Rhizophora mucronata and R. stylosa across their distributional range, by employing a suite of 20 informative nuclear SSR markers.Mangrove forests are ecologically important but globally threatened intertidal plant communities. Effective mangrove conservation requires the determination of species identity, management units, and genetic structure. Here, we investigate the genetic distinctiveness and genetic structure of an iconic but yet taxonomically confusing species complex R. mucronata and R. stylosa, and potential hybridization or introgression between them. We investigated the population genetics of each species without the putative hybrids, and found strong genetic structure between oceanic regions in both R. mucronata and R. stylosa. In R. mucronata, a strong divergence was detected between populations from the Indian Ocean region (Indian Ocean and Andaman Sea) and the Pacific Ocean region . In R. stylosa, the genetic break was located more eastward, between populations from South and East China Sea and populations from the Southwest Pacific Ocean. The location of these genetic breaks coincided with the boundaries of oceanic currents, thus suggesting that oceanic circulation patterns might have acted as a cryptic barrier to gene flow.Our results demonstrated the general genetic distinctiveness of Rhizophora species. We outlined the genetic structure and identified geographical areas that require further investigations for both R. mucronata and R. stylosa. These results serve as the foundation for the conservation genetics of R. mucronata and R. stylosa and highlighted the need to recognize the genetic distinctiveness of closely-related species, determine their respective genetic structure, and avoid artificially promoting hybridization in mangrove restoration programmes.Our findings have important implications on the conservation of mangroves, especially relating to replanting efforts and the definition of evolutionary significant units in The online version of this article (doi:10.1186/s12862-015-0331-3) contains supplementary material, which is available to authorized users. Mangrove forests are ecologically important but globally threatened intertidal plant communities . DespiteRhizophora. In the Indo-West-Pacific (IWP), Rhizophora consists of three endemic species , a variant of R. mangle from the Atlantic-East Pacific (AEP) that colonized the IWP (R. samoensis) [R. × annamalayana (R. apiculata × R. mucronata) and R. × lamarckii (R. apiculata × R. stylosa) [Rhizophora is the most popular genus for mangrove restoration in the IWP [Rhizophora species R. mucronata and R. stylosa, has remained elusive. Whereas Rhizophora apiculata is morphologically [R. mucronata and R. stylosa, the latter two species are morphologically and genetically similar, and were even suggested to be variants of the same species [Rhizophora mucronata and R. stylosa are dominant in the west and east IWP, respectively [R. mucronata and R. stylosa were reported to be dominant in Japan, even though only one common morphotype was observed [R. mucronata and R. stylosa, presenting practical challenges to effective conservation.One of the most pressing species identity issues in mangroves concerns the iconic genus moensis) , and twostylosa) -18. Rhiz the IWP -21, yet ogically ,22 and gogically ,23 distiectively ,25, withectively . These pobserved ,27,28. TR. mucronata and R. stylosa in Malaysia [Recent molecular studies are yet to resolve the taxonomic confusion between the two species. Phylogenetic analyses across the IWP with chloroplast DNA (cpDNA) and nuclear ITS sequence data did not support monophyly for either species, suggesting that they are genetically proximate and/or have experienced gene flow via introgressive hybridization ,29. ThisMalaysia . NeverthMalaysia  and nuclMalaysia  were ablR. mucronata and R. stylosa, we collected both species from their entire distribution range  across all populations was 0.108 and 0.097 for R. mucronata and R. stylosa, respectively .All loci were polymorphic, with the total number of alleles ranging from six to 16 per locus   was found in 85% and 67% of R. mucronata and R. stylosa populations, respectively  of the detected heterozygote deficits were associated with a particular population: 19 loci were from the R. mucronata population in PH1, and 18 and 15 loci were from the R. stylosa populations in MIC and FI2, respectively. Based on the null allele frequency estimated by FREENA, null alleles were potentially implicated  in 30.1% of all population-locus combinations , was comparable when categorizing populations by species or by oceanic regions Table . BetweenFST) estimated at 0.737 averaging all loci and populations (p < 0.001). All pairwise population genetic differentiation was significant at the p < 0.001 level, except between adjacent R. mucronata populations MA1 and MA2 , and R. stylosa populations VA1 and VA2 . Pairwise FST estimates are listed in Additional file R. mucronata and R. stylosa, as well as among oceanic region in each species  an