Salmo trutta) populations and a regionally used hatchery strain. The genetic differentiation, quantified as FST, was similar when measured using 16 microsatellites and 4,876 SNPs. Based on both marker types, each brown trout population represented a distinct gene pool with a low level of interbreeding. Analysis of SNPs identified half‐ and full‐siblings with a higher probability than the analysis based on microsatellites, and SNPs outperformed microsatellites in estimating individual‐level multilocus heterozygosity. Overall, the results indicated that moderately polymorphic microsatellites and SNPs from RADseq agreed on estimates of population genetic structure in moderately diverged, small populations, but RADseq outperformed microsatellites for applications that required individual‐level genotype information, such as quantifying relatedness and individual‐level heterozygosity. The results can be applied to other small populations with low or moderate levels of genetic diversity.The conservation and management of endangered species requires information on their genetic diversity, relatedness and population structure. The main genetic markers applied for these questions are microsatellites and single nucleotide polymorphisms (SNPs), the latter of which remain the more resource demanding approach in most cases. Here, we compare the performance of two approaches, SNPs obtained by restriction‐site‐associated DNA sequencing (RADseq) and 16 DNA microsatellite loci, for estimating genetic diversity, relatedness and genetic differentiation of three, small, geographically close wild brown trout ( These may include harvesting . However, microsatellite markers have limitations such as risk of homoplasy for allele size, the presence of null alleles  from the East and West coasts of the Atlantic Ocean. Despite these advances, more information on wider range of species is still needed to compare the performance and cost‐efficiency of these two marker types in determining population structure especially in small populations in need of conservation actions.The advantages of SNP markers over microsatellites include their suitability for comparisons of both strongly and weakly diverged populations, and even species, and in revealing ancestral patterns of genetic structuring compared to microsatellites due to the slower mutation rate of SNPs compared to microsatellite regions  are valuable for managing hatchery breeding strategies, and understanding demographics  and fitness is a long‐standing question in conservation and evolutionary biology: lower diversity is expected to contribute to lower fitness  populations can be genetically isolated  with both a RADseq approach and a DNA‐microsatellite panel commonly used in brown trout population genetic research , Paltamo, Finland . Wild brown trout were caught in the boreal River Oulujoki watershed from three resident brown trout populations in rivers Tuhkajoki (17 September 2013), Pohjajoki (16–17 September 2015) and Vaarainjoki  using electrofishing and transported to the Kainuu Fisheries Research station (Samples from the hatchery brood stock included in this study (Ouv) were collected from the second generation of this stock. The stock was founded in October 2000 by using adfluvial brown trout from two previous hatchery brood stocks originating from two connected rivers, River Varisjoki and upstream River Kongasjoki Figure . The for® dsDNA HS Assay Kit (ThermoFisher Scientific).Total DNA was extracted from fin clips preserved in pure ethanol or dried scales using the Omega bio‐tek E.Z.N:A Tissue DNA kit or Macherey‐Nagel NucleoSpin Tissue kit. The quality of total DNA was controlled with electrophoresis on a 1% agarose gel and with fluorometric measurements using Qubit 2.0 with Qubit2.2hw.test from pegas package  and cleaning (‐c). Orthologous tags were assembled, catalogued and matched using denovo pipeline, in which the optimal parameters were obtained following Paris, Stevens, and Catchen , after which new parameters were again passed onto STACKS’ population function. Finally, only loci present in all four populations (‐p) and in 60% of the individuals (‐r) were retained. Based on this dataset, a total of five individuals with more than 20% of missing data (Table stackr). This was done in order to remove potential sequencing errors and uninformative missing data that could potentially bias the results (see stackr package guidelines). In addition, a filter for marker heterozygosity  was appBecause the sex ratio of the samples from two of the studied populations was not known, the SNP dataset was checked for the presence of sex‐linked markers to avoid introducing bias into the analysis  using package adegenet v.2.0.1  was calculated using the method based on the linkage disequilibrium  from package Jombart,  in R. ThK value 2–7, after which the optimal K value was determined using CLUMPAK  was determined, the 20 runs of STRUCTURE using K = 4 were combined using LargeKGreedy algorithm in CLUMPP v.1.1.2 , i.e., the relationship between the heterozygous loci for an individual and the sum of observed average heterozygosity in the population , with a mean of 4–6 alleles across loci within populations Table . Nearly ss Table . Two indss Table . The estow Table . The conFST was 0.209 across populations, and was very similar to that based on SNPs (0.211). The pairwise FST values between the three rivers and the hatchery stock were highly significant, and approximately at the same level in all three datasets .According to COLONY output, the estimated number of full‐sib families was similar using microsatellite and SNP markers on the same individuals, when empirically determined error rates  were used for each marker Table . In Tuhkca. 80–100 SNP loci . The range of sMLH was much higher for microsatellites , a greater isolation‐by‐distance was identified by RADseq than by microsatellites across Northern Europe  also concluded that analyzing even a few individuals from each population with RADseq can be highly informative  using 20–412 SNPs and 5–100 microsatellites: in one population, 20 microsatellites performed as well as 75 SNPs, but in a different population more loci would have been needed from both markers. In our study, we did not find clear differences in the number of SNPs required for precise estimates of sMLH between populations. The lack of population‐specific differences in sMLH preciseness in our study suggests that the population effects in Miller et al. , thus explaining its divergence from the other wild populations. Overall, differences among populations can also be partly explained by founder effects, as they may have been initially formed by few individuals after the last glacial period since ca. 10,000 years, as well as by genetic drift, partly due to their resident life‐history strategy and small population size even in fully natural conditions. The RADseq approach applied here was also used to assess the population genetic structure of brown trout in River Koutajoki watershed in Finland , albeit higher than those of populations without migration barriers (0.043 ± 0.003; Koljonen et al., All the rivers from which the study populations originated have undergone some alterations in the environment due to logging, dams, mining or forestry‐induced decrease in water quality. These effects combined with historically high fishing pressure have further depressed the number of spawning individuals in the study populations. This explains the low genetic diversity and the Both marker types evidenced that stocked fish can disperse to the stream habitats of resident trout, as one individual of identified hatchery origin was caught in Vaarainjoki during spawning time. Hatchery fish are generally stocked directly to Lake Oulujärvi, the close‐by Lake Kivesjärvi, or to rivers Varisjoki or Kongasjoki, as 2‐ or 3‐year‐old smolts, and can thereafter disperse to Vaarainjoki. Small‐scale introductions may have been made also in Tuhkajoki, which could explain the presence of hatchery genotypes in this population. Several studies have shown that introgression from hatchery stocks to wild fish populations occurs especially when the number of stocked fish is high compared to the number of wild individuals Hansen, , as is tNe indicating a high extinction risk with the conservation of seemingly unique genetic composition. Particularly, large enough unique populations should not be mixed with hatchery stocking in line with the concept of Evolutionarily Significant Units proposed by Fraser and Bernatchez (Our results suggest that the existing wild populations in the region cannot be supported with the hatchery brood stock without risking the unique genetic composition in the wild populations. Thus, the management decisions on the wild brown trout populations need to balance the low diversity and rnatchez , while t4.3The resolution power of the marker depends on the number and frequency of alleles and loci available for each population. Microsatellites can usually carry a several‐fold higher number of alleles than SNPs, but the number of loci can be increased enormously in RADseq analysis compared to microsatellites. Thus, the abundance of SNP loci creates an inevitable advantage in resolution power, especially in cases where microsatellites may not function optimally due to high relatedness within populations or population bottlenecks. Consequently, although microsatellites have been central for characterizing post‐glacial phylogenetic relationships in salmonids (e.g., Hansen, Mensberg, & Berg, Our study indicates the benefits or RADseq can also be pronounced for small populations with limited genetic diversity due to prior population bottlenecks, which is often the case for endangered populations. Thus, the choice of marker to be used in future studies depends on the goals of the study as well as available resources; compared to microsatellite analysis, RADseq still carries higher per‐individual costs, although the overall price is decreasing with decreasing sequencing and reagent costs. There is a relatively high labor and time cost associated with establishing a new microsatellite panel, which RADseq circumvents by having similar preparation costs regardless of prior knowledge on markers. For projects aiming solely at describing population divergence across landscapes using a multiallelic microsatellites panel previously developed for the focal species, the benefits of switching to a RADseq approach can be limited. In contrast, many detailed questions on relatedness, demography, selection, genetic diversity or fine‐scale population divergence greatly benefit from an approach based on a large number of SNPs (Andrews et al., The authors declare no conflict of interest.Conceptualization: AVai, SUH, AL, JMP. Investigation: AL, MLK, JK, JMP. Formal analysis: AL, JMP. Writing –Original draft: JMP, AL. Writing –Review and editing: JMP, AL, SUH, AVas, PH, AH, MLK, JK, AVai. Visualization: JMP, AL. Supervision: AVai, SUH, AVas. Funding acquisition: AVai, AH, PH. Click here for additional data file. Click here for additional data file.


