Austruca occidentalis is a dominant species in mangrove forests along the East African coast. It enhances soil aeration and, through its engineering activities, makes otherwise-inaccessible food available for other marine organisms. Despite its importance, the habitat of A. occidentalis is threatened by human activities. Clearing the mangroves for salt farming and selective logging of mangroves trees continue to jeopardise mangrove ecosystems in the Western Indian Ocean. This study aims to use partial mitochondrial COI gene sequences and nuclear microsatellites to determine whether salt farming activities in mangroves have a negative impact on the genetic diversity and gene flow of A. occidentalis collected along the Tanzania coast. The level of genetic diversity for both mitochondrial DNA and nuclear microsatellites are relatively lower in samples from salt ponds compared to natural mangrove sites. Analysis of molecular variance (AMOVA) among all populations showed low but significant differentiation . A hierarchical AMOVA indicates lower but significant genetic differentiation among populations from salt ponds and natural mangroves sites . These results indicate that salt farming has a significant negative impact on the genetic diversity of A. occidentalis. Since higher genetic diversity contributes to a stable population, restoring the cleared habitats might be the most effective measures for the conservation of genetic diversity and hence adaptive potential to environmental change in this species.The fiddler crab  The loss of mangroves continues to increase rapidly in developing countries, where more than 90% of the world’s mangroves are located . In deveSalt production in mangroves ignores the important function of mangroves as habitat and nursery ground for many marine and terrestrial fauna , 9. Salt2 [Tanzania is one of the East African countries at the WIO with a mangrove area that covers about 1,587.44 Km2  and all 2 . This ec2 , 13–14, Patella caerulea [Ecological disturbance cause habitat disturbances once it changes food availability and predation –16. Conscaerulea . The redcaerulea –21.Austruca occidentalis has been found to be a good indicator of negative impacts of anthropogenic activities, such as sewage discharge in the mangrove ecosystem [Austruca sp. occurring at the intertidal shores of Eastern Africa [Cranuca inversa (field observation).The fiddler crab cosystem . This spcosystem –25. Theycosystem . This spn Africa . The maln Africa . It has n Africa . In the A. occidentalis larvae is about 28 days and permits extensive gene flow along the East African coast [A. occidentalis, may suffer from habitat fragmentation, resulting to genetic fragmentation between isolated populations [The pelagic larval duration of an coast . Planktoan coast . Despitean coast , 32. It ulations . UnderstThe use of molecular markers in population genetics studies has proven to be important in shedding light on several aspects related to the biology, ecology and evolution of many organisms . MoleculA. occidentalis. Conservation of species can be improved by investigating factors that can lead to evolutionary change over a short time period [Both mitochondrial DNA (mtDNA) and nuclear microsatellite data are helpful in analysing the genetic population structure of marine species –39. Howee period .A. occidentalis through inbreeding and bottleneck effects; 2) if salt farming has negative effects on the effective population size; and 3) if dispersal patterns of this species have been altered by salt farming activities. To achieve this, we assessed the degree of genetic variation and structure in populations from mangroves at salt ponds and natural mangroves. Then we examined the levels of heterozygosity and estimated effective population sizes, inbreeding and migration rates. We finally compared the genetic diversity and demographic indices between the populations at salt ponds and natural mangroves.The objectives of this study were to determine: 1) if salt farming affects the genetic diversity of No specific permissions were required for these locations/activities. The Tanzanians students are allowed by the government to conduct the scientific research but in order to export the samples abroad for laboratory analysis, the special licence/permit that is provided by the Ministry of Agriculture, Livestock and Fisheries  of the united republic of Tanzania is required. The code number of the special permit for exporting our samples to Belgium at Free university of Belgium (VUB) was 1997/2015. The field studies did not involve endangered or protected species.A. occidentalis were collected at 12 sites, of which six sites are from mangroves at salt ponds and the rest are from natural mangroves  and microsatellite analyses using either the QIAGEN  or E.Z.N.A.® Tissue DNA Kit  extraction kit. The extracted DNA was visualised in 2% TBE agarose gels.Tissue samples from a segment of the last pereiopod of 5’-TITCIACIAAYCAYAARGAYATTGG-3’) and jgHCO2198 (5’-TAIACYTCIGGRTGICCRAARAAYCA-3’) [TM thermocycler in a 50 μl reaction volume containing 0.2 mM dNTPs, 1 mM 10x PCR reaction buffer, 0.2 μM primers primer, 2.5 μl of 10 mg/ml BSA, 3 mM MgCl2, 3 μl DNA extract, and 1 U Taq polymerase. The temperature profile was set at 94°C for 5 minutes as initial denaturation, followed by 35 cycles of denaturation at 94°C for 60 seconds, annealing at 50°C for 1.5 minutes, 72°C of extension for 1 minute and 72°C of final extension for 5 minutes. PCR products were electrophoresed and visualised by using GelRed in 2% TBE agarose gels. An ABI 3770XL automated sequencer  was used for sequencing the PCR products. We successfully sequenced 138 samples from salt ponds and 136 from natural mangroves.The primer jgLCO1490 (AYCA-3’)  was usedUca mjoebergi [Ucides cordatus [A. occidentalis. The forward primer was labelled with a fluorescent dye  was performed with the software R (Version 3.1.2) to determine differences in nucleotide and haplotype diversity, observed and unbiased heterozygosity and allelic richness between populations from salt ponds and natural mangroves. Genetic diversity, heterozygosity and allelic richness were treated as dependent variables and “type of habitat “ as an orthogonal and fixed factor. Before ANOVA tests all dependent variables were tested for normality using Shapiro-Wilk and homogeneity of variances using Levene’s test and Fisher's F- test as implemented in the software R (Version 3.1.2). The values of unbiased heterozygosity were normally distributed after Ln-transformed.The sequences were first edited by using the software Chromaspro v. 1.5 (Technelysium) and compared with sequences available in GenBank using the online software BLAST in order to confirm species identity. The presences of stop codons, which indicate sequencing errors or pseudogenes, was evaluated by using the program Squint Alignment Editor v. 1.02 . A multion 1.1–2  was usedTCS 1.21  was usedTCS 1.21 –54, as wTCS 1.21 –56, wereTCS 1.21 . The witTCS 1.21 . Hierarcis) were calculated by using the software GenAlEx: version 6.5 [st-values obtained from sequences and microsatellite data. The software Fstat [Scoring of alleles was done manually with GeneMarker . All samples found to have missing values at three loci or could not be scored were removed from the data set. The final data set consists of 173 samples from salt ponds and 159 from natural mangroves. We used the software MICRO-CHECKER v. 2.23  was usedsion 6.5 . GenAlExre Fstat  was usedeμ) in microsatellite data, where Ne represents the effective population size and μ is the mutation rate per generation per locus. The same software was used to infer for the mutation-scaled migration rate M (M = m/μ) between populations from salt ponds and natural mangroves, where m represents migration rate per generation, and μ represents the mutation rate [The program MIGRATES v. 3.11.6  was usedion rate . A Brownion rate . To estiion rate , which uion rate . Seven rAfter aligning and trimming unreadable parts of sequences, we obtained an alignment of 674 bp length. All sequences are deposited at the European Nucleotide Archive (ENA) with the accession numbers LT703011-LT703284. Mitochondrial haplotype diversity for all populations was 0.23 (0 to 0.49), and nucleotide diversity 0.04% (0 to 0.08%) . Both, gThe total number of haplotypes recorded was 21. Only two haplotypes, the commonest, were shared between salt pond sites and natural mangroves. Most individuals at all sites had haplotype one . The minThe mean observed heterozygosity for all populations was 0.565 ± 0.026 and the unbiased expected heterozygosity was 0.497 ± 0.013. The allelic richness and heterozygosity were consistently higher at natural mangrove sites compared to salt pond sites. The ANOVA detected differences for observed heterozygosity , unbiased expected heterozygosity  and allelic richness  between populations from salt ponds and natural mangrove sites. The within population inbreeding coefficient ranged from –3.40 to 0.026, but all were not significant different from zero. The total number of alleles per locus was low to moderate, ranging between two and ten, with C361 having the highest and D2P3 the lowest number of alleles. Private alleles were recorded only in four populations from natural mangrove sites  . Locus Pst = 0.022, P < 0.05). The hierarchical (AMOVA) showed the existence of a low but significant genetic differentiation among samples from salt ponds and natural mangrove sites . No differentiation was observed among populations from salt ponds  and natural mangrove sites  when they were analysed separately. Analysis through contingency chi-square tables revealed differentiations in haplotype distribution between subpopulations collected from mangroves at salt ponds and natural mangroves . The pairwise Fst-values based on COI sequences for all populations were non-significant after applying sequential Bonferroni correction [The analysis of molecular variances of the COI sequences revealed a low but significant population differentiation . Hierarchal AMOVA results supported the hypothesis of genetic differentiations among populations at salt ponds and natural mangrove sites . A low but significant differentiation was observed among populations at salt pond sites based on microsatellite data , but no significant differentiation was revealed among populations from natural mangroves . Some populations have significant pairwise Fst-values adjusted through sequential Bonferroni correction  and nucleotide diversity of 0.04% (0 to 0.13%) [A. occidentalis has the lowest genetic diversity of all mangrove crabs studied so far in the WIO [Littoraria subvittata is observed at salt ponds in comparison to natural mangroves at the Tanzanian coast [A. occidentalis, can negatively affect the genetic diversity of various marine species.Both markers used revealed low genetic diversity in most populations of o 0.13%) . In that the WIO . Despite1 0 to 0. and nuclan coast . This