overlap of R. mucronata individuals from Bali Sea (IN4) with R. stylosa individuals from northwest Pacific Ocean (MIC). Model-based individual assignment via STRUCTURE was in agreement with the PCoA results. We found strong support for two genetic clusters among our samples that generally corresponded to the respective species  populations from West Indian Ocean, Arabian Sea, Bay of Bengal and Andaman Sea, and (2) populations from Malacca Strait, South China Sea, Bali Sea and West Pacific Ocean   supported two genetic clusters K = 2) for both a Figure . In R. mn Figure A. Low len Figure B. The ge for bothR. mucronata and R. stylosa. Even though the genetic proximity of these two species is unquestionable—as all 20 SSR loci were successfully amplified in both species—we are confident of the genetic distinctiveness between both species based on two lines of evidences. First, a large proportion of alleles detected in our study were unique to each species. This indicated that the detected genetic divergence was not merely a difference in allele frequency, which may be prone to the effects of population processes such as bottlenecks and genetic drift [R. mucronata and R. stylosa were collected). Therefore, these two species remained as distinct genetic entities even in close geographical proximity, either via reproductive isolation or the fixation of alleles resulting from historical vicariance.Our study provides strong evidence of the genetic distinctiveness between ic drift . Second,R. mucronata population PA1 and R. stylosa population MIC despite a clear inter-species genetic divergence in all other populations. As most of the individuals in these two populations tend to have admixed genetic characteristics despite the clear morphological assignment to one species, we inferred that hybridization and continuous introgression might be occurring in these two populations. Genetic evidence recently confirmed the presence of hybrids between R. mucronata and R. stylosa [R. mucronata and R. stylosa, though few, raise doubts on the integrity of these two species and the most appropriate species boundary applicable to them.Due to the fine resolution afforded by polymorphic SSR markers, our study also detected admixed genotypes in  stylosa ,31, even stylosa . TherefoRhizophora (estimated at 50 million years) [R. mucronata and R. stylosa were as postulated in East Africa and Australasia, respectively [et al. [R. mucronata and R. stylosa might be much wider and located further eastward, between Southeast Asia and Micronesia.Hybridization or introgression between two genetically distinct species is not uncommon among coastal organisms and is usually attributed to a recent overlap of distributional ranges following historical geographical separation ,33. The n years)  and its ectively , then thectively . Our dat [et al. , suggestR. mucronata and R. stylosa, investigated independently after removing putative hybrids, demonstrated a close association between the genetic structure and oceanic region. This supports the general genetic patterns in marine and coastal species whereby ocean currents act to maintain gene flow within an oceanic region and prevent gene flow between oceanic regions [By excluding genetically mixed individuals from the analysis, our study was able to provide a more definitive representation of the intra-species genetic structure. The phylogeography of  regions .Rhizophora mucronata, a strong divergence was detected between populations from the Indian Ocean region (Indian Ocean and Andaman Sea) and the Pacific Ocean region . The dichotomous genetic differentiation into Indian and Pacific Ocean lineages have been reported in other mangrove species [R. mucronata population IN4, located at the boundary between the two oceanic regions, was an exception to this dichotomous division. Our results showed that IN4 was included in the Pacific Ocean genetic cluster, thus indicating that it either shared the same ancestry as, or had maintained frequent gene flow with, the other populations from the Pacific Ocean. The genetic break we detected at the northern boundary of the Malacca Strait concurred with an earlier study involving a smaller geographical coverage of Southeast Asia [In  species ,37 and c species ,39, and ast Asia . An analast Asia .R. stylosa, though the genetic break was located more eastward, between populations from South and East China Sea and populations from the Southwest Pacific Ocean. This was supported by a previous study which found a genetic disjunction between populations from the Malay Peninsula and Japan [R. stylosa populations from the Pacific islands located around the equator, as well as cross-comparisons with other sympatric mangroves, such as Bruguiera gymnorhiza.A dichotomous divergence was also observed in nd Japan . The locnd Japan —that maynd Japan . The detR. apiculata, R. mucronata and R. stylosa shared similar genetic structure [et al. [et al. [R. mucronata and R. stylosa.Our findings differed from that of a previous investigation using chloroplast