I and 1.5 unit of calf intestinal alkaline phosphatase (Exo-CIAP), incubated at 37°C for 60 min, and then deactivated at 85°C for 15 min.5′-CAATGAGAATTGCACAAGCGCCTC-3′) on an ABI 3130xl automated sequencer (Applied Biosystems Inc.). Unique haplotypes were submitted to GenBank (Accession numbers JQ397722–JQ398377).Amplified DNA fragments were sequenced in the reverse direction and all unique and questionable sequences were repeated with an alternate sequencing primer . Sequences were aligned using muscle v3.6 eaView 4.2 ioEditSequences were compared and assembled using Snetwork v4.5 (Fluxus Technology Ltd.) to illustrate haplotype variability and clustering. An analysis of molecular variance (AMOVA) was conducted using Arlequin v3.5 ermanova+ a priori contrast tests following Stat et al. rlequin for these data, as determined by Modeltest 3.7 h) was calculated in Arlequin and converted to the effective number of haplotypes, following Jost h = 1 , the effective number of haplotypes cannot be calculated, so we calculated the effective number of haplotypes by assuming that the next haplotype sampled would be a duplicate (not unique). Nucleotide diversity (π) and population pairwise ΦST values were calculated in Arlequin. The statistical significance of pairwise comparisons were adjusted for family-wise false discovery rate, according to Benjamini et al. emN) were calculated from pairwise FST values in Arlequin.Median-joining haplotype networks with the default weight of 10 applied to each character were created using emN) and mutation scaled population size (θ) were conducted using Migrate v3.2.7 igrate. A nucleotide model with a transition-to-transversion ratio of 12.29∶1 was used with six regions of substitution rates and a gamma-shaped rate variation of 0.29, along with a Markov chain length = 1,000,000, sampled every 100 generations, with a 10% burn-in. Program defaults were used for all other settings. The transition-to-transversion ratio and the rate variation were calculated using Modeltest 3.7. Values for the migration rate among regions (m) and mutation scaled population size (θ) were taken from the highest peaks in the posterior probability distribution curves. The posterior probability distributions were examined to determine the credibility of each estimated parameter.Bayesian coalescent-based calculations of within- and between-region migration rates  and ranged from h = 0.83–1.00 (6–333 effective haplotypes) within sampled locations . Fifty seven percent of the variation in pairwise ΦST between samples in the NW and SC could be explained by the distance between the sites  than to either Swains (SC2) (0.27≤ΦST≤0.32) or Mo'orea (SC3*) , with the sole exception of the one-way migration from the NW into the SC region  being completely different from those of the south central Pacific  and northwestern Pacific  than did non-outbreak populations (He = 29±16), with  and non-outbreak populations in the main Hawaiian Islands  are not significantly differentiated  and NW4  and the NW2* outbreak is not significantly different from non-outbreak NW4 .In the Mariana Archipelago (NW region), outbreak  and non-outbreak populations  are significantly differentiated .Outbreak populations are significantly differentiated between archipelagos  in the NWHI at Pearl & Hermes Atoll , but Maui Nui and the Big Island are not convincingly differentiated . In contrast, pairwise analysis revealed differences between Hawai'i West (NC4) and all three other Big Island sites , though NC1-3 appeared to be panmictic . Within the Maui Nui complex, pairwise analysis also revealed genetic panmixia .Around the Big Island of Hawai'i, there was no detectable genetic difference between the 1982 collection (NC1) and the more recent collections . ST = 0.12, P = 0.001) but a trivial difference between backreef and forereef habitats . The lagoon population showed divergence from all MHI populations . Similarly, the backreef population was divergent from all MHI populations , except NC9 . However, the forereef population was genetically similar to all MHI populations , with the exception of NC4, NC8*, and NC10, . Sea stars on the forereef exhibited substantially greater genetic diversity than on the backreef and lagoon areas of PHR , represents a balance between local effective size (Ne) and the per generation migration rate (m), at least where the migration rate (m) is orders of magnitude higher than the recurrent mutation rate (μ). In keeping with that expectation, Nishida and Lucas A. planci populations in the Pacific (though they did identify restricted gene flow between Hawai'i and other locations). On the other hand, hypervariable markers, for which μ>m, typically show finer-scale structure (more divergence among populations) because population divergence is enhanced by mutations that accumulate locally, much faster than they can be spread among populations by migration. In keeping with that expectation, Yasuda et al. A. planci is a fairly common finding.Our data show that A. planci are not spreading among regions in the central Pacific via larval dispersal. Local variation is locally derived, rather than being a consequence of long-distance immigration , as evidenced by lesser genetic differentiation , though Likewise, it is improbable that the outbreak populations sampled here, within archipelagos of the SC region, are spreading via larval dispersal on the South Equatorial Countercurrent or the Southern Equatorial Current and gyre. These populations reflect an isolation-by-distance pattern, with little evidence for a distinct outbreak population spreading across the region . SimilarAcanthaster planci samples appear to represent a single population across