imThe loci C361 and P2D3 deviated from HWE mainly in populations from natural mangrove sites. The deviation of locus C361 from HWE may be explained by the effects of null alleles. A deficit of heterozygosity might be an indication for null alleles  that wasA. occidentalis at salt pond sites are subjected to human influence through clearing and selective logging of mangroves in order to establish salt ponds and construct water reservoirs. This might have contributed to unfavourable environmental conditions for larval recruitment, such as predation and extremes in local microclimate. Predators, including birds and fishes, are known to have influence on the population distribution of fiddler crabs [Poecilia reticulata was revealed in a period of four to eleven years, or 6.9 to 18.1 generations [Population bottlenecks in marine organisms can be caused by unsuitable environments and predation, which causes mortality during the larval recruitment processes . The haber crabs . Impact erations , indicatχ2 tables indicated the presence of differentiations in haplotype distribution between populations from mangroves at salt ponds and natural mangroves. The population genetic differentiation is partly supported by the results of principal coordinate analysis (PCoA). However, all pairwise Fst-values for mitochondrial data were not significant, while for microsatellite data some populations from salt ponds and natural mangroves sites were significantly different. Few significant values were also obtained between populations from mangroves at salt ponds (st-values among groups of populations [st-values based on mitochondrial and microsatellite data might be explained by the higher resolution and statistical power of microsatellite markers in detecting effects of contemporary events.The analysis based on F-statistics is congruent to previous studies, indicating that this species has extensive gene flow along the East African coast , 67. Thelt ponds . Previouulations , 67. TheA. occidentalis, show clear signals of a recent bottleneck. This bottleneck is linked to the reduction of population size due to historical event of Pleistocene period and mangrove loss or degradation by pollution [A. occidentalis, have been reported to have shrunk globally during the period of low sea-level stands [All populations from natural mangroves have significant negative Tajima’s D values, except the populations in Tanga and Mtwara, while all populations from salt pond sites have negative values that are not significant. In addition, all populations from natural mangroves have significant negative Fu’s Fs-values, but all populations from salt ponds have none-significant negative values. A negative significant value of Tajima’s D is usually an indicator of the presence of evolutionary forces that cause departures from the neutrality of the genetic marker, such as recent expansion of population size following the retraction event. A negative value of Fs is evidence of population expansion and is regarded as more powerful than Tajima’s D . The nonollution . The tesollution  was not ollution  did not ollution . The excollution . During ollution , 79. Theollution . Howeverollution –82. Thesl stands . Althougl stands –85. Habil stands . Populatl stands .A. occidentalis. Genetic diversity and allelic richness are indicators of the long-term potential for adaptability and persistence of a given species in the habitat [A. occidentalis through planting mangroves around the salt pond sites and removing the barriers such as dykes of abandoned salt ponds to enable water flow. Otherwise, if alteration of habitats through degradation and fragmentation continues, there is the possibility of driving many species to local extinction.Both, mitochondrial and microsatellite markers confirmed that salt pond activities have impacted gene flow and genetic diversity, indicating that the on-going clearing and selective logging of mangrove trees have shaped dispersal patterns and local effective population sizes of  habitat –88. The


Fallopia japonica, F. sachalinensis and their interspecific hybrid F. × bohemica are some of the most aggressive invaders in Europe and North America and they are serious threats to native biodiversity. At the same time, they constitute a unique model system for the creation of hybrids and studies of the initiation of evolutionary processes. In the presented study, we focused on (i) examining genetic diversity in selected populations of three Fallopia taxa in the invaded (Poland) and native ranges (Japan), (ii) establishing genome size and ploidy levels and (iii) identifying ribosomal DNA (rDNA)-bearing chromosomes in all of the taxa from the invaded range. We found that the genetic diversity within particular taxa was generally low regardless of their geographical origin. A higher level of clonality was observed for the Polish populations compared to the Japanese populations. Our study suggests that the co-occurrence of F. sachalinensis together with the other two taxa in the same stand may be the source of the higher genetic variation within the F. × bohemica hybrid. Some shift towards the contribution of F. japonica alleles was also observed for selected F. × bohemica individuals, which indicates the possibility of producing more advanced generations of F. × bohemica hybrids. All of the F. sachalinensis individuals were hexaploid , while those of F. japonica were mostly octoploid  and all of the F. × bohemica plants except one were hexaploid . Within the chromosome complement of F. japonica, F. sachalinensis and F. × bohemica, the physical mapping of the rDNA loci provided markers for 16, 13 and 10 chromosomes, respectively. In F. × bohemica, a loss of some of rDNA loci was observed, which indicates the occurrence of genome changes in the hybrid.The knotweed taxa  Fallopia: F. japonica var. japonica, F. sachalinensis and the hybrids of these two species, namely F. × bohemica, are considered to be one of the most aggressive plant invaders in both Europe and North America [F. japonica and F. sachalinensis, originate from Asia. The native range of F. japonica is Japan, Sakhalin Island, the Kurile Islands, North and South Korea, Taiwan, Vietnam and parts of China, whereas for F. sachalinensis the range is limited to North Japan, Korea, South Sakhalin and the Kurile Islands [th century as decorative garden plants and soon spread into natural habitats [F. japonica was brought over from Japan to Netherlands by Phillipe von Siebold, and later it was distributed to other European countries. Some records also suggest that some specimens of this species were introduced to Great Britain from China, but probably those plants did not survived [F. sachalinensis was presumably the collection from St. Petersburg, where F. sachalinensis originated from Japan and Sakhalin Island where they were grown [F. × bohemica hybrids in Europe was given only just in 1983 [The knotweed taxa  America . The kno Islands –4. Thesehabitats –7. The hsurvived . The soure grown ,6. The fFallopia japonica currently exists in most parts of the British Isles, in many parts of the European continent, also occurs in Canada and the United States, Australia and New Zealand [F. sachalinensis includes Europe, New Zealand as well as Australia and South Africa. This species also occurs in North America, Canada and the United States [F. japonica and F. sachalinensis are particularly common in ruderal habitats that have been changed by human influence and in riparian ecosystems [F. japonica with F. × bohemica) or three taxa cohabiting in the same site [ Zealand ,9–12. It Zealand . The invd States ,12,14–16osystems –19, wherame site ,21.Fallopia taxa are reviewed in Bailey et al. [F. japonica var. japonica, F. japonica var. compacta, F. sachalinensis and F. baldschuanica. Both varieties of F. japonica may be pollinated by F. sachalinensis producing the hybrid F. × bohemica, which subsequently may go through the rounds of backcrossing with either of parents. Both varieties of F. japonica and F. sachalinensis may also be crossed with F. baldschuanica, but there is only one established F. conollyana hybrid, resulting from the cross of F. japonica var. japonica and F. baldschuanica [Knotweeds constitute a unique model system for the creation of hybrids and studies on the initiation of evolutionary processes in an invaded range. The main hybridization routes of closely related y et al. . Brieflychuanica .Fallopia complex is typical in both the native and invaded ranges [F. × bohemica may represent an example of an invasion via the hybridization hypothesis, which is explained as the superiority of hybrids over their parental species, which also ensures their success in the non-native range [F. × bohemica produces significantly greater biomass than both of its parents, when grown in experimental communities of native plants, supporting the hypothesis that hybridization events increase the competitiveness of the Fallopia complex. Aside from hybridization, also multiple introductions can help to generate higher genetic diversity in the invaded range than in the native one [A large intraspecific ploidy variation for the d ranges . In the d ranges  postulatve range –26. In ative one ,28.The genetic diversity of a species has consequences for various processes, such as colonization, many life history traits , population history, the impact of environmental factors and anthropogenic disturbances –33.Genetic diversity in the populations of different plant species, in and out of their native and invaded range, has been the subject of several studies –38. ThesGenetic diversity studies can also indicate the origin of populations, the routes of their introduction and can explain the mechanisms of their spreading and adaptation on a local scale ,41.Fallopia complex has been investigated using morphological, cytogenetic and molecular markers, including RAPDs, ISSRs [Fallopia taxa have small and morphological undifferentiated chromosomes makes an analysis of karyotype morphology difficult. As indicated for other genera, the application of FISH with repetitive sequences is often useful in cytogenetic analysis [Fallopia taxa. The preliminary chromosome identification and the dynamics of chromosome rearrangements are necessary to understand the evolution of Fallopia genomes, especially to identify the parental chromosomes in the F. × bohemica hybrid genome and to elucidate the extent of genomic changes that may have occurred in the F. × bohemica genome after hybridization. This genomic analysis is complementary to other molecular approaches that support comprehensive studies on a genome evolution within the invasive Fallopia complex.The genetic diversity in the s, ISSRs –44, SNPss, ISSRs ,46 and As, ISSRs ,48 as wes, ISSRs . Howevers, ISSRs ,50,51. Tanalysis . To the F. × bohemica hybrid and its parental taxa, thereby restoring fertility by enabling chromosome pairing during meiosis. We would like to testify its assumptions by