DNA and nuclear ribosomal DNA, which showed that tructure . Lo et a [et al.  reported [et al.  represenR. mucronata and R. stylosa provide a basis for the definition of ESUs in these species. Even though the exact levels of molecular phylogenetic distinctiveness required for the definition of ESUs are still debatable [The genetic clusters we identified in ebatable ,45, the ebatable  to verifR. mucronata and R. stylosa populations. Excessive homozygosity has been shown in Rhizophora [Rhizophora species have been shown to be self-compatible [R. stylosa, which exhibits a typical fertilization rate of only 3-4% under natural conditions [Rhizophora. Inbreeding in R. mucronata and R. stylosa could be similarly expected in naturally fragmented mangrove habitats, as fragmentation reduces pollen availability and the number of pollen donor in wind-pollinated plants [Rhizophora, we interpret this as a result of inbreeding rather than the presence of null alleles.In this study, low levels of genetic diversity and heterozygosity were widespread in izophora , and is izophora ,47, the izophora ,49 or inizophora -52. Sincizophora . Inbreedizophora . Indeed,mpatible ,55,56, pmpatible ,57. For nditions . Thus, pnditions , may be d plants ,61. SincR. mucronata and R. stylosa. By employing a suite of 20 informative nuclear SSR markers, we demonstrated the general genetic distinctiveness of R. mucronata and R. stylosa, and potential hybridization or introgression between them. Since inter-species gene flow was implicated, we investigated the population genetics of each species without the putative hybrids, and found strong genetic structure between oceanic regions in both R. mucronata and R. stylosa. Both species showed a dichotomous genetic divergence among their respective populations. Even though the locations of the genetic break were different for each species, they coincided with the boundaries of oceanic currents, thus suggesting that oceanic circulation patterns might have acted as a cryptic barrier to gene flow.Our study represents the first population genetic studies covering the entire distributional range of the species complex Rhizophora species. Previous studies on Californian seagrass revealed that unintentional anthropogenic mixing of two genetically distinct species in a transplantation effort might have promoted recent hybridization and introgression between them [R. mucronata and R. stylosa by outlining their respective genetic structure and identifying geographical areas that require further investigations.Our findings have important implications on the conservation of mangroves, especially relating to replanting efforts and the definition of ESUs in een them . This hiR. mucronata and R. stylosa samples were collected from a total of 24 and 12 populations, respectively, from the entire Indo-West Pacific region   and observed heterozygosity (HO) were calculated using GenAlEx 6.5 software [AR) and heterozygote deficiency (estimated by FIS and assessed at the P < 0.05 significance level). Allelic richness was rarefied to the minimum sample size of 16 individuals.To estimate the genetic diversity within population, expected heterozygosity , from K = 1 to K = 15. Each run consisted of 106 replicates of the Markov chain Monte Carlo (MCMC) after a burn-in of 105 replicates. The most likely number of population clusters was estimated by the ∆K parameter [The pattern for genetic differentiation was visualized via one individual-based analysis, the Principal Coordinate Analysis (PCoA), and two population-based analyses, an unrooted consensus neighbor-joining (NJ) tree and a Bayesian model-based clustering method implemented in the software STRUCTURE . PCoA waulations . The STRarameter  using tharameter .AMOVA analysis was conducted to determine the partitioning of genetic variation under two scenarios: 1) populations were grouped according to species; and (2) populations were grouped according to oceanic region regardless of species (refer to Table  populatiFST) averaged across all loci, and pairwise FST estimates between all population pairs [For each species, population differentiation  between two species were identified and removed from the following analysis. The hybrids were defined as individuals with < 90% of its genotype having an inferred ancestry from either species when K = 2 for the inter-species STRUCTURE analysis. The genetic clusters were determined using STRUCTURE  with thehttp://datadryad.org/resource/doi:10.5061/dryad.42711/1.The data set supporting the results of this article is available in the Dryad repository , http://