Information on genetic diversity and population structure are very important in any breeding programme for the improvement of traits of interest and the development of outstanding products for commercialization. In the present study, we assessed the genetic diversity of 94 early-maturing white and yellow tropical maize inbred lines using single nucleotide polymorphism (SNP) markers. The larger number of SNP markers used in this study allowed a clearer inference of the population structure of the 94 inbred lines. Cluster analysis resolved the inbred lines into different clusters based on their pedigree, selection history and endosperm colour. However, three heterotic groups were revealed by population structure analysis, but additional field evaluation could be more informative to confirm the heterotic groups identified. Nevertheless, wide genetic variability existed among the inbred lines making them unique with the potential to contribute new beneficial alleles to maize breeding programmes in the tropics, especially in the West and Central Africa (WCA) sub-region. For good progress from selection in any crop improvement programme, information on the genetic diversity and population structure of the base germplasm is crucial. Therefore, plant breeders routinely resort to newly available tools to make informed decisions on selection. In the past, a number of researchers have highlighted the importance and the need for accurate assessment of genetic diversity in applied breeding programmes. This allows the examination of issues relating to the assignment of inbred lines to heterotic groups, selection of efficient testers for testing of inbred lines in hybrid combinations, drawing inferences on the introgression of desirable genes from diverse germplasm sources into available genetic base, identification of diverse parental combinations to create segregating progenies with maximum genetic variability for further selection and the estimation of genetic diversity loss during conservation or selection –6. StudiDNA based markers are preferred over morphological and biochemical markers because they are not affected by environmental factors and/or by the developmental stage of the plant ,10. ConsWith the advances in marker technology, single nucleotide polymorphism (SNP) markers have become the choice due to their low cost per data point, high genomic abundance, locus-specificity, co-dominance, the potential for high throughput analysis and lower genotyping error rates ,19–24. SStriga, and streak resistant/tolerant) broad-based populations such as TZE-W Pop DT STR and TZE-Y Pop DT STR in the early maturing  group, and TZEE-W Pop DT STR and TZEE-Y Pop DT STR in the extra-early  maturing group during the first decade of the inception of the programme [Striga resistant and/or drought-tolerant maize inbred lines derived from different source populations selected from the panel of inbred lines of the IITA maize improvement programme using SNP markers.In the 1990s, the maize improvement programme of IITA initiated a breeding programme for the extra-early- and early-maturing groups which resulted in the development of some multiple stress tolerant , and/or tolerance to drought , with tolerance or resistance to  drought . The inb drought . The firTM Sample Prep 2010 Geno/Grinder . Total genomic DNA extraction was performed using the DArT protocol (www.diversityarrays.com/files/DArT_DNA_isolation.pdf). The quality and quantity of DNA in each sample was determined on 2% agarose gel followed by quantification using an ND-1000 Spectrophotometer (Nanodrop Technologies). For genotyping, DNA samples were sent to Diversity Arrays Platform [Fresh leaves samples were collected from three weeks old seedlings within each genotype and stored in a deep freezer at -80°C. Prior to genomic DNA extraction, each sample was dried in a Labconco Freezone 2.5L System lyophilizer  followed by grinding using SpexPlatform . LibrarySNP markers with more than 20% of missing data, 20% of heterozygosity and the minor allele frequency lower than 0.05 were eliminated resulting in 15,047 SNP markers which were used for further analysis . The polThe data from the 15,047 SNP markers were subjected to population structure analysis based on the admixture model-based clustering method using the software package STRUCTURE 2.3.4 . The besFollowing the determination of the number of clusters using STRUCTURE, the 15,047 SNP marker data were analyzed using DARwin software . The neiThe results of the summary statistics of the SNP markers are presented in 0, which has yellow endosperm colour  of the genetic distances fell between 0.32 and 0.42 . The lowm colour .ST values of 0.83, 0.49 and 0.01, respectively. The highest allele frequency divergence of 0.15 was recorded between sub-populations 2 and 1, followed by sub-population 3 and 1 with 0.11, while the least allele frequency divergence of 0.07 was recorded between sub-populations 2 and 3.Based on 15,047 SNPs, population structure analysis revealed three distinct sub-populations in the 94 inbred lines . The subOP STR 106 and were characterized by yellow endosperm maize lines. The second cluster generated by the phylogenetic analysis included 32 inbred lines classified into several sub-clusters. However, it is clear that the grouping of 20 maize inbred lines based on the phylogenetic analysis was similar to that based on the population structure analysis  , WEC STR and TZE-Y Pop STR Co (S2). All the inbred lines in this phylogenetic cluster belong to sub-population 3 (red colour) except 12 lines which were classified by the population analysis into the admixture group  Figs  and 3. Nre group .Genetic diversity studies are very essential in the selection of individual genotypes among closely related groups for initiation of new breeding activities. It is particularly important that the genetic similarity or dissimilarity among inbred lines are established to facilitate the development of productive hybrids which are often a product of crosses involving inbred lines of opposing heterotic groups. Heterotic populations are also developed from inbred lines sharing common ancestry. New set of inbred lines are derived from the heterotic populations and the inbred lines are expected to combine well with inbred lines developed from other opposing heterotic populations. Genotyping has proved to be one of the reliable approaches of establishing such phylogenetic relationships among a set of inbred lines . In the Genetic distance provides a measure of the degree of relatedness between individuals in a population . The resThe SNP markers clustered the inbred lines based on their ancestry, selection history and endosperm colour Figs . HoweverPopulation structure analysis is a process of inferring individual ancestry of inbred lines from genotypic information . The stuST values obtained for sub-populations and the moderate allele frequency divergence observed between sub-populations indicates that these inbred lines are fixed and can be classified into genetically distinct groups (heterotic groups). These characteristics make them a valuable resource for genetic studies in maize and association mapping where uniformity of inbred lines and genetic divergence are required. For future development of productive hybrids, crosses should be made between parent lines from different sub-populations, particularly between inbred lines in sub-populations 1 and 2.The lower levels of heterozygosity observed among inbred lines within the three sub-populations suggested that the SNPs were effective in forming homogeneous sub-populations. The very large FHigh genetic distances obtained among paired inbred lines revealed the uniqueness of the studied lines and existence of substantial genetic variability that could be exploited for the development of productive hybrids. The inbred lines were assigned into heterotic groups based on similarity of ancestry, selection history and endosperm colour. This study showed that SNP markers were more reliable in categorizing maize inbred lines into groups on the basis of shared phylogeny. The low heterozygosity observed among inbred lines within sub-populations and the moderate divergence among sub-populations suggested that the inbred lines could be used in the development of productive hybrids breeding or heterotic populations for the West African Sub-region.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.


Vitex rotundifolia is an important coastal and medicinal plant, and is recorded in the List of the Important Wild Plants for Conservation in China and Japan. However, an effective conservation strategy is lacking. In the present study, the genetic diversity and population structure were analyzed using phylogeographical methods based on the trnH-psbA and trnG-trnS intergenic spacers of the chloroplast DNA (cpDNA) sequences from 157 individuals from 25 sampling sites for V. rotundifolia and V. trifolia plus the internal transcribed spacer (ITS) of the nuclear ribosomal DNA (nrDNA) sequences of 177 individuals from 27 sampling sites. The results showed that V. rotundifolia and V. trifolia had eight cpDNA and two nrDNA haplotypes, respectively, and the V. rotundifolia has a low level of genetic diversity , a more pronounced genetic differentiation among populations (population differentiation at the species level (GST) = 0.201, population differentiation at the allele level (NST) = 0.462), and an insignificantly different phylogeographical structure . In addition, haplotype network analyses indicated that V. rotundifolia and V. trifolia have distinct haplotypes. Divergence dating based on BEAST software analyses showed that most cpDNA clades diverged in the late Pleistocene era. Demographic analysis indicated that V. rotundifolia underwent a rapid demographic expansion. Some scientific strategies are suggested for resource conservation of V. rotundifolia based on its genetic diversity and population structure. Vitex rotundifolia, which often grows on beaches and sand dunes, is a widely distributed shoreline shrub of the family Lamiaceae,  markers. Their fine-scale spatial autocorrelation analysis showed a clear within-population structure, with gene clusters of approximately 20 m, which could act as a guide for sampling strategies. They also reported the overall genetic diversity (GD) of V. rotundifolia on China was moderate (GD = 0.190) and the genotypic diversity was greater than the average values for a clonal plant, indicating its significant reproduction through seedlings. Pharmacological research discovered that the genetic variation pattern was closely associated with the chemical constituents in the fruit of V. rotundifolia  haplotype sequences from 23 sampling sites covering most distribution ranges of V. rotundifolia and V. trifolia from 25 sampling sites for cpDNA and 177 individuals of V. rotundifolia and V. trifolia from 27 sampling sites for ITS were sampled from distribution areas in East Asia .A total of 157 individuals of ast Asia . Leaf satrnG (encoding tRNA glycine)-trnS (encoding tRNA serine) and trnH (encoding tRNA Histidine)-psbA (encoding photosystem II protein D1) and ITS intergenic spacer region DNA sequences were examined to analyze the genetic variation in V. rotundifolia and V. trifolia from different geographical locations  PCR SuperMix . Double-stranded DNA was amplified after 3-min incubation at 94 °C; followed by 30 cycles at 94 °C for 30 s, 54/56 °C for 30 s, and 72 °C for 30 s; with a final extension at 72 °C for 15 min. To examine the geographical distribution of cpDNA and nrDNA in V. rotundifolia and V. trifolia, we amplified two noncoding regions of cpDNA trnG-trnS and trnH-psbA and ITS intergenic spacer regions using primers that successfully amplified the expected DNA fragments and exhibited some variations in our preliminary experiments  was used to obtain absolute values of TMRCA (The program BEAST version 1.5.3  was usedof TMRCA .hd) and