focusing on the following aspects: (i) an analysis of genetic diversity in selected invasive populations of three Fallopia taxa from Poland and its native (Japanese) range; (ii) an analysis of the possible relationship between the level of genetic diversity within each taxon and the taxonomic constitution that is characteristic for the stands that were studied, i.e. stands consisting of one taxon  or that were composed of two or three taxa cohabiting in the same area ; (iii) an analysis of the number and chromosomal location of rDNA loci in all of the taxa from the invaded range and (iv) the genome size and ploidy of the plants collected in Poland.Our hypothesis assumes that the taxa composition within a particular location may influence on the genetic diversity within the population as the presence of several taxa cohabiting together may allow their interspecific hybridization and/or backcrosses between the Fallopia japonica, F. sachalinensis and F. × bohemica. The classification of each taxon was based on their morphological characters such as leaf shape and size, trichome type and morphology, which are well described as traits that are diagnostic for the identification of Fallopia taxa [Three invasive knotweed taxa were used in the study: pia taxa . Our stuF. japonica  and from four stands in Japan (JP). In the introduced range (PL), two stands were ‘heterogeneous’–comprised of two or three taxa co-occurring in the same area and one was ‘homogeneous’ and consisted of one taxon–japonica .Fallopia presents the most adverse impact on local biodiversity. In the native range (JP), the samples were collected from four ‘homogeneous’ stands–two composed of F. japonica and two consisting of F. sachalinensis . The nuy et al. .Leaf tissues from 200 ramets, including five ramets from each of the populations in Japan (Itami (It), Osaka (Os), Toma-cho (To), Obira-cho (Ob)) and 30 from each Polish population from the Jasieniczanka River (CDJ) and Biała River (CDB), were used for DNA extraction and further examination using AFLP markers .A piece of rhizome was cut from each individual originating from the Polish populations, potted in universal potting soil, periodically and cultivated in a greenhouse at 20°C and a 14-h daily photoperiod. Newly developed root tips from this greenhouse material were used for the chromosome counts, genome size and rDNA-FISH analysis. Of the 200 individuals used for AFLP analysis, 167 were examined using flow cytometry (FCM) and 30  were used for chromosome counts and chromosome identification using 5S and 35S rDNA sequences.EcoRI cutting site was used for the further visualization of the AFLP products. To ensure reliable band pattern analysis, the entire AFLP procedure was performed in two technical replications for each sample and the fragments were separated using a denaturing polyacrylamide gel electrophoresis (6% acrylamide/bis-acrylamide 19:1 solution (Sigma), 7 M urea (Amersham Pharmacia), 1 × TBE). A Li-Cor sequencer was used for electrophoresis with the following parameters: 1300 V, 30 mA, 30 W and medium speed of laser scanning. In order to calculate the length of amplified products, a 50–350 bp size marker  was loaded on to the gel. The ten most polymorphic AFLP primer combinations were used, which were selected after previously pre-screening a set of 15 selective primer pairs . Total genomic DNA was extracted from 100 mg of silica-dried material using the micro-CTAB method . The AFLer pairs .ij/Bij × 100) /D; where: A = the number of polymorphic loci for a pair of samples i and j, B = the total number of loci for a pair of samples analyzed and D = the number of pairs of samples analyzed. The percentage of polymorphisms within and between the populations was calculated based on the number of polymorphic loci with a minimum frequency of 5%.In order to describe the level of polymorphism among the individuals, only the unambiguous bands in each primer combination were counted. The entire set of bands was transformed into a 0–1 matrix consisting of monomorphic and polymorphic products . The levD = 1 – Σ (ni(ni− 1)) / (N(N– 1)), where ni is the number of ramets of the ith genet and N is the total number of ramets sampled [The clonal diversity within populations was calculated using Simpson’s index of diversity  sampled . For thee) and Nei’s index of diversity were used to measure the genetic variation among particular taxa using AFLPSURV 1.0 software [The expected heterozygosity , 60 plants of F. × bohemica  and 20 plants of F. sachalinensis (from heterogeneous stand the Jasieniczanka River) using FCM. Vicia villosa cv. ‘Minikowska’  was used as the internal standard. Nuclear samples were prepared as was previously described [2 isolation buffer  Triton X-100) supplemented with 1% (w/v) PVP-10, propidium iodide  and ribonuclease A (50 μg ml-1). For each sample, 3000–5000 nuclei were analysed immediately after preparation using a CyFlow SL Green  flow cytometer, equipped with a high-grade solid-state laser with a green light emission at 532 nm, a long-pass filter RG 590 E, DM 560 A as well as with side (SSC) and forward (FSC) scatters. Histograms (CV = 3.23–5.41) were evaluated using FloMAX software . Nuclear DNA content was calculated using the linear relationship between the ratio of the Fallopia/Vicia peak positions on the histogram of fluorescence intensities. Additionally, the 1Cx (DNA content of one non-replicated monoploid genome with chromosome number x) was calculated.The genome size in the young leaves of escribed  using a F. japonica, F. × bohemica and F. sachalinensis selected from Polish populations were collected in ice water. They were then pretreated with 2 mM 8-hydroxyquinoline for 4 h, fixed in ethanol with glacial acetic acid  and then stored at -20°C until use. Fixed roots were washed in a 0.01 M citric acid-sodium citrate buffer (pH 4.8) and digested in an enzyme mixture of 20% (v/v) pectinase (Sigma), 1% (w/v) cellulase  and 1% (w/v) cellulase ‘Onozuka R-10’ (Serva) for 2–2.5 h at 37°C. A single root tip was washed in cold distilled water and transferred into a drop of 45% acetic acid on microscope slide and squashed. The coverslips were removed after freezing and slides were air-dried. Chromosome analysis was carried out on 3–5 well-spread metaphases. Each chromosomal preparation was derived from a different single root tip, thus each preparation corresponded to one individual.The root tips of ten individuals of BamHI sub-clone of the 5S rDNA from the wheat clone pTa794 [ClaI sub-clone of the 26S rDNA coding region of Arabidopsis thaliana [Two kinds of probes were used–(i) 5S rDNA probe was generated by the PCR amplification of a 410-bp e pTa794  and labethaliana  with digThe pre-treatment and denaturation of chromosome slides subjected to the FISH experiments were carried out as follows: RNase treatment (37°C for 1 h), 0.1× SSC at room temperature for 1 min, incubation of slides in 2× SSC at 65°C for 20 min, 0.1× SSC at room temperature for 1 min, denaturation of slides in 0.07 N NaOH at room temperature for 1 min, 0.1× SSC at 4°C for 1 min, 2× SSC at 4°C for 1 min and then the slides were dehydrated in an ethanol series at room temperature  and flowers of both sexes (Jasieniczanka River–heterogeneous stand). We also recorded the presence of both sexes of F. sachalinensis–females and hermaphrodites were observed at the Jasieniczanka River. No flowers were observed in the Fallopia japonica and F. sachalinensis populations from Japan at the time the material for the presented study was collected.The types and fertility of flowers were recorded for each Fallopia populations from the introduced and native ranges based on AFLP markers and ten selective primer combinations resulted in a total of 679 loci of good quality and 323 (47.6%) of these were polymorphic across all of the samples in the study. These primer combinations generated 150 552 bands and the majority of those exhibited a consistent pattern in both technical replications of the AFLP analysis. Only 21 bands produced ambiguous results, resulting in an error rate of 0.0139%.The analysis of genetic diversity of the F. × bohemica population from the Jasieniczanka River in Poland was characterized by the occurrence of 17% polymorphic AFLP loci and the population of F. japonica from Itami in Japan had about 10% polymorphic loci. These values were also accompanied by the highest values of the expected heterozygosity from among all of the populations studied  was observed in m Poland . MoreoveF. × bohemica population from the Jasieniczanka River, and the lowest, only one genet, within the population of F. japonica from the Biała River  and the population of the same taxon located along the same river valley, but which was cohabiting with individuals of F. × bohemica. Comparable level of variation, of around 5%, was found between different populations of F. japonica, from both the Biała and the Jasieniczanka Rivers as well as between F. japonica from the Biała River (homogeneous stand) and F. × bohemica population from the same river valley. The highest diversity (around 10–11%) was noticed between F. sachalinensis from the Jasieniczanka River and all other taxa from invaded range.In the introduced range the lowest value of polymorphism, 2.76%, was observed between F. sachalinensis populations from Obira-cho and Toma-cho (3.75%). Higher polymorphism was present between F. japonica from Itami and Osaka (8.74%).Similar comparisons for the populations from native range showed that the less variation was observed between two F. japonica population from Itami and the F. sachalinensis population from Obira-Cho in Japan.The highest value of polymorphic loci, 25.22%, was observed between the F. japonica populations differ between each other on average in 10–12% of AFLP loci. The hybrid F. × bohemica from invaded range was more polymorphic in comparison to F. sachalinensis (19.81%) than to F. japonica from native range (11–13%). The highest polymorphism was noticed between F. sachalinensis populations from Poland and Japan  were the most similar (Nei’s genetic distance of 0.0158). A comparison of the Fallopia populations from Poland showed that the genetic distance between the different F. japonica populations, between the F. japonica and F. × bohemica populations or between different populations of F. × bohemica was similar and usually lower than the distance between the population of F. sachalinensis and any of the populations of remaining taxa   . ConsideF. × bohemica from that location using FAMD software. The results showed that the hybrid index for the majority of these individuals was near 0.5, thus indicating the equal genetic contribution of the parental alleles in the genomes of the hybrid .Because a stand from the Jasieniczanka River was composed of three taxa cohabiting together, the calculation of the maximum likelihood-based hybrid index was performed for the individuals of e hybrid . Some shPartitioning of the molecular variance