Stuckenia, the wide geographic range of S. pectinata and S. filiformis make them suited for examination of topographic and climatic effects on plant evolution. Using nuclear ITS sequence and ten chloroplast sequences, we conducted comparative phylogeographical analyses to investigate their distribution regions and hybrid zones in China, and compare their phylogeographical patterns and demographical histories. These two species were allopatric in China. S. filiformis occurred only on the Qinghai-Tibet Plateau (QTP), whereas S. pectinata occupied a wide range of habitats. These two species formed hybrid zones on the northeastern edge of QTP. Most of the genetic variance of S. filiformis was between the southern and eastern groups on the QTP, showing a significant phylogeographic structure. The geographical isolations caused by the Nyenchen Tanglha Mountains and the Tanggula Mountains promoted intraspecific diversification of alpine plants on the QTP. This study revealed the lack of phylogeographic structure in S. pectinata, due to the continued gene flow among its distribution regions. The ecological niche modeling showed that the distribution ranges of these two herbaceous species did not contract too much during the glacial period.In the aquatic genus  Phylogeographical patterns in China did not show an expected contraction-expansion pattern, which is in accordance with the geological records showing that no unified ice sheet had developed in China during the QuaternaryChina consists of the following five major regions: north China, subtropical  China, southwest China (Yungui Plateau), northwest China and Qinghai–Tibetan Plateau (QTP). In north, subtropical and southwest China, the predominant response of temperate plant species to Quaternary climatic change appeared to be one of range fragmentation, vicariance, and population isolation with limited or no spatial demographic expansion6 km2) plateau on earthin situ survival on the central platformSpecies distributions in the western high mountain areas of China were affected by drastic changes in topography and altitude due to the uplift of the QTPCeratophyllum demersum and Stukenia pectinata. Most of the widespread aquatic species excel in asexual reproduction with a variety of clonal propagules. They are also capable of long distance dispersal by seeds and specialized vegetative structuresStuckenia (Potamogetonaceae) is characterized by long stipular sheathes, tubular leaves with air channels bordering the midrib, flexuous peduncles, and subterranean tubersS. pectinata (L.) Börner, while S. filiformis (Persoon) Börner is mostly distributed in America and Europe, and extends to Western, Northern and Central AsiaFew phylogeographical studies in China were conducted on herbaceous aquatic species. Aquatic plants are widespread compared to terrestrial plants. Many aquatic species are globally distributed, such as Stuckenia species show a wide range of morphological variation. The most reliable characters which separate these two species are the structure of the leaf sheaths and the size of the fruitsS. pectinata the margins of the sheaths are free and often overlap, and the fruits are longer than 3.3 mm. S. filiformis, by contrast, has sheaths with margins which are united to form a tube, and fruits which are usually shorter than 3 mm, and do not exceed 3.2 mmS. × suecicus has been described by Hollingsworth et al.et al.Stuckenia hybrids are extremely difficult to distinguish from their highly variable parental species. Thus molecular techniques have been used to investigate the nature of some putative Stuckenia hybrids8S. pectinata and S. filiformis make them suited for examination of topographic and climatic effects on plant evolution and speciation. Herein we report on the comparative phylogeography of S. pectinata and S. filiformis in China. Chloroplast (cp) DNA is maternally inherited in Potamogetonaceae by experimental hybridsS. pectinata and S. filiformis across their distribution ranges in China.Phylogeography is concerned with principles and processes governing the geographical distributions of genealogical lineages, especially those within and among closely related speciesS. pectinata were mainly distributed in the temperate regions and the Yungui Plateau (YGP), whereas its specimens were rare in subtropical China and the QTP. In contrast, S. filiformis were mainly distributed on the QTP. However, its specimens were scarcely found on the high altitudinal and arid northwestern QTP, and were mainly restricted to the middle altitudinal and moist southern and eastern QTP. Our specific objectives were to use DNA barcoding, ecological niche modeling and phylogeographical analysis to (1) investigate the distribution regions of these two species in China and find whether they have sympatric regions and hybrid zones, and (2) compare their phylogeographical patterns and demographical histories as two closely related aquatic species.The specimen records showed that these two species were allopatric in ChinaS. filiformis and S. pectinata were 682 bp and 673 bp respectively. A total of 31 base substitutions and two length variants were found between these two species . Three heterozygous genotypes  with additive pattern of ITS sequences were also generated in S. pectinata by cloning their ITS sequences. Each population of S. filiformis and S. pectinata only had one ITS genotype. All hybrid individuals of S. × suecicus (S. filiformis × S. pectinata) possessed an