nucleotide diversity (πd) based on the cpDNA and nrDNA sequences of V. rotundifolia and V. trifolia , total diversity (hT), geographical total haplotype diversity (VT), geographical average haplotype diversity (VS), the level of population differentiation at the species level (GST), and an estimate of population subdivisions for phylogenetically ordered alleles (NST). GST and NST are often used to assess the geographical structure affecting population differentiation.The analyses of molecular variance (AMOVA) in ARLEQUIN 3.5 were performed to calculate the genetic variation between utations . The proutations  was usedD test and Fu’s Fs test in the ARLEQUIN 3.5 software, with 1,000 permutations, were applied to detect historical demographic range expansions of V. rotundifolia and V. trifolia , and the nucleotide diversity (πd) within each population are presented in V. rotundifolia, and five haplotypes were obtained from four sampling sites of V. trifolia  ranged from 0 to 0.639, and the nucleotide diversity  from 0 to 0.00065 in the 21 sampling sites for V. rotundifolia. At the species level, hd,cp and πd,cp were 0.360 and 0.00010, respectively. When excluding six poorly sampled populations, hd,cp and πd,cp slightly changed to 0.363 and 0.00010, respectively. Within the four V. trifolia sampling sites, hd,cpranged from 0 to 0.639 and πd,cp from 0 to 0.00065, and at the species level, hd,cp was 0.812 and πd,cp was 0.00039. These results indicated that both V. rotundifolia and V. trifolia have relatively low genetic diversity. The highest haplotype diversity occurred in population WN for V. rotundifolia and 3_YN for V. trifolia, indicating that Wanning of Hainan Province and Xishuangbanna of Yunnan Province may be the centers of biological diversity of V. rotundifolia and V. trifolia, respectively.Based on the 157 samples of trifolia . The valV. rotundifolia and V. trifolia  was 0.440, the range of haplotype diversities within population was from 0–0.667; the nucleotide diversity  was 0.00000; and the range of nucleotide diversities within population was 0–0.00200. When we excluded six poorly sampled populations, the ITS total hd,nr and πd,nr slightly changed to 0.443 and 0.00000, respectively. Within the four V. trifolia sampling sites, the total values of hd,nr and πd,nr were 0.516 and 0.00000, respectively. These results indicated that based on the ITS nuclear sequence, V. rotundifolia and V. trifolia both have a low genetic diversity.The length of the ITS sequence was 334 bp for 177 individuals, but only two haplotypes and one polymorphic site were found in trifolia , Table 3hT, VT, hS, and VS values for V. rotundifolia were 0.281, 0.285, 0.225, and 0.285, respectively, and those for the V. trifolia populations were 0.869, 0.921, 0.690, and 0.553, respectively, indicating that V. rotundifolia populations also have low genetic diversity, and that the genetic diversity of the V. trifolia populations was much higher than that of V. rotundifolia populations. The parameters of hT, VT, hS, and VS based on ITS sequence could not be calculated because only two haplotypes were found in V. rotundifolia and V. trifolia.Furthermore, PermutCpSSR_1.2.1 analysis based on cpDNA sequences deduced that the V. rotundifolia and V. trifolia populations, and 80.36% of the total cpDNA variation existed within V. rotundifolia populations. In addition, 78.25% of the variations based on cpDNA were found between V. rotundifolia and V. trifolia populations , showing a noticeable phylogeographical structure across 25 V. rotundifolia and V. trifolia populations, and the eight haplotypes displayed a clear geographical pattern. However, the NST value was higher than its corresponding GST value  in 21 V. rotundifolia populations, indicating that there was an unclear cpDNA phylogeographical structure. As shown in V. rotundifolia and V. trifolia populations. Furthermore, the TCS 1.21 network analysis of cpDNA haplotypes revealed the relationship of the interior  and the tip (derived) haplotypes. Haplotype H4 was inferred as the ancestral haplotype, as determined by outgroup weight based on haplotype positions in the network. H1, H7, and, H8 were shared only by V. rotundifolia. H2–H6 were shared only by V. trifolia. H1 was the most frequent and widely distributed haplotype in 17 of the 21 populations of V. rotundifolia.The phylogeographical analysis based on the cpDNA sequences found that the V. negundo as an outgroup. The result showed that the phylogenetic tree of the eight cpDNA haplotypes had a comb-like structure )  value ranged from 919 to 1,070 for all the nodes, the divergence time between y (HPD)) . Hence, V. rotundifolia populations and V. trifolia populations separately. The results found that V. rotundifolia and V. trifolia populations each had a relatively smooth unimodal distribution, which was consistent with distribution under an exponential growth rate , indicating that V. rotundifolia has not passed through a recent demographic expansion. In the four V. trifolia population, Tajima’s D  and Fu’s Fs  tests also showed no demographic expansion (trnG-trnS and psbA-trnH).Based on the cpDNA data, mismatch distribution analysis was performed to test whether the current expansion occurred in wth rate , indicatxpansion . This reV. rotundifolia is native to seashores from eastern India to Hawaii and from Japan to Australia, and can also be found in Brazil, Mauritius, Bangladesh, and Sri Lanka  compared with most other plants (Rhizophora (Rhizophoraceae), with nucleotide diversities for trnG-trnS and trnH-rpl2 being 0.04 ± 0.02 and 0.03 ± 0.02, respectively, and πnr = 0.02 ± 0.01  Long-distance dispersal (LDD) by sea-drifted fruit: long-distance dispersal can result in the homogeneous distribution of DNA haplotypes at the spatial scale  and JP (Japan), shared the common haplotype H1. This wide distribution of haplotypes may be caused by long-distance dispersal of this plant’s fruit. The V. rotundifolia fruit is covered with a thick hydrophobic coating that can resist water penetration, rendering it able to float in the ocean, drift onto the beach, and disperse over long distance via sea drift (V. rotundifolia fruit may be responsible for the low genetic diversity of this plant. (2) The similar ecological environment: life history traits and environmental factors affect the genetic diversity and structure of species  The asexual reproductive mode: this species proliferates by sexual reproduction, as well as clonal propagation through the elongation of rhizomes and root systems . The current distribution of haplotypes may be caused by climate oscillation during the Quaternary period, which resulted in further large-scale migration of most plants and animals, and the subsequent accumulation of genetic variations and specific phylogeographical structures . Furthermore, AMOVA analysis showed low genetic differentiation among V. rotundifolia populations, indicating that rapid spread by sea currents may have played a key role. The unclearly phylogeographical structure and the low genetic differentiation in V. rotundifolia populations may relate to the rapid long-distance dispersal by sea drifts. This is not consistent with the report by GST = 0.587) among populations of V. rotundifolia; the authors stated that such characteristics of V. rotundifolia are likely attributed to its sexual/asexual reproduction and limited gene flow. These contradictory results may have been caused by the different gene markers used (ISSR vs. cpDNA).Based on the cpDNA data, the results showed that all populations of ructures . V. rotuV. rotundifolia and V. trifolia occurred about 0.23 mya , indicating a relatively short divergence time, corresponding to the late Pleistocene era, before the onset of the last glacial maximum (LGM). Following this divergence, long-distance dispersal may have been a major historical process for V. rotundifolia, because they are present throughout the Pacific, including the coastal areas of the continents and many islands. Additionally, we detected a recent population expansion of V. rotundifolia based on the apparent mismatch distribution, showing that V. rotundifolia had undergone a long-distance dispersal expansion in the short term.Estimated divergence times indicated that the genetic divergence between V. trifolia. Populations 3_WC and 3_YN are from the southern part of China and have higher haplotype diversities in all populations. Moreover, the results of the TCS network analysis also showed that the haplotypes shared by V. rotundifolia were deduced from those of V. trifolia. Thus, we speculated that Yunnan and Wenchang regions might be two origin areas of V. rotundifolia. During the Quaternary period, V. rotundifolia expanded to eastern China rapidly.The ancestral haplotypes are located in a central position within the phylogeographical network . HaplotyV. rotundifolia and V. trifolia populations have no shared haplotypes. In addition, analysis of molecular variance (AMOVA) showed a significant difference in genetic differentiation between V. rotundifolia and V. trifolia . These findings supported the view that V. rotundifolia and V. trifolia are two separate species.The analysis of cpDNA data in this study showed that the V. rotundifolia’s habitat, reducing the number of individuals in some populations. Therefore, V. rotundifolia resource protection is urgently required. The management priority is to conserve the population of the species that has the greatest diversity. Our results showed that the WN, XL, and QD populations have the highest haplotype diversity and nucleotide diversity in V. rotundifolia; therefore, conservation priority should be considered for these populations. V. rotundifolia from Jiangxi province of China had a considerably high genotypic diversity index and should be prioritized for protection measures.It is essential to study the morphological variation, genetic diversity, and population structure to provide basic information on plant conservation . ClimateV. rotundifolia populations in China face a serious threat of extinction because of the limited number of individuals. Therefore, initial conservation measures for V. rotundifolia should be considered to increase the population size and genetic diversity. Among the extant populations, there are fewer than 10 individuals in the DL, LYG, and XL populations. Hence, we need to increase the population size for in situ conservation with high priority in these populations. In addition, habitat protection is also an important concern in V. rotundifolia conservation because of habitat damage by increased human activities and over-exploitation of the plant as a medical resource. Therefore, it is necessary to establish local nature reserves, especially for those small-sized and seriously disturbed populations. Ex situ conservation strategies, including germplasm collection, culture in botanical gardens , or reintroductions at appropriate areas with similar habitats, are also effective measures to preserve the genetic resources of V. rotundifolia. When the ex situ conservation is put in practice, samples should be collected from as many individuals as possible, because a large portion of the genetic diversity exists within, rather than among, populations. Sample collection from the WN, XL, and QD populations is recommended. Our experience, together with the results from previous studies, suggested that individuals in a population should be collected at >20 m spatial intervals to avoid collecting individuals with identical or similar genotypes  should be protected. A balanced and dynamic conservation strategy for V. rotundifolia has been proposed.In summary, the present studies revealed the intraspecific genetic variation pattern of 10.7717/peerj.6194/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.6194/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.6194/supp-3Supplemental Information 3Click here for additional data file.