using hierarchical AMOVA in which all of the populations were divided into two groups based on their range (introduced or native) showed that the majority of the variance (about 65%) was explained at the among-population level. An important part of the observed variance (about 22%) was contributed by groups. The smallest amount of the observed variance resulted from the diversity within each population . When poFallopia species  with only 3% hexaploid (2C = 6.45 pg). Ninety-eight per cent of the F. × bohemica plants were hexaploid (2C = 6.46 pg) and only one plant out of 59 was octoploid and had 2C = 8.96 pg DNA. The 1Cx DNA content of F. sachalinensis was 1.00 pg and it differed slightly for the other two species depending on the ploidy; it was 1.08 pg for hexaploids and 1.11–1.12 pg for octoploids.FCM analysis and chromosome counts confirmed the occurrence of hexaploid and octoploid plants among the  species . It reveFallopia species , were located in the proximal part of the chromosomes  where this taxon cohabitates with both of its parental taxa. Because this population contained multiple genotypes and the level of clonality was very low, it raises the possibility that sexual reproduction may take place at this stand. The values of the hybrid index calculated for the individuals from this population suggest that most of them were formed de novo after the hybridization events of the parental taxa. The observations of the types of flowers produced by those taxa also allow for such a possibility and suggest that some individuals of F. sachalinensis were the donors of pollen for the fertilization of F. japonica flowers, which is the general way in which the F. × bohemica hybrid is formed in the invaded range [F. × bohemica hybrid may be possible, which may result from the pollination of F. japonica flowers by F. × bohemica or by sexual reproduction among F. × bohemica individuals. These events may possibly be accompanied by the elimination of some part of the F. sachalinensis chromosomes, thus resulting in progeny with an allelic composition that is skewed towards the F. japonica parent  were found. The presence of tetraploids, however, was observed within the F. sachalinensis taxon in our preliminary study of a population from the Jasieniczanka River. That preliminary study was limited to seven individuals and was not combined with AFLP analysis. However, it resulted in the identification of one tetraploid and six hexaploid plants (data not shown). This finding is in contrast to the F. sachalinensis ploidy in Czech populations, which are predominantly tetraploid with some representatives of hexa- and octoploid forms [F. japonica individuals were octoploid and the F. ×bohemica hybrids were predominantly hexaploid. It is probable that the hexaploid F. × bohemica originated after the hybridization of the 8x F. japonica and 4x F. sachalinensis parents, although the occurrence of 4x F. sachalinensis individuals appeared to be very rare in the population in this study. This observation also raises the question on the possible advantage of a hexaploid constitution of F. sachalinensis in the habitat that was studied.The chromosome number of the 6 and 88 . In thisid forms . In the F. japonica analyzed here, only two hexaploid plants (encoded PL_CDJ_FJ_13 and PL_CDJ_FJ_18) occurred in the octoploid population. Since no hexaploid forms of this species were found previously and the 1Cx of those plants was the same as that of F. × bohemica , it can be assumed that they were hybrids rather than F. japonica. Similarly, the single octoploid plant (encoded PL_CDJ_FB_13) that was found in the hexaploid population of F. × bohemica, which had 1Cx = 1.12 pg, seems to belong to another taxa, possibly F. japonica. This finding points to the genetic identity of these individuals as measured by AFLP analysis. The comparison of their fingerprints with other samples shows that they represent genets that differ from all of the other individuals from the taxa under study. In a dendrogram, the PL_CDJ_FJ_13 and PL_CDJ_FJ_18 individuals are grouped together within the F. japonica cluster from the Jasieniczanka River, although they are placed farther from other individuals of this population. Two individuals from F. × bohemica are located at a short genetic distance from this cluster, which may indicate their relatively close genetic kinship. It may also be evidence of advanced backcrossing events between an initial hybrid and its parental species, in this case F. japonica. This could result in a common cytological constitution of Polish F. × bohemica plants being retained with some admixture of F. japonica loci, which was also reflected at the phenotypic level. A similar explanation may be the case for the PL_CDJ_FB_13 individual from the Jasieniczanka River population. Its morphology resembles F. × bohemica, but its genome size is closer to F. japonica and the dendrogram shows that it is slightly more distant from the other F. × bohemica individuals from the Jasieniczanka River population and its genet is also different than all of the other individuals from the taxa studied.In populations of Fallopia taxa [F. japonica and F. × bohemica individuals and also a group of F. × bohemica that is close to the F. japonica individuals. The latter group may represent successive generations of Fallopia amphiploids or introgression [Fallopia complex, including a comparison of resynthesized (newly formed) and relatively recent Fallopia allopolyploids, might be necessary to address the question of whether genome changes following allopolyploidy are random or whether there is a predisposition to change of individual loci.Other reports point to the existence of a heterogeneous constitution of the hybrids  and showpia taxa . Such grgression , which mFallopia taxa have small and morphological undifferentiated chromosomes makes an analysis of karyotype morphology difficult. As indicated for other genera, the application of FISH with repetitive sequences is often useful in cytogenetic analysis [Fallopia taxa. The chromosome complement of the three Fallopia taxa delivered four to ten chromosomes with a locus 5S rDNA and six chromosomes with a locus 35S rDNA. None of chromosomes identified bear the fraction of rDNA at which both rDNA loci were colocalized. Double-FISH with 5S and 35S rDNA provided markers for 10–16 out of 66–88 chromosomes of the Fallopia taxa. A comparative study of the number and position of chromosomal rRNA genes showed some similarity between all of the genomes that were studied; this early stage of Fallopia comparative cytogenetics might indicate a complex phylogenetic relationship (origin) of the Fallopia taxa. It is worth mentioning that only three pairs of 35S rDNA-bearing chromosomes were observed in the F. japonica individuals (2n = 8x = 88). We would expect eight 35S rDNA loci to be present since each subgenome (1x) harbours a single rDNA locus. One possible interpretation is that a homozygous deletion of two 35S rDNA loci occurred during or after the autoalloploidization of the F. japonica octoploid stabilization.The fact that analysis . To the F. × bohemica plants, the rDNA loci polymorphism might be a proof of its spread via clonal reproduction, [F. × bohemica genome-like chromosomes. This genome exhibited the same number of 35S rDNA loci that were observed in both parental species, although some variation in the number and position of 5S rDNA loci were revealed. Locus loss has previously been detected after allopolyploid and autopolyploid formation [F. × bohemica hybrid plants. The rapid loss or de novo generation of NORs affects the homogenization of rDNA in natural hybrids [F. × bohemica hybrid plants might also be mediated by the uniparental elimination of (r)DNA, which may be part of a broader process of DNA deletion that is induced by interspecific hybridization [Brassica species [F. japonica octoploid individuals); the deletion of a few pairs of 5S rDNA loci probably also occurred in F. × bohemica. In the F. sachalinensis and F. × bohemica genomes, the main 5S rDNA loci are embedded next to centromeric heterochromatin, which is typically rich in transposable elements, and therefore a transposon-mediated rearrangement might also contribute to the loss or transposition of 5S rDNA sequences [F. japonica. This suggests chromosome rearrangements resulting from a genome imbalance during polyploid formation as was recently shown in Tragopogon allotetraploids [Fallopia plants, so further studies are required for critical FISH analyzes on the variation patterns of rDNA loci.In the case of the duction, , but thiduction, ,71, whatormation , althougormation . Variatiormation ,74,75, tormation , i.e. a  hybrids . The unedization , especia species ,80, thusequences ,82 that Fallopia complex. Based on a genetic diversity study, we found that significant differences exist between the populations from both the invaded and native ranges. The Polish populations of the study are characterized by a different reproduction strategy with a very high level of clonality compared with the native populations. Our cytological studies show the occurrence of genome rearrangements in F. ×bohemica as indicated by the rDNA-FISH analysis where the gain or loss of rDNA loci together with some rDNA movements might be a possible trend of chromosome evolution in this genus. We also found a relatively high uniformity of genome ploidy within the taxa studied with only a few exceptions that may represent successive generations of hybrids or introgression forms of the parental species F. japonica or F. sachalinensis in the background of F. ×bohemica. The findings of the presented study open another questions on genome composition and its evolution within the Fallopia complex. They relate to local phenomenon such as the occurrence of F. sachalinensis in the heterogeneous stands and its influence on the overall genetic diversity of taxa that cohabitate together. An interesting question also relates to the details of the genomic changes in F. ×bohemica assuming that this taxon retains the ability to reproduce sexually and if so, what are the precise factors that may limit such sexual reproduction and move the population towards clonal spread.The combination of several molecular methods used in the presented study made it possible to give a broader picture of the genetics and evolution of the invasive S1 TableX–primer combinations used in the analysis of heterogeneous and homogeneous populations.(PDF)Click here for additional data file.S2 Table(XLS)Click here for additional data file.S3 TableFallopia japonica; FB–F. ×bohemica; FS–F. sachalinensis. a The levels of polymorphism were calculated after pairwise analyses of individuals from each taxon.Codes of populations–see (PDF)Click here for additional data file.S4 TableF. sachalinensis parental species; expected h = 1 for F. japonica parental species; lnL–likelihood values.Expected h = 0 for (PDF)Click here for additional data file.