additive pattern of ITS sequences. When sequences were cloned from them, these hybrid individuals yielded two types of ITS sequences, which were identical to either S. filiformis or S. pectinata.The nuclear genotypes of each sampling population were recorded in  species . Only onS. filiformis and S. pectinata were shown in S. filiformis did not occur outside of the QTP. In accordance with the specimen records, S. filiformis had a discontinuous distribution on the QTP. It scarcely distributed on the high altitudinal and arid northwestern QTP. This species mainly occurred at intermediate altitudes ranging from 3000–4800 m on the southern and eastern plateau. S. pectinata occupied a wide range of habitats in northwest China, north China and southwest China  and heterozygous genotypes  of S. pectinata were common in China (S. × suecicus (S. filiformis × S. pectinata) occurred in the northwestern YGP and the northeastern and southern QTP  of S. filiformis and S. pectinata were estimated to be 0.797 and 0.776 respectively.Thirteen and 12 haplotypes were revealed in S. filiformis were structured into two geographical regions: the southern and eastern QTP (S. pectinata haplotypes were shown in The haplotype distributions of tern QTP . NineteeP < 0.001). The ecological niche models under current climate conditions differed greatly between species  comprised seven haplotypes (Hap1-7) located at the southern plateau, whereas the rest two clades (E & F) comprised six haplotypes (Hap8-13) situated in the eastern plateau. In the haplotype network, these two geographical lineages were separated by 14 steps  to the eastern plateau (clades E & F). The reconstruction results showed that S. pectinata migrated from northwest China (clades G & H) to north and southwest China . It diversified in southwest China , and then dispersed from southwest China to north and northwest China (clade O). The migration routes of the widespread haplotypes  were annotated on the distribution map of S. pectinata  was 0.01 individual per generation. The AMOVA results indicated that 75.54% of the total genetic variation of S. pectinata occurred among populations and 24.46% within populations. When considering groupings based on geographical range , we detected little variation among groups (7.46%). The FST and Nm values of S. pectinata were 0.755 and 0.16 respectively.Results of the AMOVA analyses were presented in S. filiformis showed that NST (0.982) was significantly higher than GST , indicating significant phylogeographical structure across the species’ entire distributional range. Spatial genetic analysis of S. filiformis using SAMOVA revealed the presence of two groups of populations with an FCT value of 0.882. This grouping is mostly consistent with genealogical relationships of haplotypes (NST was not significantly higher than GST (P < 0.05). This result implies that if any phylogeographical structure was present within either group of S. filiformis, it was very weak. The permutation test of S. pectinata showed that NST (0.977) was higher than GST (0.954), but not significantly (P > 0.05), implying a lack of correlation between haplotypes and geographical distributionS. pectinata.Permutation test of plotypes . Populatplotypes . HoweverFs test and Tajima’s D test were presented in Fs value  and non-significant Tajima’s D value  supported that S. filiformis did not experience rapid range expansion. Its 95% confidence intervals of growth rates included zero  and Tajima’s D value was non-significant . In addition, its 95% confidence intervals of growth rates included zero  and the adjacent hills . These results suggested that some populations of S. pectinata might disperse into the southern valley of QTP along the Yarlung Tsangpo River and hybridize with S. filiformis there. The cpDNA haplotype of this S. × suecicus population (Hap 1) is identical to the southern populations of S. filiformis. Because no S. filiformis population on the northeastern QTP has the same haplotype, this population of S. × suecicus could not be the descendents migrated from the hybrid zone on the northeastern QTP.In the southern valley of QTP , the hybrid he model  predicteS. × suecicus lived with S. pectinata, while S. filiformis was missing. The postglacial retreat of S. filiformis from the northwest YGP was predicted by the ecological niche model  was identical to the northwest YGP populations of S. pectinata. Because no S. pectinata population on the northeastern QTP is genotype BB, this population of S. × suecicus could not be the emigrants from the hybrid zone on the northeastern QTP.In the northwestern YGP , he model . The cpDS. pectinata were sympatric, which led to the extensive hybridization and introgression among these genotypes , whereas the average haplotype diversity within population was low (hS = 0.092 for S. filiformis and 0.04 for S. pectinata). This is because most of the S. filiformis and S. pectinata populations only contain a single haplotype, which is common in clonal aquatic plants78S. filiformis was assigned