Myocastor coypus) are large semiaquatic rodents native to subtropical and temperate South America. Nutrias have been introduced on all continents, except Oceania and Antarctica, and have become invasive in many countries. This study carried out a survey of nutrias in South Korea under the Nutria Eradication Project from January 2013 to August 2015. Out of 1509 habitat traces, 1497 tracks were observed in the Nakdong River basin. Based on the ecological field data, we identified concentrated areas of nutria populations. Tissue samples were collected from captured nutria for genetic analysis. According to the microsatellite marker analysis, the estimated genetic diversity of the nutria populations was low, which suggests that nutrias in South Korea originate from a single population.Nutrias (Myocastor coypus) is an invasive alien species that have had major adverse effects on biodiversity and the agricultural economy in wetland habitats. Since 2014, the Ministry of Environment in South Korea has been carrying out the Nutria Eradication Project, and we investigated nutria distribution and genetic diversity of nutria populations in South Korea. We estimated that 99.2% of nutria habitats are in the mid-lower Nakdong River regions. To further analyze the genetic diversity in eight major nutria populations, we performed a genetic analysis using microsatellite markers. Genetic diversity levels of the eight nutria populations in South Korea were relatively lower than those in other countries. The probability of migration direction among nutria populations was predicted from genetic distance analysis. Genetic structure analysis showed little difference among the nutria populations in South Korea. These results suggest that nutrias in South Korea originated from a single population. Our results provide important data for establishing management strategies for the successful eradication of nutria populations in South Korea, as well as in other countries with alien invasive species.The nutria ( Myocastor coypus) is a large, semi-aquatic rodent that originates from the southern regions of South America and has been introduced to every continent except Oceania and Antarctica [Environmental concerns regarding invasive alien species have been rapidly increasing with the expansion of global trade. Invasive mammalian species, especially rodents, have adverse effects on the biodiversity of wetlands, such as reducing biodiversity, disrupting ecosystems, human activities, and human welfare ,2. The ntarctica ,4,5,6. Itarctica ,11,12,13tarctica ,16,17,18Nutrias were first introduced to South Korea in 1985. Commercial nutria farming was initiated in 1987, and the number of nutria breeding farms rapidly increased to 450 by the early 2000s ,19; the To reduce the number of nutrias in the natural environment, the Korean government implemented The Nutria Eradication Project in 2014. Several government bodies are currently involved in this program—including the Ministry of Environment, local environmental agencies, the National Institute of Ecology (NIE), local governments, and private organizations. Furthermore, the Ministry of Environment organized a strategic committee to establish relevant policies and detailed plans . The locPrior to the initiation of the Nutria Eradication Program, the Korean government focused on the three strategic approaches: (1) monitoring of nutria individuals and habitats; (2) predicting the population distribution by analyzing the genetic structure; and (3) developing eradication programs based on nutria ecology. In a recent study, Kim and colleagues have focused on characterizing habitat location and distribution patterns to provide fundamental data for estimating habitat change in South Korea . HoweverTo improve the nutria eradication program, genetic information on populations in highly concentrated habitats near the Nakdong River basin is needed to estimate migration patterns for preventing the re-colonization of nutria. Microsatellite markers are broadly available and have been applied to study the population structures and dynamics in many invasive rodents, such as nutrias, ship rats, and Norway rats ,25,26,27http://pro.arcgis.com) and observed habitat traces within a 100 m × 100 m area (1 ha) which were visualized by one habitat trace [The nutria distribution investigation was conducted in South Korea from January 2013 to August 2015 based on the information on nutria breeding farm locations and four watershed basins: the Han River, Geum River, Yeongsan River, and Nakdong River basins. The locations of the farms, survey area, and tissue sample collection sites are presented in at trace ,21. ThisNutria habitats were also analyzed using the kernel density tool, which utilizes kernel functions to estimate the degree of concentration per unit area, in ArcGIS 10.3.1. The kernel density tool was used based on the method described by Silverman . The kerNutria tissue samples  were collected from eight regions containing large nutria populations in the Nakdong River basin A–C: BusaMcoD214, McoD271, McoD59, McoD69, McoC124, McoC203, McoD60, McoD60, McoB17, McoC118, McoA04, and McoD228) were amplified with fluorescent-labeled primer sets  according to the manufacturer’s instructions and DNA concentrations were measured by NanoDrop . To analyze the microsatellite variability, 100 ng/µL DNA was used and 12 microsatellite markers  and observed heterozygosity (OH) were estimated using GenAlEx v6.5 [http://www2.unil.ch/popgen/softwares/fstat.htm) was also used to calculate the inbreeding coefficient (ISF) and allelic richness (RA) among populations and loci [http://cmpg.unibe.ch/software/arlequin35/) [https://web.stanford.edu/group/pritchardlab/structure.html) with K ranging from 1 to 10 and 100,000 Markov chain Monte Carlo (MCMC) steps under an admixture model. The value of ΔK was calculated using the method described by Evanno [http://www.helsinki.fi/bsg/software/BAPS/) [STF values of genetic divergence and Nei’s genetic distance were calculated using the statistical program GenAlEx v6.5 (http://biology-assets.anu.edu.au/GenAlEx/Welcome.html) [https://www.mybiosoftware.com/tfpga-1-3-analysis-allozyme-molecular-population-genetic-data.html) was performed to identify correlations between geographical distance and genetic distance among the nutria populations [Expected heterozygosity  ,43. To iy Evanno . The pope/BAPS/) ,46,47. Tme.html) ,48. A Maulations .Representative habitat traces of nutria distribution were observed in two water systems in the Han River and Nakdong River basins and one in Jeju Island. Among the 1509 habitat traces, 1497 were observed in the Nakdong River basin, five in the Han River basin, and seven in Jeju Island A.The kernel density was used to estimate a smoothing phase of location coordinate data. Nutria samples in the 0.1% high density distributed area were collected using the CVh value in the kernel density calculation E. The ecMcoD228, which had no heterozygosity. Some microsatellite markers were presented as monomorphic within populations, namely McoD59 and McoC203 in CHA, McoC203 in JIN, McoD214 in MIO, and McoC203 and McoD60 in MIY. These results indicate that the genetic diversity among nutria populations in South Korea is relatively low.Previous studies have reported successfully settled nutria populations in the middle to lower reaches of the Nakdong River basin, as well as in other sites, such as the Han River and Jeju Island ,50. EstiRA) and observed heterozygosity (OH) are generally used to estimate the genetic diversity among populations [RA), observed heterozygosity (OH), and expected heterozygosity (EH). The average values of R, HOA, and EH were 1.806, 0.298, and 0.305, respectively (2 = 0.0137). Nutria populations in South Korea had relatively lower levels of OH (0.298) than those from other locations, such as Louisiana, USA  [RA values (1.806) than those in other locations, such as Louisiana, USA (3.8) [OH and RA indicate that nutria in the Nakdong River basin have a low level of genetic diversity. The low level of genetic diversity and lack of heterozygosity in the McoD228 marker may suggest that nutria in South Korea spread from one population source. This means that, even though nutrias were imported twice, only the secondary import from Bulgaria successfully settled in the Nakdong River, and the population from France [Invasive species, established in a relatively small population, can undergo bottlenecks that may decrease its genetic diversity . Geneticulations ,34,39,53ectively . The valmarkers) ; Czech Rmarkers) ; and theSA (3.8)  and the ISF) were calculated to estimate the population size and possibility of migration. The calculated ISF values were 0.256 and 0.232 in the MIO and YAN populations, respectively  A. BAPS, STF) show the degree of genetic relationship between populations. STF has been used to infer migration rates and represent genetic differentiation among populations [STF of nutrias in South Korea. A low value of STF between two populations means similar genetic structures and little population differentiation. We estimated the values of STF using eight nutria populations in South Korea. The values varied from 0.013 between the BUS and YAN populations to 0.292 between the JIN and MIO populations  between DAE and MIO imply that nutrias can migrate between the two regions through the Miryang River and the Cheongdo Stream. As shown in ISF (-0.119) in the DAE population imply that the population may have established a sufficiently large population for outbreeding, whereas the MIO population may have a relatively small population according to the high value of ISF (0.256), as shown in ISF, the MIY population may have established a sufficiently large population for outbreeding (The analysis of ly small . The YANbreeding .From our results, we confirm nutria population groups in the Nakdong River basin and decided that capturing each nutria group using genetic data on migration direction in the area would be a more efficient approach than a broader eradication strategy that does not use prediction data. Having implemented this strategy to effectively capture individual nutria groups, the Government started operating additional maneuver capture teams, as well as the existing nutria capture teams in the assigned Nakdong River basin area. These strategic modifications from the primary plans have enabled a more efficient nutria elimination strategy in South Korea that reduced 88% of the nutria habitat from 2014 to 2018 .We estimated the genetic origin of and relationships among nutria populations that successfully settled in South Korea as an invasive alien species. The Nakdong River hydrosphere is densely interconnected and resembles a net-like structure. This prevents the complete eradication of nutria, despite the progress in nutria capture techniques and ongoing efforts at the national level. For the successful nutria eradication project, fundamental genetic information on nutria population dispersion and habitat changes with respect to the Nakdong River hydrosphere is necessary for South Korea. In this study, genetic diversity and structural analysis indicated that the populations of nutria in the middle-low regions of the Nakdong River might originate from one population source, possibly from the second nutria import, and the populations can be classified into four groups using pairwise genetic distance data. These genetic analyses can be used to estimate the population structure and genetic diversity of invasive species in general, especially vertebrates. We investigated the establishment process and the population composition of nutria prior to the initiation of the Nutria Eradication Project. Using genetic diversity analysis, population structure and geographical migration were inferred, establishing a foundation for estimating habitat change and creating detailed long-term eradication plans.