Danaus plexippus (L.), from Mexico were assessed based on analyses of mitochondrial cytochrome c oxidase subunit I  and subunit II  gene segments and 7 microsatellite loci. The sample of 133 individuals included both migratory monarchs, mainly from 4 overwintering sites within the Monarch Butterfly Biosphere Reserve (MBBR) in central Mexico (states of Michoacán and México), and a nonmigratory population from Irapuato, Guanajuato. Haplotype (h) and nucleotide (π) diversities were relatively low, averaging 0.466 and 0.00073, respectively, for COI, and 0.629 and 0.00245 for COII. Analysis of molecular variance of the COI data set, which included additional GenBank sequences from a nonmigratory Costa Rican population, showed significant population structure between Mexican migratory monarchs and nonmigratory monarchs from both Mexico and Costa Rica, suggesting limited gene flow between the 2 behaviorally distinct groups. Interestingly, while the COI haplotype frequencies of the nonmigratory populations differed from the migratory, they were similar to each other, despite the great physical distance between them. Microsatellite analyses, however, suggested a lack of structure between the 2 groups, possibly owing to the number of significant deviations from Hardy–Weinberg equilibrium resulting from heterzoygote deficiencies found for most of the loci. Estimates of demographic history of the combined migratory MBBR monarch population, based on the mismatch distribution and Bayesian skyline analyses of the concatenated COI and COII data set (n = 89) suggested a population expansion dating to the late Pleistocene (~35000–40000 years before present) followed by a stable effective female population size (Nef) of about 6 million over the last 10000 years.Population genetic variation and demographic history in  Danaus plexippus (L.)  are best known for their impressively long distance annual migrations (up to ~4000 km) from their summer feeding and breeding ranges in the eastern USA and southern Canada to overwintering sites in the Monarch Butterfly Biosphere Reserve (MBBR) in the mountains of central Mexico (states of Michoacán and México), their huge numbers (tens to hundreds of millions) in the overwintering sites, and their return migration north in the spring after breaking reproductive diapause (Asclepias spp. (Apocynaceae), and their incredible migratory ability hold clues to basic biological processes observed in a wide range of organisms. Monarchs from western USA also undergo a shorter migration from the Rocky Mountain region to overwintering sites along the California coast   . In addigratory) .D. plexippus from eastern North America has experienced large declines in recent years  and 503 bp of the mitochondrial c cytochrome oxidase subunit II gene (COII), together with analysis of a subset of the microsatellite loci developed for worldwide genetic studies in monarchs  What are the levels of genetic variability in monarchs from the major overwintering sites in the reserves and other sites in Mexico and elsewhere in North America? 2) To what degree are the migratory and nonmigratory populations genetically connected? 3) What can genetics tell us about their effective population sizes and historical demography? 4) Which genetic markers appear to be the most informative? We address these questions here by analyzing data obtained from direct sequencing of 658 base pairs (bp) of the barcode region of the mitochondrial cytochrome monarchs . We alsoms BOLD;  in our aD. plexippus are currently recognized from mainland North America , northern South America and the Caribbean, although 2 of these assignments are questionable , and then distinguish between “migratory” and “nonmigratory” populations. Although “nonmigratory” D. plexippus from Costa Rica are known to undergo short regional migrations  of migrating butterflies was also obtained on 31 October and 1 November, 2015 from a threatened coastal sand dune habitat near Guaymas, Sonora  and 2 in the state of Mexico  , were sa, Sonora . Butterf GenBank  and BOLD GenBank .Total genomic DNA was extracted from 2 legs of each butterfly using the DNeasy™  protocol. The polymerase chain reaction (PCR) was used to amplify the 2 mitochondrial gene segments using primer pairs and standard assay conditions described previously for COI  and COIID. plexippus from the MBBR  in COI and 22.0% (n = 123) in COII. Together these results indicate that the barcode sequences analyzed represent mtDNA, and are not nuclear mitochondrial pseudogenes (numts).Translation of COI and COII sequences in MEGA version 5.0.5  revealedD. plexippus. The selected loci included 5 dinucleotide repeats  and 2 trinucleotide repeats (loci 1679 and 165). In the MBBR sample (n = 27) of The DNA extract described above was used to amplify 7 of the polymorphic microsatellite markers developed by Primer sequences for each locus are given in Microsatellite genotypes were analyzed with the microsatellite plugin of Geneious software (Biomatters Limited) and Microsatellite Analyzer (MSA) version 4.05 . ObserveD and Fu’s (1997)FS, were carried out in ARLEQUIN. We analyzed COI and COII separately, as well as concatenated, in order to compare our results on COI with sequences of other nonmigratory populations from North America mined from GenBank and BOLD in which corresponding COII sequences were not available. The concatenated data set was used for tests of historical demography of migratory monarchs from the MBBR described below.Calculations of genetic diversity indices of COI, COII and the concatenated data set for MBBR samples were performed in DnaSP version 5.10.01 . NeutralTajima’s (1989)D. plexippus populations from Mexico and Costa Rica. For this analysis, we assumed that each of the 4 MBBR collection sites represented separate populations. The Guaymas population was omitted owing to small sample size (n = 5). In addition, only the 19 complete (656–658 bp) barcode sequences from Costa Rica listed in the ΦST), among localities within groups (ΦSC), and among the migratory and nonmigratory groups (ΦCT). The calculation of signiﬁcance of the ﬁxation indices ΦST, ΦSC, and ΦCT (α = 0.05) was based on 10000 permutations of the data matrix. Pairwise estimates of ΦST among populations, and estimates of number of migrants per generation (Nm) among populations assumed to be in mutation-drift equilibrium  the concatenated COI and COII data set from the 4 MBBR sites, and 3) the microsatellite data set from Mexico (excluding Guaymas).Hierarchical analysis of molecular variance AMOVA;  performeilibrium , were alHaplotype networks of both COI and COII sequences were constructed using statistical parsimony implemented in TCS version 1.21 . The conD. plexippus from the MBBR  which were collected during a single overwintering period. Changes in effective female population size (Nef) over time in monarchs from the MBBR were estimated with 1) analysis of the distribution of pairwise differences among haplotypes (mismatch distribution) (SSD) statistic and the raggedness statistic (rg), and their corresponding P values. The sudden expansion model is rejected at P < 0.05.For analyses of demographic history we used the concatenated COI and COII data set from ibution)  performeibution) . For theμ) of 2.9 × 10−9 based on an estimated 2.3% pairwise sequence divergence rate per million years for mtDNA  sampling of sequence data to estimate a posterior distribution of effective population size through time . The modor mtDNA  and 4 geor mtDNA .http://dx.doi.org/10.5061/dryad.64m4n (All unique haplotypes of the new COI and COII sequences reported here are available in GenBank (accession numbers KX110357–KX110382). Microsatellite genotypes have been deposited in Dryad: ad.64m4n .h) and nucleotide diversity (π) in the COI gene segment were generally low in each of the 7 populations sampled from Mexico, with overall values of h = 0.466 and π = 0.00073 in the combined sample . Three of the 6 haplotypes  were singletons each consisting of a single nucleotide substitution. All nucleotide substitutions were synonymous third codon position transitions.Haplotype diversity , only 6n = 7) of nonmigratory monarchs from 7 sites on the Yucatán Peninsula, Mexico  was 74% but was reduced to approximately 22% in the nonmigratory populations from Costa Rica, Irapuato, Mexico, and the Yucatán Peninsula, Mexico (n = 37).A comparison of the frequencies of COI haplotypes 1 and 2 for all samples analyzed from Mexico and Costa Rica is shown in tana Roo ) locatedh = 0.629; π = 0.00245; n = 123) was higher than that seen in COI, and was comprised of 11 variable sites and 10 haplotypes  reflected the higher diversity values present in COII. Neutrality tests were not significant for the separate or concatenated gene segments (Genetic diversity of the concatenated COI and COII data set (segments .D. plexippus from Mexico is shown in n = 200) and their sample from Mexico (n = 27) each showed significant deviation from HWE at locus 165. Overall, however, 8 of the 11 loci analyzed in detail by Variation in the 7 microsatellite loci over all populations of Microsatellite analyses of the 7 loci in each of our 7 populations from Mexico, together with results of D. plexippus from Mexico and Costa Rica showed significant population structure , with 38.61% of the genetic variation distributed between migratory and nonmigratory groups  . The remaining 60% of the genetic variation was found within populations.The hierarchical AMOVA of the combined migratory and nonmigratory COI data set of = 0.047) . Less thΦST suggested no significant structure among the migratory populations in Mexico, or between the 2 widely separated nonmigratory populations in Irapuato, Mexico, and Costa Rica  of the 5 values of ΦST for Irapuato and the migratory populations in Mexico were significant. Nonetheless, the 3 nonsignificant values of ΦST in Irapuato were relatively high and similar to those seen in Costa Rica. In addition, P values of the 3 ΦST values were < 0.05. All estimates of Nm (0.32–1.64 migrants per generation) between migratory and both nonmigratory populations were low . Pairwise comparisons of ΦST again suggested no significant structure among the migratory populations in Mexico  from central Mexico, indicated that 10% of the genetic variation occurred among populations and that significant structure was present  was not significant (P = 0.15). None of the pairwise comparisons of ΦST among the 4 MBBR sites was significant (not shown).The AMOVA of the concatenated data set from the 4 MBBR sites indicated that most (97%) of the genetic variation occurred within populations and that the overall FST between the 6 populations of D. plexippus from Mexico, including the nonmigratory Irapuato population, were significant , however, was only slightly higher than the cutoff for significance using the Bonferroni correction (P = 0.0033). An analysis of the discrepancies in results of population structure between mtDNA and microsatellite markers is provided in the Discussion.In contrast to the mtDNA results, but in agreement with nificant , indicatSSD  and rg  were small and not statistically signiﬁcant at the 0.05 level indicating that the sudden expansion model could not be rejected. The value found for τ, the time to the population expansion, where τ = 2ut, and u is the mutation rate for the entire gene segment, and t is the number of generations since the expansion (−8) or 