the 590 km stretch of the MHI, thus outbreaks may spread via larval dispersal here. High density outbreak populations of A. planci exhibited comparable haplotypic diversity and were genetically indistinguishable from low density populations, with the exception of the West Hawai'i sample (NC4). As in vermetid snails  Zebrasoma flavescens) A. planci was slightly differentiated from the other MHI samples, hinting that finer-scaled population partitioning may occur. It is difficult to predict whether and exactly how an outbreak might propagate through the archipelago. The prevailing ocean currents that run along the 590 km stretch of the MHI are haphazard, due to eddys, mesoscale instability, and seasonal variability The majority of Support for the secondary outbreak hypothesis along a 730 km stretch of the Mariana Archipelago is somewhat equivocal. Following the predictions of this hypothesis, the Mariana outbreak populations are (1) genetically similar to one another and (2) genetically dissimilar to the non-outbreak populations ; Fig. 4.Barriers to gene flow across small channels between islands and groups of islands have been identified in a number of high dispersal species A. planciAcanthaster, suggests that the lagoonal population has not reached equilibrium  and the genetic similarity between outbreak and non-outbreak populations found within archipelagos is inconsistent with the secondary outbreak hypothesis. However, the lack of divergence between outbreak and non-outbreak populations within archipelagos indicates that populations are exchanging gametes via dispersal.To be conservative, one should assume that outbreaks might spread between locations that do not exhibit genetic population structure, while realizing that a lack of structure does not prove evidence that outbreaks are spreading in this fashion. Our recommendation to managers is to consider seriously the role that environmental conditions and local nutrient inputs play in driving outbreaks.A. planci populations from different regional and archipelagic zones investigated, suggesting that outbreaks in the central Pacific are not triggered by mass dispersal events, as previously proposed A. planci population density, larval production, and the number of available migrants.In examining the secondary outbreak hypothesis with mtDNA control region markers in the central Pacific, we discovered substantial genetic differentiation in all A. planci larvae. Since outbreaks are not spreading among archipelagos, the efficiency and effectiveness of coral reef conservation efforts to control the spread of A. planci in the central Pacific can be greatly improved by focusing efforts within archipelagos and islands.The phenomenon of outbreaks occurring at similar times among vastly disjunct areas is probably due to similar climatic, ecological, or anthropogenic conditions, rather than the planktonic dispersal of Figure S1Median-joining haplotype network of Acanthaster planci samples color coded by region. Corresponding location numbers are in parenthesis. Each circle represents a unique haplotype connected by a line to those that differ by one or more base pairs. Those lines that represent ≥5 bp differences were labeled by barred increments; however, lines are not drawn to scale. Nodes on the lines indicate missing haplotypes. The smallest colored circles represent a singleton haplotype, and the largest circle represents 25 individuals.(TIF)Click here for additional data file.Figure S2Median-joining haplotype network of Acanthaster planci samples color coded by island with the exception of north central Pacific (NC), which is color coded by the subregions MHI (main Hawaiian Islands) and NWHI (Northwestern Hawaiian Islands). Corresponding location numbers are in parenthesis. Each circle represents a unique haplotype connected by a line to those that differ by one or more base pairs. Those lines that represent ≥5 bp differences were labeled by barred increments; however, lines are not drawn to scale. Nodes on the lines indicate missing haplotypes. The smallest colored circles represent a singleton haplotype, and the largest circle represents 25 individuals.(TIF)Click here for additional data file.Figure S3Median-joining haplotype network of Acanthaster planci samples color coded by outbreak and non-outbreak. Corresponding location numbers are in parenthesis. Each circle represents a unique haplotype connected by a line to those that differ by one or more base pairs. Those lines that represent ≥5 bp differences were labeled by barred increments; however, lines are not drawn to scale. Nodes on the lines indicate missing haplotypes. The smallest colored circles represent a singleton haplotype, and the largest circle represents 25 individuals.(TIF)Click here for additional data file.Figure S4Median-joining haplotype network of Acanthaster planci samples color coded by coded by habitat at Pearl & Hermes Atoll. Corresponding location numbers are in parenthesis. Each circle represents a unique haplotype connected by a line to those that differ by one or more base pairs. Those lines that represent ≥5 bp differences were labeled by barred increments; however, lines are not drawn to scale. Nodes on the lines indicate missing haplotypes. The smallest colored circles represent a singleton haplotype, and the largest circle represents 25 individuals.(TIF)Click here for additional data file.Table S1M and θ posterior probability distributions as calculated by Migrate using a Bayesian MCMC simulation.(DOCX)Click here for additional data file.Table S2M and θ posterior probability distributions as calculated by Migrate using a Bayesian MCMC simulation.(DOCX)Click here for additional data file.