Arundo donax L. represents one of most studied one characterized by very low genetic biodiversity. Although it is a perennial rhizomatous tall grass native to eastern and southern Asia, it spreads only asexually in the invaded range all over the world thriving very well in a large array of pedo-climatic conditions. This ability to morphologically or physiologically adapt to a broad array of conditions could be attributed to epigenetic mechanisms. To shade light on this relevant issue, 96 stems of A. donax from spontaneous populations distributed across the Italian invaded range  were analysed. Leaf DNAs were extracted and processed through AFLPs and MSAPs for defining either genetic and epigenetic profiles. Both analyses clearly showed that the A. donax populations of Sardinia island are genetically distinct from those of Italian mainland; AFLPs showed an extremely low genetic biodiversity due to vegetative reproduction, whilst, epi-biodiversity, estimated through MSAP marker, increased within the analyzed populations. These results suggest that the capability of A. donax to invade and thrive in diverse environmental conditions can be, at least, partially attributed to a higher epigenetic variability. Therefore, the different DNA methylation status may have significant and important biological meaning, in particular, in the case of invasive clonal plants such as A. donax, also for the biodiversity definition, and MSAP marker can be considered an useful and cost effective marker to reveal it.Among the most widespread plant species with clonal reproduction  Arundo donax L. , also known as giant reed, is a perennial rhizomatous tall grass, native to eastern and southern Asia  were calculated using Arlequin [st value following the methods of Excoffier, Smouse [One of our aims was to compare the genetic biodiversity, calculated considering the Arlequin  and GeneArlequin , 36. In , Smouse  and Peak, Smouse . The bin, Smouse . Clustere.g., two dimension scatterplots).The multivariate AFLP or MSAP datasets were further analysed by Principal Coordinate Analysis (PCoA) using a covariance matrix. The major axes of variation in each dataset were identified and the assessments of the main trends in the data were graphically represented in a reduced-dimension space  to belong, partially or fully, to one or more populations under investigation, it is possible to speculate if and how the DNA methylation status alters the population structure. The number of populations (K) was estimated by performing 10 runs for each population, from K = 1 to K = 10. Each run consisted of 100,000 MCMC (Markov Chain Monte Carlo) permutations with a burn period of 10,000, assuming no a priori information on population affiliation, the admixture and correlated allele frequency methods. The K values were estimated using the method of Evanno, Regnaut [A Bayesian approach was applied, using Structure ver. 2.2 , to infe Regnaut  with 20  Regnaut .MseI profile), and of the MSAP (MspI and HpaII profiles), the following subtractions between genetic distance (GD) matrices were performed: GDMSP-GDAFLP; GDHPA-GDMSP; GDHPA-GDAFLP. It might happen that two distances are roughly similar ; other cases where the AFLP or MspI distances are greater than those of MspI or HpaII, showing somewhat smaller values, or, if the opposite is true , positive values would be obtained.To quantify the differences between the genetic distances, calculated on the basis of the AFLP  reflected DNA methylation variability.The ninety-six specimens of A. donax was very low (i.e. for Pop9 and Pop13. The highest number of common bands (< = 50%) was found in the populations collected in mainland Italy (from Pop8 to Pop13); in contrast, in the other populations (from Pop1 to Pop7 and Pop14), they were absent or close to zero. When the number of common bands (< = 25%) was considered, a maximum value of four was reached within Pop10 and Pop13, three within Pop9, two within Pop12, and one within Pop8 and Pop14.The main biodiversity indices, calculated for the AFLP profiles, demonstrated that the genetic biodiversity of the 14 investigated populations of very low ; in partIn general, the Shannon index (I) was very low ; only in the case of Pop10 and Pop13, which were collected in Emilia Romagna , it was 0.065 and 0.056, respectively. However, the molecular variance calculated among populations, equal to 89%, was much higher than that estimated within populations, equal to 11%. This result was due to a higher number of polymorphic loci found in the Pop8, 10, 13, and 14  with respect to those obtained by analysing all the other populations, which showed a percentage of polymorphic loci equal or close to zero.A. donax, Bayesian analyses were performed on the AFLP data set using the Structure software [A. donax from Sardinia and Campania region (red), whilst, the second, the samples collected in Emilia Romagna region (green).To investigate the whole Italian population of software . The delA. donax, Jaccard Dissimilarity Coefficient (JDC) was calculated, and the output UPGMA bootstrap dendrogram  including: 1E) samples ADE10_3 and ADE10_4; 2E) samples from ADE13_1 to ADE13_4; 3E) sample from ADE9_1 to ADE9_4, ADE10_1, ADE10_2, and from ADE12_1 to ADE12_4; 4E) samples from ADE10_5 to 10_12, and ADE11_1 to ADE11_4. The highest JDC was reached in the case of the node that grouped samples ADE10_3 and ADE10_4, which can be considered, therefore, as an out-group. However, the AFLP analyses confirm that A. donax adopts vegetative reproduction and, in particular, the population are constituted by mono-clonal stands.In order to estimate the genetic similarity among the analysed specimens of ndrogram  confirmeA. donax to invade and thrive in diverse non-native habitats, and in Sardinia in particular, and the fact that it shows different phenotypes and phenology in clonal populations [The ability of ulations , 43, mayA. donax populations. Given that the specimens collecting from the 14 populations shown very similar AFLP genetic profiles and that the statistical analyses divided all the samples in just two groups (meta-populations), we fixed the a priori number of groups equal to two for the analyses performed on MSAP data, in agreement with the estimated number of groups obtained by the Structure software and UPGMA analyses for AFLP data.The “msap” algorithm, developed in the R environment , was useMspI  was the most abundant methylation status.HpaII and MspI profiles. The results obtained were 0.51 and 0.35, respectively. A Wilcoxon rank sum test with continuity correction was significant (P<0.01). The percentage of polymorphic methylation sensitive loci, revealed by MspI and HpaII digestions, was 15% and 90%, respectively.In order to estimate the biodiversity due to different DNA methylation status, the Shannon diversity index was calculated separately for MspI and HpaII) have been processed together, as we made above. However, in our opinion, in the case of clonal populations, it could be possible to consider the two profiles independent; in this way each difference in the biostatistical indices will be attributed to DNA methylation effect, or to error rate. The main biodiversity indices are reported in Tables A new approach was applied to analyse the MSAP data; until now the two profiles obtained during the MSAP analysis  for MspI profile. In the case of HpaII, Pop2, Pop3, and populations from 9 to 13 showed the highest number of common bands (< = 50%); the highest number of common bands was detected in Pop13. Nevertheless, the percentage of polymorphic bands ranged between 0.0% (Pop11 and 12) and 39.0% (Pop3) in the case of the MspI profile, and between 0.0% (Pop11 and 12) and 51.0% (Pop3) in the case of HpaII, and, in general, it increased when the latter profile was considered. The inter- and intra-population molecular variance was 53% and 47% for MspI profile, and 58% and 42% for HpaII profile.The number of private bands in each of the 14 populations was zero in the case of the A. donax populations was analysed with no a priori information, using the Structure software [MspI or HpaII). The statistical model described by Evanno, Regnaut [MspI at the K value two and three , and the other cluster included all of the other A. donax specimens collected in mainland Italy (red bars). In contrast to the AFLP result, ten specimens, collected in Emilia Romagna region , were assigned both to red cluster  and to green cluster (with the probability of roughly 20%). When K = 4 was considered  were assigned to the yellow one; the specimens from ADE12_1 to ADE13_4 constituted the green cluster.The bar plot obtained for the se K = 2  was quitnsidered , the SarHpaII profile was considered. In this case, K was equal to three, and several specimens showed an ambiguous membership  to be assigned to the green one. Furthermore, the samples collected in Emilia Romagna were clearly assigned to the red cluster.The optimal population number and the cluster membership changed when the mbership . In the A. donax specimens, the JDC was calculated for both MspI and HpaII data sets, and the two UPGMA dendrograms are reported in Figs MspI and HpaII profiles showed an increased epigenetic diversity when compared to those determined by AFLP. In the case of MspI profile (A. donax collected in Sardinia Island plants are separated in 4 clusters with the highest JDC ≅ 2: 1S) sample ADS3_1 and ADS3_2; 2S) from ADS1_1 to ADS1_8, ADS6_3 and ADS6_4, ADS8_1 to ADS8_4, and ADS14_3 and 14_4. In the case of A. donax collected in Emilia Romagna, the specimens were subdivided into five subgroups (JDC≅ 3) including: 1E) samples ADE10_3 and ADE10_4; 2E) from ADE13_1 to ADE13_4; 3E) ADE9_1 to ADE9_4, ADE10_1 and ADE10_2, from ADE12_1 to ADE12_4; 4E) from ADE11_1 to ADE11_4; 5E) from ADE10_5 to ADE10_12.In order to visualize the level of similarity among the  profile , we founHpaII profile   matrices were performed: GDMSP-GDAFLP; GDHPA-GDMSP; GDHPA-GDAFLP (as reported in section 2.2). In our study, the GDHPA was greater than that calculated in the case of AFLP and n green) . A similn green) . In thisn green) . When GDn green) , the GDHA. donax in Italy, we analysed 96 individuals from 14 naturalized populations using AFLP (EcoRI/MseI–genetic diversity) and MSAP (EcoRI/MspI and EcoRI/HpaII–epigenetic diversity).Plants in general, and in particular the invasive riparian one, may have developed molecular processes to adapt themselves in order to colonize new and frequently changing habitats. Epigenetic mechanisms are, very likely, the trigger of a series of complex molecular mechanisms that allow plants to thrive in sometimes harsh conditions. In order to understand whether epigenetic biodiversity, rather than genetic diversity, might better explain the invasive success of the archaeophyte In general, plants use epigenetic mechanisms to counteract environmental variations and habitat changes , or copeDifferent epi-phenotypes, although related to the same genome, are hypothesized to promote significantly environmental adaptation, because they promote phenotype diversification and increase the probability to win the “war for life” in new or highly competitive ecosystems, or in relation to environment changes. The benefits of phenotype diversification induced by epigenetic variation are particularly noticeable in plant species that adopt vegetative reproduction, which may explain the “invasion paradox” , or the A. donax, and to compare it with genetic diversity, a set of genetic biodiversity indices, estimated on the basis of AFLP data (genetic profile), were compared with those obtained with MSAP markers (epigenetic profiles). The Jaccard Distance Coefficient (JDC) revealed a very low genetic biodiversity in the case of A. donax collected in Sardinia (ADS) and in Emilia Romagna (ADE). The AFLP analyses confirmed the particular tendency for vegetative reproduction and, at the same time, demonstrated that the specimens of A. donax collected in Sardinia are genetically isolated from those thriving in Emilia Romagna. A high similarity value was expected; in fact, Mariani, Cabrini [A. donax, also asserted that this species, originating in Asia, where it is not sterile [A. donax populations of the Mediterranean basin are characterized by a very low genetic diversity, and by a low incidence of rare polymorphic fragments. Despite the low genetic biodiversity of A. donax found in Sardinia and in southern Italy, Pilu, Cassani [etc.) and biomass production, the results reported by Pilu et al. [A. donax