1.335 × 10−5. Based on these values, and assuming 4 generations per year, the estimated time to the population expansion in D. plexippus  was 34597 (5084–61742) years ago.A plot of the distribution of pairwise differences among haplotypes in the concatenated data set  showed rxpansion , was 3.6xpansion , the meaNef) in D. plexippus beginning about 40000 years before present, which then remained stable over the last 10000 years at a median estimate of about 6 million individuals. The estimated date of the beginning of the population expansion agrees remarkably well with that obtained from the mismatch distribution. Although large negative FS values are also typically found in expanding populations .Bayesian skyline analysis  showed aulations , the negD. plexippus across the overwintering sites in central Mexico, which is consistent with other genetic studies utilizing a variety of markers showing panmixia in the entire North America migratory population of monarchs . The apparent lack of structure between nonmigratory and migratory populations suggested by microsatellites is in contrast to the evidence for structure from mtDNA (present study) and whole genome analysis  and USA (n = 3) all possessed haplotype 1. Haplotype 1 also is the only haplotype currently recorded in individuals from Australia (n = 7) and Spain (n = 4), countries which were colonized by migratory monarchs  of the COII gene segment among migratory monarchs in Mexico (haplotype A predominates) and the nonmigratory Irapuato population (haplotype B predominates) which contributes to the significant population structure observed among the 2 groups . Further America . This inD. plexippus to central Mexico (n = 5), data on this population are important because so little is known about migratory corridors in Sonora and possible interactions among monarchs in other populations found farther south. As shown in Abronia maritima Nutt. ex S. Watson (Nyctaginaceae), and desert palafox Palafoxia arida B.L. Turner & Morris (Asteraceae).We have included several migratory monarchs from Guaymas, Sonora, Mexico in this study. Coastal Sonora is roughly 1000 km west of the fall migratory route of eastern USA populations of l Mexico . Monarchl Mexico . AlthougD. plexippus from the MBBR dating to the late Pleistocene (35000–40000 before present) are in general agreement with results obtained from whole genome analysis which showed a population expansion in monarchs dating to about 20000 years ago (μ = 8.4 × 10–9) were slightly different than we used. The relatively stable, long-term pre-expansion effective population female size (Nef) of about 200000 shown in Ne = about 400000) for the North American population, assuming an equal sex ratio, although estimates of effective population sizes after the expansion differed in the 2 studies. The lack of pre-expansion population fluctuations in the distant past , which are typically lower than census population sizes, are important parameters in conservation biology in that they provide a means to evaluate threats to the genetic health of populations (Ne utilizing a variety of methods (Ne, but in the potential loss of the genetic diversity so critical to conservation management and planning.Reliable estimates of effective population size (ulations . Also, uulations , emphasi methods . A recen methods  can be e


DNA (mtDNA) d‐loop region in 309 individuals from 13 populations, we reveal the maternal genetic variation and demographic dynamics of Ethiopian indigenous goats. A total of 174 variable sites that generated 231 haplotypes were observed. They defined two haplogroups that were present in all the 13 study populations. Reference haplotypes from the six globally defined goat mtDNA haplogroups show the two haplogroups present in Ethiopia to be A and G, the former being the most predominant. Although both haplogroups are characterized by an increase in effective population sizes (Ne) predating domestication, they also have experienced a decline in Ne at different time periods, suggesting different demographic histories. We observed seven haplotypes, six were directly linked to the central haplotypes of the two haplogroups and one was central to haplogroup G. The seven haplotypes were common between Ethiopia, Kenya, Egypt, and Saudi Arabia populations, suggesting common maternal history and the introduction of goats into East Africa via Egypt and the Arabian Peninsula, respectively. While providing new mtDNA data from a historically important region, our results suggest extensive intermixing of goats mediated by human socio‐cultural and economic interactions. These have led to the coexistence of the two haplogroups in different geographic regions in Ethiopia resulting in a large caprine genetic diversity that can be exploited for genetic improvement.The Horn of Africa forms one of the two main historical entry points of domestics into the continent and Ethiopia is particularly important in this regard. Through the analysis of mitochondrial  These have been further categorized into four family groups based on geographic location, and two production systems across three agro‐ecological zones , a large number of which are of indigenous types kept mainly for subsistence. They inhabit a wide range of habitats and production systems ranging from the cool highlands to hot arid lowland environments , C, D, F, and G) with a weak phylogeographic structure have been reported in domestic goats  was amplified using nested PCR , 0.2 μM of each primer, 1.5% Hi‐Di™ formamide , 0.005 mg of Bovine Serum Albumin (ThermoScientific), and 50 ng of template DNA. A two‐stage touchdown PCR involving an initial denaturation at 95°C for 3 min followed by the first stage of amplification of five cycles , and a second stage that involved the same profile but 30 cycles of amplification and an annealing temperature of 53°C, was performed. A final extension step at 72°C for 7 min completed the PCR. The PCR products were purified using the QIAquick® PCR Purification Kit  following the manufacturer's instructions. The purified products were sequenced using the BigDye Terminator v3.1 Cycle Sequencing Chemistry (Applied Biosystems) and the ABI Prism 3130XL automatic capillary sequencer  following the manufacturers protocols.The entire mtDNA CR Table . The PCR2.3C. hircus reference sequence (Genbank accession number GU223571). We generated 309 sequences and determined the haplotypes with DnaSP v5 . The sequence fragments were edited manually using MEGA 6  network . All the mutations and character states were weighted equally. To visualize the variation in Ethiopian goats in the context of the global caprine diversity, 229 sequences of domestic goats from 20 countries and representing the six globally defined mtDNA d‐loop haplogroups  were retrieved from the Genbank .To visualize the genetic relationship between individuals and populations, a phylogenetic tree was constructed using all the haplotypes generated in Ethiopian goats with the neighbor‐joining (NJ) algorithm implemented in MEGA6. The level of confidence associated with each bifurcation was evaluated with 1,000 bootstrap replications. To obtain further insights into the genetic relationships between the haplotypes and determine the number of distinct mtDNA nk Table  and inclviz (i) assuming no clusters in the dataset, (ii) between the three groups of populations as defined by FARM‐Africa (ϕ) statistics representing haplotype correlations at various hierarchical levels  were calculated. Levels of significance of the variance components associated with the hierarchical clusters were evaluated with 1,000 nonparametric bootstrap coalescent simulations in Arlequin v3.5.To partition genetic variation among populations and groups of populations, analysis of molecular variance (AMOVA) was performed following 1,000 permutations in Arlequin v3.5. The analysis was limited to Ethiopian goats and various hierarchical clusters were tested M‐Africa , and  from the simulated model of expansion  following 1,000 coalescent simulations. To complement the mismatch distributions, Fu's FS . This analysis was limited to Ethiopian goats and the 481 bp HVI of the mtDNA d‐loop region. We calibrated the BSPs using the molecular rate of evolution (μ) of the HV‐I region in goat mtDNA d‐loop of 2.73 × 10−7 substitutions/site/year (s/s/yr) following Nomura et al. , 174 variable sites  were observed. These defined 231 haplotypes  sequences of Ethiopian goats and 229 HV‐I haplotypes retrieved from the GeneBank and representing the six main haplogroups observed in goats to construct a NJ tree to assess genetic relationships. The NJ tree revealed three well‐resolved clusters; two were specific to Ethiopian indigenous goats and the third one clustered together the haplotypes representing haplogroups B, C, D, F, and wild  bp seques Figure . We also3.3AMOVA analysis incorporating the 13 populations assuming no hierarchical clusters, as well as, the three groups proposed by FARM‐Africa  showed t3.4D and Fu's FS statistics; both were negative and significant for each population, with the exception of Abergelle and Gondar whose Tajima's D was negative but not significant, for the global dataset of 13 populations, and for the two haplogroups. The bimodal peaks observed in the two haplogroups were surprising and unexpected. We therefore counterchecked all the sequences against their respective chromatograms for base calling errors. The sequences turned out to be correct and there was no mix‐up between the sequences used to generate the datasets for the mismatch distribution analysis. The bimodal peaks also appear not to be a unique feature of haplogroups A and G in Ethiopian goats. We also observed two peaks in haplogroup A in Sudanese goats. Chen et al.  over time by generating BSP's for the two haplogroups . This average value is similar to that of Iberian (0.996) and European (0.994) goats but its higher than that of Sicilian (0.806–0.969), South and Central American (0.963), Atlantic  exceeding those observed in other populations  and edges (n = 335) that represent subpopulations and population subdivisions, respectively  that lacked a clear phylogeographic structure. As observed in other populations, haplogroup A was the most diverse and geographically widespread. Human‐mediated translocations through commercial trading, socio‐cultural exchanges, and seasonal migrations in search of forage and water resources could explain the lack of phylogeographic structure. The initial introduction of the two haplogroups and their subsequent intermixing has created a genetic treasure‐trove of caprine genetic diversity that can be exploited in breeding programs aimed at improving the species.None declared.KT, OAM, AD, TD, and JB conceived the study. GM did the sampling; OAM, AD, TD, and JB provided guidance during sampling and laboratory analysis; GM, SO, NZ, AA, GA, JT, CM, and JN performed laboratory analysis and data management. GM analyzed the data; JMM guided data analysis and interpretation; GM and JMM wrote the manuscript. All authors read and approved the final manuscript.All the animals used in this study are owned by small‐scale farmers and pastoralists in Ethiopia. Prior to sampling, the objectives of the study were communicated to all the farmers, in their local languages for ease of understanding, following which permission was obtained to sample the animals. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.