to invade and colonize different environments, although the Italian population of giant reed is characterized by a very limited genetic biodiversity due to primary asexual reproduction and seed sterility [In order to evaluate the epigenetic status of an invasive alien plant species such as  Cabrini , suggest sterile , spread  Cassani  and Cose Cassani , respectu et al.  and Coseu et al.  showed aterility . This abA. donax to diverse habitats, and might be responsible for the different phenotypes and physiological responses, as highlighted by Pilu, Cassani [A. donax is a good model organism for studies comparing biodiversity and population structure as obtained by AFLP and MSAP because of its vegetative reproduction. The differences observed in the populations studied here, using these two molecular approaches, are probably related to the different environments in which the populations are now thriving. Presumably, as plants cannot move around to find the best habitat, they had to adopt molecular mechanisms, such as epigenetic changes, to counteract diverse external stimuli  and, consequently, modify gene expression [A. donax is highly methylated (ca. 50% of the amplified loci were sensitive to methylation). It is known that epigenetic modifications are crucial for promoting phenotypic variation in living organisms [Ranunculus reptans L. and Rhamnus frangula L., respectively, to explain the late appearance of the invasive features with respect to the start of their habitat colonization. After each generation, the specimens would be better adapted to the local conditions, independently of their genetic profile [Our data are support the hypothesis that epigenetic mechanisms, and in particular DNA methylation status, may be involved in the adaptation of  Cassani . This st Cassani , 47–49;  Cassani , 50–52. pression , 56, 57.pression ), but thpression  and Haubpression  found eppression  demonstrrganisms –64, as arganisms . Epigenerganisms , 65, sugrganisms . Such a rganisms  and Fraprganisms  in the c profile .A. donax. Bayesian and non-Bayesian analyses showed consistent results for each profile analysed. In particular, when HpaII profiles were considered, the optimal number of populations and the recognized clusters present in the dendrogram were different compared to those obtained using AFLP and MspI profiles. Therefore, the different optimal population numbers underlie diverse memberships on the basis of DNA methylation status and a higher biodiversity. In general, when MspI and HpaII dendrograms were considered, the similarity coefficient decreased because of the different DNA methylation status of each A. donax specimen. Similar to the bar plot, the AFLP dendrogram well separated ADS and ADE specimens into clusters, although the similarity value and the cluster memberships varied when the two different molecular profiles were considered, revealing a variation of the clustering when compared with AFLP (genetic), or MspI (epigenetic) profiles.Our results, here reported, shed light on the effects of DNA methylation on biodiversity and on the invasive ability of This observation suggests that pedo-climatic conditions may generate variations in the DNA methylation status that drive the mechanisms of convergence and/or divergence of populations experiencing similar/dissimilar habitat; as suggested by Schulz et al. 2014 , this maA. donax, present in diverse regions in Italy, is very low. Moreover, the populations of Sardinia are genetically isolated and clearly differentiated from those growing in Emilia Romagna, suggesting different introduction routes of this plant species in the two Italian regions. The estimated indices and population structure show that the similarity decreases when MspI and HpaII profiles are considered, and, furthermore, that the population structure is altered by the different DNA methylation status. Our results suggest that the ability of A. donax to invade and thrive in diverse environmental conditions can be attributed, at least, in part to a higher epigenetic variability as previously hypothesized by Pilu, Cassani [In conclusion, our study confirms that the genetic biodiversity of the archaeophyte  Cassani  in the c Cassani  and by o Cassani , 26, Gua Cassani , 69, 70.S1 Table(DOCX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S1 Document(PDF)Click here for additional data file.S2 Document(PDF)Click here for additional data file.S3 Document(PDF)Click here for additional data file.


Mnemiopsis leidyi, and used genotyping‐by‐sequencing (GBS) to explore the spatial population structure in its nonindigenous range in the North Sea. We analyzed 140 specimens collected in different environments, including coastal and estuarine areas, and ports along the coast. Single nucleotide polymorphisms (SNPs) were called in approximately 40 k GBS loci. Population structure based on the neutral SNP panel was significant , and a distinct genetic cluster was identified in a port along the Belgian coast . Remarkably, no population structure was detected between geographically distant regions in the North Sea (the Southern part of the North Sea vs. the Kattegat/Skagerrak region), which indicates substantial gene flow at this geographical scale and recent population expansion of nonindigenous M. leidyi. Additionally, seven specimens collected at one location in the indigenous range  were highly differentiated from the North Sea populations . This study demonstrates the utility of GBS to investigate fine‐scale population structure of gelatinous zooplankton species and shows high population connectivity among nonindigenous populations of this recently introduced species in the North Sea.Nonindigenous species pose a major threat for coastal and estuarine ecosystems. Risk management requires genetic information to establish appropriate management units and infer introduction and dispersal routes. We investigated one of the most successful marine invaders, the ctenophore This article has earned an Open Data Badge for making publicly available the digitally‐shareable data necessary to reproduce the reported results. The data is available at: The DNA sequences generated for this study are deposited in the NCBI sequence read archive under SRA accession numbers SRR6950721–SRR6950884, and will be made publically available upon publication of this manuscript. Mnemiopsis leidyi in its nonindigenous range in the North Sea was investigated. Two genetically distinct clusters were identified.The population genomic structure of the gelatinous zooplankton species  We investigated the presence of distinct source populations and estimated their contribution to the coastal population. Based on source–sink dynamics, we expected to find higher genetic diversity in the three putative source populations compared with the population(s) in the North Sea. Second, we compared geographically distant regions; the Southern part of the North Sea and the Kattegat/Skagerrak region (DK) in the north. Third, sampling covered one location in the indigenous species range . We expected low levels of population structure among the regions in the Southern part of the North Sea, due to local, annual migration, and increasing genetic differences between individuals from the Southern part of the North Sea, the Baltic Sea, and Chesapeake Bay due to isolation by distance. Our main aims were to develop a GBS procedure for M. leidyi with a focus on marker density and data completeness, and to describe spatial variation in SNP frequencies after the introduction and secondary spread in the North Sea. Specific goals were to (a) identify outlier SNPs putatively under natural selection; (b) determine the population structure based on all SNPs and neutral SNPs separately; (c) quantify genomewide diversity and structure among environmentally distinct regions in the Southern part of the North Sea and the geographically distant regions of Kattegat/Skagerrak (nonindigenous) and Chesapeake Bay (indigenous); and (d) identify recent migrants between regions by population assignment.In the current study, we used high‐density SNP markers to investigate the spatial population structure of 22.1Mnemiopsis leidyi specimens were collected from 23 sampling locations , MspI (C|CGG), ApeKI (G|CWGC), EcoRI (G|AATTC), EcoT22I (ATGCA|T), and PstI (CTGCA|G). Ten libraries were prepared for each enzyme using a set of ten specimens. The performance of the enzymes was evaluated by gel electrophoresis of the restriction digest and a pilot sequencing run. MspI was selected for genotyping all other samples (see Section http://www.deenabio.com/services/gbs-adapters). Barcode sequences were 4 to 9 bp, differed from each other by at least 3 substitutions and contain homopolymers of maximum 2 bp. Restriction digests and adapter ligations were performed according to the enzyme manufacturer's recommendations (New England Biolabs). Individual libraries were purified with 1.6× MagNa magnetic beads . All samples were genotyped individually with a single‐enzyme GBS protocol modified from Elshire et al. . The pro2.3GNU parallel  scripts  with the exact test implemented in Plink 1.9  and does not require population priors. Clustering of the zero‐inflated genotype data was reduced by applying the SNP thinning algorithm, with default window size and R2 threshold. The test statistic is based on regression of the scaled genotypes and k PCs that represent relevant population structure. The distribution of this test statistic is expected to follow a chi‐square distribution with k degrees of freedom when there are no outliers present. The genomic inflation factor (GIF) is the ratio of the observed and expected median of the test statistic, and can be used for rescaling inflated distributions. The FST statistic of the analysis with OutFLANK was based on the geographical regions as prior populations  were calculated for the geographical regions  implemented in Adegenet 2.1.0 . The read depth distribution of the three remaining enzymes was evaluated either using saturation curves showing the number of loci per sample as a function of the number of reads mapped per library or, alternatively, by analyzing the number of loci shared between libraries  with increasing total number of reads mapped. These curves show that about 30 k loci are expected if at least 600 k reads per library are mapped, but that mapping higher numbers of reads may be required to reach saturation in the number of independent GBS loci for ApeKI or MspI. The number of loci shared between samples was determined per enzyme , approximately 1 k SNPs with excessive heterozygous genotype calls and significantly deviating from HWE .The SNP thinning algorithm of s Figure a,b. The C Figure c,d, and 3.4He and Ar showed similar patterns among geographical regions  for both panels  and both the datasets including and excluding Chesapeake Bay . The results of DAPC based on all SNPs (Figure Mean s Figure . The higay Table . Pairwisay Table . Pairwiss Figure  confirm s Figure . In conts Figure . Two spes Figure .44.1M. leidyi by comparing the performance of six restriction enzymes and opted for the frequent cutting enzyme MspI, based on marker density and data completeness.SNPs have proven to be effective for genetic characterization of marine populations (Benestan et al., 4.2M. leidyi in the Southern part of the North Sea indicates the establishment of a persistent population (Vansteenbrugge et al., Mnemiopsis leidyi has been observed during the winter in the Scheldt estuary and ports along the Belgian coast, and the presence of larvae and high population densities suggested that reproduction events in these areas might populate the adjacent coastal zone (Vansteenbrugge et al., The recurrent presence of Adaptation to the local environment is an important driver of population differentiation in marine organisms (Gagnaire et al., Our data further show that Ostend port is probably not an important source area for the North sea, because no individuals collected in the adjacent coastal area were assigned to this population. The two other potential source areas, the Scheldt Estuary and Dunkirk port, were inhabited by the prevalent genetic cluster and are better candidate source populations. However, we did not detect a higher genetic diversity in the Scheldt Estuary and Dunkirk port compared to the coastal area. This can be explained by population expansion during the seasonal reproduction events.4.3M. leidyi over a large north–south range across the North Sea. We compared specimens collected in the Southern part of the North Sea in 2014 with specimens of the Kattegat/Skagerrak region that were collected in the same year. Although these regions are 500 km apart, both groups were indistinguishable. The lack of population differentiation in marine invertebrates is commonly explained by large population sizes (Deagle et al., M. leidyi, patterns of population subdivision concord with well‐established oceanographic boundaries. Distinct populations were found along the coastal zones north and south of the oceanographic front of Cape Hatteras (ca. 550 km apart; Bayha et al., M. leidyi.We investigated the population structure of 4.4M. leidyi showed that the Northwestern European invasion originated from the indigenous species range along the Atlantic coast of North America. Nonindigenous populations often experience decreased genetic diversity due to founder effects. The ability to self‐fertilize and establish new populations in nonindigenous areas with an extremely small number of individuals could intensify this phenomenon. However, previous studies found no evidence of decreased genetic diversity of M. leidyi in the Northwestern Europe. This was attributed to the introduction of large numbers of ctenophores, possibly from recurrent introductions (Bayha et al., Previous molecular studies of We showed the presence of extensive population structure across the North Atlantic Ocean, with significant neutral differentiation between all individuals of the nonindigenous area of the North Sea and the indigenous population of Chesapeake Bay. Furthermore, genetic diversity among regions in the nonindigenous range was lower than in the indigenous population, despite the much lower sampling size in the latter population. This suggests founder effects and limited reintroduction pressure from the indigenous range. However, these results should be interpreted with care. First, comparison among studies is complicated, since sampling dates span several years. The population in Chesapeake Bay may no longer represent the original population introduced in Northwestern Europe. This scenario is likely because local extinction and recolonization were also observed in the nonindigenous range (Jaspers et al., 5M. leidyi populations. We found evidence for the presence of two genetically distinct populations in the North Sea, with a secluded population present in a single port and a prevalent population present in environmentally distinct and geographically distant regions of the North Sea. Dispersal via ocean currents and anthropogenic transport are important factors in secondary spread of M. leidyi and a better understanding of these mechanisms is crucial for developing effective control measures and management strategies. Both nonindigenous populations were genetically distinct from a population sampled in the indigenous species range. More elaborate sampling in both the indigenous and the nonindigenous area would deepen our insight of introduction pathways and establishment of M. leidyi.Our results show that GBS is a powerful method to investigate fine‐scale structure among None declared.CV, LV, HM, OH, TR, IRR, and KH planned the experiment. CV and TK carried out the laboratory experiments. CV and TR analyzed the NGS data. CV, LV, SD, and KH conducted the population genetic analysis. CV, LV, SD, OH, TR, IRR, and KH wrote the manuscript. All authors read and approved the final manuscript. Click here for additional data file.


Sepia officinalis) and its dicyemid parasite, based on a homologous molecular marker (cytochrome oxidase I). We show that the population of common cuttlefish in the Mediterranean area is fragmented into subpopulations, with some areas featuring restricted level of gene flow. Amongst the studied areas, Sardinia was genetically the most diverse and Cyprus the most isolated. At a larger scale, across the Mediterranean, the population structure of the parasite shows co-diversification pattern with its host, but a slower rate of diversification. Differences between the two counterparts are more obvious at a finer scale, where parasite populations show increased level of fragmentation and lower local diversities. This discrepancy can be caused by local extinctions and replacements taking place more frequently in the dicyemid populations, due to their parasitic lifestyle.Population structure and biogeography of marine organisms are formed by different drivers than in terrestrial organisms. Yet, very little information is available even for common marine organisms and even less for their associated parasites. Here we report the first analysis of population structure of both a cephalopod host ( The lack of dispersal barriers should, in theory, lead to the maintenance of large effective populations sizes, spanning vast areas of suitable habitats and showing low level of inter-population genetic variation2. However, while empirical data confirmed this view for some organisms , others showed surprisingly high diversification on a smaller scale than would be expected . Palumbi et al.1 listed several factors possibly responsible for such diversification. They include biological traits as well as physical barriers (mainly ocean/sea currents). Since then, many studies were carried out on a broad taxonomic range of marine organisms in different oceans and seas, revealing a high variety of reconstructed genetic patterns and their relationships to the oceanographic conditions, showing that genetically homogeneous populations are not the only option in marine organisms8.In marine organisms, genetic structure is usually supposed to be determined by various extrinsic factors unique to this environment. Among the most typical factors are the lack (or rare occurrence) of obvious dispersal barriers or boundaries compared to terrestrial systems10 and their possible influence on population genetic connectivity has been investigated for many organisms (e.g. reviews focused on fish and benthic invertebrates12). Mediterranean Sea has been traditionally divided into Western and Eastern Basins (here shortened as WB and EB), connected by the Strait of Sicily and possibly also by the Strait of Messina. Water currents in the WB are defined by two major oceanographic fronts, the Almeria-Oran front (AO front) near Gibraltar and the North Balearic front (NB front) near Balearic islands. In the EB, the two major barriers are represented by the Otranto Strait, separating Adriatic Sea, and the Greek islands, forming the Aegean front10. In a recent meta-analysis Pascual et al.12 showed that the relationship between population structure and oceanographic features in Mediterranean Sea varies considerably across different species and is largely determined by the life history of the given organism. Particularly, the presence/absence of a pelagic larva, and duration of this phase, is an important factor in the dispersal capacity and therefore determines population structure. For example, the organisms with low dispersal capabilities show significant genetic differentiation, but their population structure is not determined by the oceanographic fronts12. High variety of the reconstructed genetic patterns for different organisms shows that understanding genetic differentiation and gene flow in marine conditions will require number of genetic studies on a rich variety of biologically different organisms.Mediterranean Sea, with its extremely rich biodiversity and a long history of research interest, belongs among the best mapped marine regions. As a consequence, the oceanographic processes (currents and discontinuities) are well known14), marine surveys are limited to a few studies, usually involving complex multi-host systems15, or parasites of sessile hosts, lacking opportunities for co-dispersal16. The studies performed so far indicate that the answer to this question is likely to be dependent on the particular model and its biological traits. For example, Blasco-Costa and Poulin17, reviewing studies on 16 trematode species, concluded that the host mobility is the main determinant of the parasite genetic diversification. A slightly different view was presented by Maze-Guilmo et al.18. Based on their meta-analysis of a broader spectrum of parasites, they demonstrated that the outcome of such parasite-host comparison is dependent on various biological traits . For example, they suggest that, generally, parasites tend to show lower genetic differentiation than the host, particularly in hermaphroditic parasites with asexual reproduction phase, whereas for gonochoristic groups the genetic differentiation is often the same or even higher than in the host. Maze-Guilmo et al.18 explain this discrepancy by a lower number of dispersal events required for successful host colonization by hermaphroditic parasites.Considering strong dependence of parasites on their hosts, and obvious role of the host in parasites’ dispersal, the factors driving population diversity of free-living marine organisms should, in turn, affect the genetic diversity and structure of their parasites. However, the degree of such interdependency is yet unclear. While in the terrestrial systems, comparative studies on both host and parasite population structures are more common  and its parasites from a rarely studied group Dicyemida. This model allows for addressing the general issues of genetic diversification in marine environment but also specifically the relationship between diversities of the host and parasite. To our best knowledge, this study introduces the first single-host model entirely bound to the marine environment and involving a free-living host.Analysing the impact of host population structure in multi-host systems, such as in digenean parasites, is complicated by the presence of intricate networks often including migratory or terrestrial hostsS. officinalis) is an important species for fisheries in the Mediterranean Sea and the neighbouring Atlantic coast, which makes it one of the few marine organisms for which considerable amount of data is available. Considering the span and connectivity of the suitable habitats, together with the mobility of adults, cuttlefish could in theory maintain large continuous population, with isolation by distance as a main pattern of the genetic structure. However, the known biological features of the cuttlefish suggest that more complex population structure, affected by other factors than mere geographic distance, might be expected. Cuttlefish is a benthic cephalopod, that, unlike most octopuses and squids, does not possess an obvious pelagic early phase of life (e.g. larvae) as a mean of dispersal. Moreover, its lifespan of only one to two years suggests a quick turnover of the residing population. These life history traits may predispose cuttlefish to formation of fragmented populations (e.g. a network of relatively isolated subpopulations with poor genetic exchange). The few studies carried out for this species in general support this view, indicating substantial level of structuring across the whole Mediterranean Sea and the effect of Isolation By Distance (IBD). However, they do not provide any conclusive view, since based on the used molecular marker, they provide slightly different pictures. The first indication of significant subpopulation structuring in this organism came from the allozyme analysis carried out by Perez-Losada et al.20, which revealed clinal changes of the analyzed allozymes between Mediterranean and Atlantic localities but did not indicate any clear subdivision associated with any oceanic front in the Mediterranean. A significant increase in the genetic distances across the AO front was indicated by the following microsatellite study, which, however, covered only the coasts of the Iberian peninsula21. The third study, using mitochondrial gene for COI, brought the most complex analysis covering a large area from Greece to the Atlantic coast of Portugal. It confirmed the general picture of a highly fragmented population with restricted gene flow and possible effect of some oceanographic factors22.Common cuttlefish  and do not transmit vertically (see26) their population structure could deviate from the host’s pattern within local populations. Almost no information is currently available on dicyemid population structure or genetic diversity to test these questions. Up to now, a little more than 120 species of dicyemids have been described27, whereas only a few 18S rDNA sequences from dicyemids associated with eastern Pacific cephalopods28 and from Dicyemennea