Pinctada margaritifera were examined across its ~18,000 km Indo-Pacific distribution.Genetic structure in many widely-distributed broadcast spawning marine invertebrates remains poorly understood, posing substantial challenges for their fishery management, conservation and aquaculture. Under the Core-Periphery Hypothesis (CPH), genetic diversity is expected to be highest at the centre of a species’ distribution, progressively decreasing with increased differentiation towards outer range limits, as populations become increasingly isolated, fragmented and locally adapted. The unique life history characteristics of many marine invertebrates such as high dispersal rates, stochastic survival and variable recruitment are also likely to influence how populations are organised. To examine the microevolutionary forces influencing population structure, connectivity and adaptive variation in a highly-dispersive bivalve, populations of the black-lip pearl oyster Fst = 0.2534–0.4177, p < 0.001), compared to Pacific Ocean oysters, where basin-wide gene flow was much higher . Partitioning of genetic diversity  attributed 18.1% of variance between ocean basins, whereas greater proportions were resolved within samples and populations (45.8% and 35.7% respectively). Visualisation of population structure at selectively neutral loci resolved three and five discrete genetic clusters for the Indian and Pacific Oceans respectively. Evaluation of genetic structure at adaptive loci for Pacific populations , revealed five clusters identical to those detected at neutral SNPs, suggesting environmental heterogeneity within the Pacific. Patterns of structure and connectivity were supported by Mantel tests of isolation by distance (IBD) and independent hydrodynamic particle dispersal simulations.Analyses utilising 9,624 genome-wide SNPs and 580 oysters, discovered differing patterns of significant and substantial broad-scale genetic structure between the Indian and Pacific Ocean basins. Indian Ocean populations were markedly divergent  contains supplementary material, which is available to authorized users. C) versus marginal (M) populations [C-M) hypothesis, also known as the core-periphery hypothesis . Whi. WhiC) vis  and gene flow (m) should be highest at the centre, and lowest at range margins. Consequently, central populations are expected to be less genetically differentiated and possess higher levels of genetic diversity, than those existing at range margins [C-M cline, local adaptation may be observed between populations existing at the core and range peripheries.It is expected that the interplay of microevolutionary forces, , will largely determine the magnitude and extent of population structure and connectivity, although the spatial distribution and demographic characteristics of the species could also exert strong influences , 6. The us topic , 9. One  margins , 7. FurtC-M genetic patterns in terrestrial taxa [C-M patterns compared to terrestrial taxa may differ from expectations under the CPH, as the homogenising influence of gene flow may maintain high connectivity across the C-M cline [While several studies have examined ial taxa , 10, comial taxa , and marial taxa . Marine ial taxa –16. Cons-M cline . FurtherC-M trends [Among marine invertebrates, species which are either completely sessile as adults , or possess very limited mobility , present additional challenges for assessment of M trends , 18. As M trends , 20. FurM trends , 22.C-M patterns involving terrestrial taxa, include examination of the geographical direction of the periphery studied, latitudinal effects, the effects of species-range geometry (e.g. shape and size), as well as sampling strategy [Given the complex nature of the biological and environmental influences at play, it is important to consider multiple sources of information for range-wide investigations in the marine environment, particularly when the species being examined is extensively distributed across heterogenous habitats. Considerations that have been highlighted in previous analyses of strategy , 5, 10. strategy , 23–26.Incorporation of environmental data such as dispersal modelling into range-wide studies is capable of offering unprecedented insights into larval dispersal limits , 27–29, Pinctada margaritifera (Pteriidae), is a marine bivalve mollusc that has a broad Indo-Pacific distribution , Cook Islands and French Polynesian individuals as P. m. var. cummingi , and all Central and Western Pacific specimens as P. m. var. typica . Indian Ocean populations are represented by P. m. var. persica , P. m. var. erythraensis  and P. m. var. zanzibarensis .Fig. 1MP. margaritifera at nuclear markers , at various sites in the Western and Central Pacific [Significant genetic heterogeneity has been reported for  Pacific , 42, 47, Pacific . More re Pacific . Since t Pacific , and useP. maxima, which is consistent with CPH assumptions. However, the natural distribution of this species is considerably less extensive than that of P. margaritifera [P. margaritifera is the Akoya pearl oyster, currently recognised as the P. fucata/martensii/radiata/imbricata species complex [Previous studies of range-wide genetic structuring in Pteriid pearl oysters have produced mixed results. Lind et al.  reportedritifera , 35. A b complex . While t complex , 35, 51.P. margaritifera occurs over 26–30 days in captivity [P. margaritifera across the extent of its Indo-Pacific distribution spanning over 18,000 km, and compare our observations with expectations under the CPH and regional morphological subdivisions. Independent population genomic and hydrodynamic approaches were utilised to assess population genetic structure, adaptive variation and larval connectivity. Through the use of independent biological and environmental datasets, this work sheds light on the links between genetic structure, ecology and oceanography, to reveal how populations of a broadcast spawner can be organised and maintained in the marine environment.Larval development of aptivity , 53, howaptivity . The higP. margaritifera (n = 580) between 5 and 18 cm in dorso-ventral measurement (DVM) were collected from 14 sites across the species distribution , the Persian Gulf  and Post Office Island in the Abrolhos Islands group, Western Australia (n = 50). All Indian Ocean samples consisted of wild individuals with the exception of the Abrolhos Islands collection, where oysters were hatchery-produced from wild-caught broodstock. In the Western Pacific, oysters were sampled from Checheng, Taiwan (n = 24), Nha Trang, Vietnam (n = 47) and Manado, Indonesia (n = 48). Central Pacific locations were represented by Kavieng, Papua New Guinea (n = 38), Gizo Island in the Solomon Islands (n = 50), the Great Barrier Reef, Australia (n = 35), the Fiji Islands (n = 61) and Tonga (n = 28). In the Eastern Pacific, oysters were collected from Manihiki Atoll in the Cook Islands (n = 45), and Arutua, French Polynesia (n = 50). All Pacific Ocean samples consisted of wild oysters, with the exception of the Cook Islands and French Polynesian samples that were sourced from pearl farm stocks.Adult and juvenile Proximal mantle and adductor muscle tissues  were removed and transferred to tubes containing 20% salt saturated dimethyl sulfoxide  preservative . GenomicP. margaritifera. Briefly, genome complexity reduction was achieved with a double restriction digest, using a PstI and SphI methylation-sensitive restriction enzyme (RE) combination, in a joint digestion-ligation reaction at 37 °C for 2 h with 150–200 ng gDNA. Because P. margaritifera like other bivalve species is highly polymorphic [Diversity Arrays Technology (DArT PL) proprietary genotyping by sequencing (DArTseq™) reduced-representation libraries were prepared as described by Kilian et al.  and Sansymorphic , 60, higymorphic .PstI-compatible (forward) adapter incorporated an Illumina flowcell attachment region, sequencing primer sequence and a varying length barcode region [SphI cut-site overhang. Samples were processed in batches of 94, with 15% of all samples in a batch randomly selected for replication, to provide a basis for assessing region recovery and genotyping reproducibility. Target “mixed” fragments [SphI and NlaIII cut-sites were selectively amplified using custom designed primers for each sample, under the following PCR conditions: initial denaturation at 94 °C for 1 min, then 30 cycles of 94 °C for 20s, 58 °C for 30s and 72 °C for 45 s, followed by a final extension step at 72 °C for 7 min. Amplified samples were subsequently cleaned using a GenElute PCR Clean-up Kit , on a TECAN Freedom EVO150 automated liquid handler.Custom proprietary barcoded adapters (6–9 bp) were ligated to RE cut-site overhangs as per Kilian et al. , with the region , 62. TheTo examine fragment size concordance and digestion efficiency, all samples were visualised on a 0.8% agarose gel stained with EtBr, and quantified using the ImageJ software package . SamplesRaw reads obtained following sequencing were processed using Illumina CASAVA v.1.8.2 software for initial assessment of read quality, sequence representation and generation of FASTQ files. Filtered FASTQ files were then supplied to the DArT PL proprietary software pipeline DArTtoolbox, which performed further filtering, variant calling and generated final genotypes in sequential primary and secondary workflows . Within The secondary workflow employed the DArTsoft14 and KD Compute packages along with the DArTdb database, to identify polymorphisms by aligning identical reads to create clusters across all individuals sequenced. These clusters were then catalogued in DArTdb, and matched against each other to create reduced-representation loci (RRL), based on their degree of similarity and size. SNP and reference allele loci were identified within clusters and assigned the following DArT scores: “0” = reference allele homozygote, “1” = SNP allele homozygote and “2” = heterozygote, based on their frequency of occurrence. To ensure robust variant calling, all monomorphic clusters were removed, SNP loci had to be present in both allelic states (homozygous and heterozygous), and a genetic similarity matrix was produced using the first 10,000 SNPs called to assess technical replication error , and excOnce SNP markers had been confidently identified, each locus was assessed in the KD Compute package for homozygote and heterozygote call rate, frequency, polymorphic information content (PIC), average SNP count, read depth and repeatability, before final genotype scores were supplied by DArT PL. Following the receipt of genotype data from DArT PL, the dataset was further filtered to retain only a single, highly informative SNP at each genomic locus. This was achieved by filtering out duplicate SNPs , according to call rate and Minor Allele Frequency (MAF). Subsequently, loci were screened for call rate, average Polymorphic Information Content (PIC), MAF and average repeatability, to retain SNPs suitable for population genomic analyses. All loci were then tested for departure from Hardy-Weinberg Equilibrium (HWE) using Arlequin v.3.5.1.3 , using aHo) and average expected heterozygosities corrected for population sample size (Hn.b.) were computed. Inbreeding coefficient (Fis) calculations and estimation of effective population size based on the linkage disequilibrium method (NeLD), were also carried out for each population, all using Genetix v.4.05.2 [R package Rhh [A) using the diveRsity [R package. The number of private alleles (Ap) was computed using HP-RARE v.1.0 [Ar, <5% MAF) was computed manually for each population.For assessment of genomic diversity within and between populations, allelic diversity indices including average observed  standard genetic distances (DS) between populations were also computed in Genetix v.4.05.2 with 10,000 permutations [R package adegenet 1.4.2 [divMigrate function of the R package diveRsity, utilising the Nei’s Gst method [Pairwise utations , along wge Poppr . The AMOge Poppr , based outations , and broet 1.4.2 –78. The et 1.4.2 , 80. To t method , 81.Fst outlier detection [To first create a selectively neutral dataset for population genomic analyses, a filtered dataset containing 10,683 SNP loci was used as the starting point for this step. Both BayeScan v.2.1 , 83 and etection –87. Candetection , 48, 88,Fst outlier tests were used to detect local adaptation, with population pairs tested at FDRs of 0.001, 0.005, 0.01, 0.05 and 0.1 and 0.2. However, testing for Fst outliers was restricted to populations sampled from the Pacific Ocean basin, as they were the least differentiated amongst themselves , while all Indian Ocean populations were significantly more divergent. Comprehensive descriptions of the settings used for both software packages were as per Lal et al. [R package GWASTools v.1.14.0 [Further population-specific l et al. , 48. Resv.1.14.0  was usedhttps://github.com/CyprienBosserelle/DisperGPU). Larvae of P. margaritifera remain in the plankton for 26–30 days prior to settlement [To independently evaluate larval connectivity using oceanographic data for comparison with population genomic analyses, larval transport pathways between sampling locations were simulated using the particle dispersal modelling software DisperGPU  data , 91. HYCmulation , 93, whex represents particle position (latitude and longitude), Δx is particle displacement during a time step Δt (which was set at 1 h), and up is the surface current speed at the location of the particle. K is the eddy diffusivity which takes account of the random displacement of the particle, due to turbulent eddies at a scale smaller than the hydrodynamics model resolution. K is calculated after Viikmäe et al. [Here e et al.  as folloEh is a horizontal turbulent diffusion coefficient, and RNA with RNB are normally distributed random numbers. The horizontal turbulent diffusion coefficient is unknown, but assumed to be 5 m2s−1 [up  were ree season , 99. ThiParticle positions were extracted at time intervals of 60 and 90 days post-seeding for the first and second spawning seasons respectively, per year, and particle displacement visualised using the Generic Mapping Tools package . Explicip < 0.009), and 99 loci were monomorphic across all 14 populations, which were subsequently removed together with 107 loci under significant LD (p < 0.0001). These steps collectively resulted in the retention of 10,683 SNPs  SNPs at genomic loci resulted in the removal of 8,079 SNPs (41% loss), after which the dataset was filtered for call rate (65%), average PIC (1%), MAF (2%) and average repeatability (95%). A total of 7 loci were found to deviate from HWE (A) and rare allelic richness  were similar, and appeared to vary by Ocean basin (Table A for Pacific Ocean populations ranged from 1.6256 (Cook Islands) to 1.8067 (Indonesia), whereas Indian Ocean populations produced values of 1.3934–1.5649 . Trends in the total numbers of private alleles (Ap) reflected the divergence between ocean basins and support very limited inter-basin gene flow, with more than 25% of total SNPs genotyped containing private alleles within each basin; . Within ocean basins, little difference  was seen among Pacific populations (Ap range of 188–205), while greater differences  were observed among the Abrolhos Islands, both Tanzanian, and Iranian sites .Patterns observed in the mean numbers of alleles per locus  than average expected heterozygosities for all populations), and displayed similar variability with the trends observed for A and Ar values. Pacific Ocean populations displayed generally higher values , than did Indian Ocean populations . These patterns also extended to individual average multi-locus heterozygosity (MLH) computations, and measurements of standardised heterozygosity (SH). Average MLH was relatively uniform within Pacific Ocean populations, ranging from 0.0844 (French Polynesia) to 0.1030 (Fiji Islands), which was mirrored in the SH results of 0.9777–1.2189 for the same populations respectively. Within Indian Ocean samples, oysters collected from Tanzanian and Iranian sites showed lower values , than animals sampled from the Abrolhos Islands .Average observed heterozygosities were significantly lower (Fis) values displayed a similar partitioning by region, with values for Pacific Ocean populations ranging from 0.5372 (Fiji Islands) to 0.6433 (Taiwan), while Indian Ocean animals , returned higher values from 0.6795  to 0.7008 (Iran). Very similar patterns were evident in related homozygosity by locus (HL) and internal relatedness (IR) multi-locus metrics , while Cook Islands individuals produced higher values . The lowest estimates were obtained from Abrolhos Islands oysters , indicating a possible bottleneck, as these animals were F1 hatchery-produced offspring of wild-caught parents.Inbreeding coefficient (Fst estimates (Table p < 0.001) for all population comparisons, with the exception of the two Tanzanian sites (0.0007), and PNG with the Solomon Islands (0.0059). A clear separation in population structure between ocean basins is evident, with pairwise estimates between sites all >0.25, ranging from Tanzania, Mtwara and Indonesia (0.2894), to Iran and the Cook Islands (0.4684). Within the Pacific, populations appear to be isolated by geographic separation, e.g. pairwise estimates for the GBR and Solomon Islands (0.0078) indicate greater homogeneity than more distant population pairs, such as the Cook Islands and Taiwan (0.1090). Higher degrees of separation are apparent within Indian Ocean populations, with pairwise estimates between Iran, and Mafia Islands with Mtwara being 0.2444 and 0.2534 respectively. The greatest level of differentiation among Indian Ocean sites was detected between the Abrolhos Islands and Iran (0.4177), with oysters from the Abrolhos Islands demonstrating greater similarity with Pacific populations (Abrolhos Islands and GBR pairwise Fst = 0.1311).Pairwise es Table  were higDS) described a similar pattern to the pairwise Fst estimates (Table p < 0.05). Partitioning between these populations however, was less evident, with DS = 0.071 and 0.074 respectively (Iran with Mafia Islands and Mtwara). Distances between all Pacific Ocean populations conversely indicated greater homogeneity, ranging from 0.005 , to 0.044 (Cook Islands with Indonesia and Taiwan pairwise comparisons). Oysters collected from the Abrolhos Islands were similarly differentiated, with DS = 0.056 when compared to GBR individuals, and up to DS = 0.082 with French Polynesian animals.Pairwise Nei’s standard genetic distances , as well as for all populations considered together , although additional sampling within each region is needed to confirm the strength of these results. Further Mantel tests within the two largest Pacific Ocean population groupings did not detect significant IBD patterns (p > 0.05). Visualisation of population structure with a DAPC (α-score optimised to retain 22 PCs), revealed clear differentiation between all Pacific Ocean, and both Tanzanian and Iranian populations , and between samples within populations (35.74%). Variation between populations within ocean basins was estimated at just 0.36%, indicating that genotypic variability at the individual oyster level accounted for the majority of the observed variation. Mantel tests indicated isolation by distance dispersal patterns both within each ocean basin . Among the 37 balancing loci, average Fst = −0.0066 (range = −0.0114 to −0.0031), demonstrating that diffuse population structure . Strong population structure was evident within and between both ocean basins, however, due to the relative isolation of populations between these regions, each is discussed separately.At a broad scale, Fst estimates reaching a maximum of 0.1090 (Cook Islands and Taiwan), over a distance of approximately 9,900 km. Despite the high degree of admixture among populations, visualisation of population structure  is evident despite the considerable distance involved . This observation does not support generalised CPH predictions, and is likely a result of greater connectivity of this population pair through ocean current circulation [For the present study, at the geographical limits of the species distribution in the Pacific, decreased differentiation between Taiwan and French Polynesia . Becausell size , 119, thell size , 120–122DS estimates , Madagascar, Arabian Sea, Bay of Bengal, Andaman Sea and Sumatra may resolve these questions, and potentially ascertain the presence of a genetic break between the Indian and Pacific Oceans. Pairwise C-M trends were absent, and may mean that the homogenising influence of basin-wide current circulation patterns disrupts any obvious patterns. However, ocean currents together with isolation by geographic distance are also likely to maintain sub-regional population structure .The CPH predicts that genetic diversity and connectivity should be highest at the centre of a species’ range and decrease towards the periphery, however, our data indicate the presence of patterns which are substantially more complex than generalised CPH predictions. For Pacific populations, easily discernable r et al.  for the P. margaritifera [Sample collection for the current study was organised according to the published theoretical distribution of ritifera , and theritifera  warrant ritifera , 5, 6.P. margaritifera are complex. In the Pacific Ocean, our data indicate that ocean currents, seascape features and geographic distances are major influences on population connectivity which both disrupts C-M clines, and simultaneously stabilises population structure according to basin sub-regions [C-M trends, ocean currents may play a large role in maintaining divergent populations. It is possible that a genetic break between the Indian and Pacific Oceans may exist at the South-East Asian archipelago, and further investigation of these populations could provide answers to this question, as it has for other marine invertebrates [P. margaritifera would not have been possible without simulations of larval dispersal, and we suggest that oceanographic and/or ecological modelling data is an indispensable component of range-wide investigations of genetic structure in marine organisms, which possess passively dispersing planktonic larvae [It is evident that the biological and physical processes governing population structure and genetic diversity in -regions . Broad-stebrates , 129. Gac larvae , 132.P. m. var. typica and P. m. var. cummingi as sub-species classifications in the Pacific Ocean, given the level of broad-scale admixture detected and absence of evidence for distinct ESUs. Unfortunately, as Hawaiian populations could not be sampled, no conclusion as to the status of P. m. var. galstoffi may be drawn. However, given the ability of larvae to disperse across the Pacific basin over the span of several generations, it is possible that Hawaiian populations may not be as divergent as previously thought [P. m. var. zanzibarensis and P. m. var. persica in the Indian Ocean may constitute distinct ESUs, given their substantial divergence from all other populations, although denser basin-wide sampling is required for verification. A comprehensive range-wide phylogenetic analysis of P. margaritifera is also needed to assess how many ESUs may be present, and to determine if the black-lip pearl oyster represents a true species complex. Because there are discernable regional morphological differences within P. margaritifera, there may be parallels with the Akoya species complex, which also displays morphological variability, high levels of gene flow and has a similarly extensive Indo-Pacific distribution [Data presented here do not support  thought . Conversribution , 50.P. margaritifera, with the discovery of five distinct genetic stocks in the region. Given the economic importance of pearl oyster aquaculture for several Pacific Island nations [Our findings hold regional fishery management implications for Pacific populations of  nations , 134, th