eledones associated with Eledone cirrhosa in the Mediterranean29 have been published. Despite the fact, that dicyemids were also suggested as suitable tags in phylogeographic parasitological research31, only several sequences usable for population studies are publicly available in GenBank, but has not been published .Even less information is available on intraspecific genetic diversity of the organisms associated with the cuttlefish as a host. Perhaps the closest topic addressed for the Mediterranean cephalopods is the study of symbiotic 24) to dislodge attached dicyemids. Liquid with the dislodged dicyemids was transferred into a micro-tube and centrifuged at low speed to concentrate dicyemids in the solution. Supernatant was removed, additional amount of liquid with dicyemids was added and the centrifugation was repeated. Finally, supernatant was removed and 1 ml of pure ethanol was added for storage.Samples were collected at 13 localities in the in the Mediterranean area and the Atlantic coast of Portugal  was fixed and stained for confocal microscopy to provide illustrative photos of dicyemids from 33 and H700534 were used for amplification of a 1100 bp fragment of the mitochondrial cytochrome oxidase I (COI) gene. For dicyemids, new specific primers were designed based on COI gene sequence extracted from a preliminary draft assembly of a cuttlefish renal tissue transcriptome (see Supplementary Methods for details). To supplement COI based species delimitation of the studied dicyemid parasite with a nuclear locus, we amplified and sequenced 1330 bp fragment of 18S rDNA in 23 samples across our population sets with primers F3 and R2 published in28. Standard PCR protocol with Taq polymerase was followed (for PCR conditions and primer sequences see Supplementary Table S2O per reaction added and incubated for 15 minutes at 37 °C in thermocycler) and sequenced with PCR primers on the ABI analyzer (Thermo Fisher). Each sample was sequenced in both directions. Where necessary , specific sequencing primers were used to obtain the full length of the sequence . For the parasites, pooled samples  were extracted by QIAamp DNA Micro Kit (QIAGEN). In both cases, the protocol provided by the manufacturer was followed. For cephalopod hosts, universal primers F1490https://www.geneious.com/). Mafft v. 735 was used for translation guided alignment of sequences. To evaluate Sepia diversity in a broader context, publicly available sequences were added to the sequences obtained in this study  of Sepia officinalis covering the whole length of the alignment was added to the sequences obtained in this study to verify correct assignment of all samples to S. officinalis species in the population network. To show S. officinalis population structure across its dispersal area, publicly available sequences were added to the alignment, which was however shorter (473 bp) due to only partial overlap between the sequences (Accession numbers in the Supplementary Table 41). Portuguese populations were omitted from AMOVA analyses due to the lack of parasite data.To visualize the geographic diversity of populations, haplotype networks were designed in PopArt software42), neutrality tests (Tajima’s D43), Fu and Li’s D* and F*44 and Fu’s Fs45) and several population size change statistics (Raggedness46), Ramos-Onsins test47 were obtained in DNASP ver. 548. Pairwise Fst values and their significance was obtained by 10000 permutations in Arlequin49. Due to the fact that population sample sizes vere highly unequal in some cases  in R software50. As in AMOVA, Portuguese population was not included in the correlations due to the lack of parasite data. Due to the extremely small sample size Greek population was excluded from the regression analysis. Mantel test implemented in adegenet R package51 was used to test for Isolation By Distance (IBD) between individuals, separately for parasite and host (again excluding Portuguese population). This test compared the similarity of matrices of genetic (Gst52) and geographic  distances for individual samples (R code available in the Supplementary Methods). To control for the effect of distant populations with small sample size on the results of Mantel test, we re-calculated the test on a dataset excluding Greek and Cyprus populations.To test for correlation between population divergence of the host and its parasite, we estimated a linear regression of their pairwise population genetic distances  sequence of parasite are stored in GenBank . Overview of all sequences used in this study with corresponding accession numbers (both from public database and produced in this study) are also provided in Supplementary Table In this study, we successfully sequenced and examined 60 individuals of the cephalopod host and 50 samples of their dicyemid parasites.S. officinalis specimens in this study and from available S. officinalis sequences in public databases  were found to be genetically distinct from the rest of the Sepia officinalis reference sequence (see the methods), the other includes a few samples from the Eastern Mediterranean in Cyprus and Aegean sea in Greece  the resulting sequences seemed to belong to a different dicyemid species, possibly Dicyema sp. or Dicyemennea sp. (Accession numbers MN310702-MN310704). In three cases  the resulting sequencing chromatogram showed double peaks in one position (a different position in each sequence) which suggests that more than one COI haplotype may be sometimes present in the sample, possibly because multiple individuals were used for DNA extraction. We arbitrarily chose the higher peak for nucleotide assignment for further analyses. We verified the species identity of our COI sequenced samples with 18S sequencing. All 18S sequences, except one , were identical to each other and were also identical to one of the Pseudicyema truncatum sequences available in genbank . However, there is only 97% match to the other available Pseudicyema truncatum sequence . SOIR11 sample showed only 94% match to other acquired 18S sequences and was not closely related to any available dicyemid 18S sequence, supporting the view that this cuttlefish specimen was infected by a different dicyemid species to the one this study focused on. A list of samples for which 18S sequence is available is provided in Supplementary Table Based on morphological examination of the stained preparations, 18S sequence and confocal microscopy Fig. , we tentPopulation structure of the parasite showed an interesting difference in comparison to the host. In majority of the comparisons, the basic population characteristics  revealed lack of low and high frequency polymorphisms, which is usually interpreted either as a sign of past population bottlenecks or population admixtureue Table .Regression statistics of pairwise population distances of the host and parasite showed very high level of co-diversification Fig. . The sloIn the case of parasites, we observed higher molecular variance between populations than within populations in AMOVA , the inclusion of several distant localities  allowed for assessing the diversity along the whole West-East axis of the Mediterranean Sea. At this large perspective, the distant localities are genetically completely disconnected and do not share common haplotypes . Although the number of samples is low for the east localities (Greece + Cyprus), this picture is consistent with the biological predispositions of 54). Second, the potential of the AO front as major determinant of population structure is suppressed by biological traits of the organisms. In such case we would expect to see either mixed population across the whole area  or strong fragmentation which would “hide” the effect of any physical barrier. However, in our data, we did not see any of these two possibilities. Instead, we found two distant genetic lineages covering disjunct geographic areas but overlapping at the Sardinian coast. In fact, the Sardinian samples proved to be genetically the most diverse. As shown in the haplotype network . It might also be relevant to mention that in their analysis on connectivity among various Mediterranean areas, Andrello et al.56, although working with biologically very different model, detected Sardinian coasts as the localities with the highest “betweenness centrality”.This scenario suggests a prominent position of the Sardinian coast as a determinant of genetic diversity, independent on the known oceanographic factors (i.e. the currents and fronts). While the current data do not allow for identification of this spot as a contact zone or source of the genetic diversity, it is interesting to note that similar picture of the diversity at Sardinian coast was obtained for the octopusges Fig.  in55. It57. However, we believe this was not the case in our dataset. Based on the lack of diversity in 18S dicyemid data and the congruent pattern of increased local diversity in Sardinia for both the host and its parasite we believe that results of the correlations were not affected by artificial pooling of spatially subdivided populations. To increase the power of IBD tests it is recommended to complement the analyses with multilocus nuclear datasets to increase power of the test57. Addition of such markers, despite difficult for the small bodied parasites nested in host tissue, would be highly beneficial for future studies.The comparison between the host and the parasite population structures reveals two conspicuous patterns. First, there is no straightforward correspondence between the genetic origin of the host and the parasite, and second, the overall degree of genetic diversification is lower in the parasite than in the host , the local diversities might be affected by demographic processes. Particularly, parasites may regularly undergo bottlenecks removing considerable portion of the diversity. In our data, a convincing example is provided by the best sampled population from the Adriatic Sea between the Croatian and Italian coasts. The host sample from the Croatian coast is genetically diversified, encompassing 11 different haplotypes, which are evenly distributed  and most of them are shared with the host samples from the Italian coast Fig. . In cont44, the whole picture  strongly suggest a bottleneck followed by an expansion. This view is also well compatible with the narrower geographic distribution of the parasite’s eastern Adriatic cluster  in comparison with the host’s genetic cluster 3, which encompasses the Croatian as well as the Italian and Sardinian samples, often with shared haplotypes. The resulting scenario thus includes a diversified host population, genetically interconnected between the Croatian and Italian coasts, and a recent recolonisation of the local Croatian population with a single genetic lineage of the parasite (i.e. bottleneck with early stage of the following diversification). This replacement/recolonisation scenario is also well compatible with the high degree of the incongruence between the geography-genetic patterns in the host and the parasites  values of the neutrality tests statistics Table . Althoug57, we use these statistical parameters rather as an accessory evidence while the main population patterns are derived from the haplotype networks. When summarized, these patterns show that at the big scale, both the hosts and the parasites are strongly diversified and their populations are genetically rather fragmented. This is documented not only by the lack of shared haplotypes among the distant localities, but also by considerable genetic distances among the haplotypes (and many missing haplotypes). In this sense, the character of the parasite’s genetic structure reflects the basic fragmentation and diversity of the host’s populations. At the finer  scale, the parasite’s genetics/geography pattern only partially reflects the distribution of the host. This shows that in local populations the extinctions and replacements take place regularly, leading to the genealogical incongruencies between the hosts and the parasites, and to the decrease of genetic diversity in the parasite. This scenario of reduced genetic diversity is in line with assumptions made by Maze-Guilmo et al.19 for hermaphroditic parasites with an asexual stage, which seems to be a common mode of reproduction in all dicyemids23.Considering possible bias of the population statistics due to the small sample sizes for some localities and a potentially low power of Mantel tests and mtDNA to uncover IBD patternsSupplementary Information