between the east and south groups. Because of the marked geographical structure, a very high estimate of interpopulation differentiation was recorded (FST = 0.981) in S. filiformis. In contrast, the genetic differentiation was lower (FST = 0.755) in S. pectinata, due to the lack of differentiation among its distribution regions . Only 7.46% of the cpDNA variation in S. pectinata was distributed among the three distribution regions. Comparing to the extremely low level of gene flow in S. filiformis (Nm = 0.01), S. pectinata showed a higher level of gene flow (Nm = 0.16).Both species had high total genetic diversity at the species level . SAMOVA identified that most of the genetic variance was between the south and east groups (S. filiformis comprises two major lineages. The deep lineages within S. filiformis were dated to occur during the Pliocene (3.93 Ma) far before the LGM. Populations from the southern plateau were found to harbour ancestral haplotypes. Divergence time estimation and ancestral area reconstruction suggested that S. filiformis migrate from the southern plateau to the eastern plateau during the Pliocene , and Hap23 was widely spread in north China and southwest China.S. pectinata , which suggested that considerable gene flow occur among the three distribution regions. All the haplotypes of S. pectinata occurred before the LGM, but only the populations from northwest China were found to harbour ancestral haplotypes (Hap 19 & 21). Molecular dating and RASP analyses suggested that their descendent haplotype (Hap15) migrate from northwest China to north China and southwest China during the Pliocene  during the Pleistocene, and then one new haplotype (Hap 16) migrated backwards from southwest China to north China and northwest China. Followed by this migration, some new haplotypes were formed in northwest China (Hap 22) and north China (Hap 14 & 18). The lack of phylogeographical signal was mostly due to the facts that S. pectinata exhibited a continuous distribution of dominant haplotypes  among its distribution regions, and the gene flows among these regions continued from the Pliocene to the Pleistocene. The lack of geographical isolation among these regions and the ability of long distance dispersal triggered these gene flows among populations of S. pectinata.PERMUT analysis recorded no significant phylogeographical structure in Pliocene . After tS. filiformis maintained in the southern and eastern QTP during the LGM , whereas new haplotypes could diversify in the YGP, and migrate backwards to north China and northwest China. Because the northern part of China and the whole QTP were not covered by large ice sheets during the Quaternary glaciations, some plant species could survive in the north region of China (temperate species) and the QTP (cold-tolerant species) during the glacial period. This comparative study would provide insights into the evolutionary history of plants with similar life histories and distribution patterns in China.This comparative study of Stuckenia species were collected throughout the entire range of this genus in China. For each population, 5–10 randomly selected individuals were sampled at intervals of at least 10 m apart to avoid collecting clones. Fresh leaves were immediately dried in silica gel. Voucher specimens were deposited at Wuhan Botanical Garden, Chinese Academy of Sciences. The latitude, longitude, and altitude of each sampling site were recorded in Stuckenia was recently elevated from a subgenus in Potamogeton to the rank of genusPotamogeton for divergence time estimations, we selected four Potamogeton species  as outgroups for phylogenetic analyses.A total of 87 populations of trnL-trnFtrnD-trnTrpl20-rps12, trnS-trnGrpoB-trnC, psbM-trnDtrnL-rpl32, trnQ-rpS16, ndhAx1-ndhAx2atpF-atpHet al.Total genomic DNA was extracted from silica-gel-dried leaf tissue using a modified CTAB methodNASP v.5.0 to extract nuclear genotypes and chloroplast haplotypesHd) were calculated using DNASP. The geographical and attitudinal distributions of genotypes and haplotypes were plotted on a relief map of China using DIVA-GISWe used DThe global climate database from WorldClim8 generations under the GTR model with gamma, as inferred from MrModeltest v.2.0Genealogical relationships among haplotypes were inferred by TCS v.1.21< 0.05). Thus, an uncorrelated lognormal model was used to describe the relaxed clock. The root of the tree, which is the split between Potamogeton and Stuckenia, was constrained between 23.03–20.44 Ma based on the known fossils of Potamogeton428 generations under the GTR nucleotide substitution model with gamma. We checked convergence of the stationary distribution using Tracer v.1.5.1BEAST v.1.5.4We employed the Statistical Dispersal-Vicariance Analysis (S-DIVA) in RASP v.3.0 FST) and gene flow (Nm) among geographical regions were calculated. The Kimura two-parameter model was chosen, and significance of the variance components was tested with 1000 random permutations.Hierarchical analysis of molecular variance (AMOVA) was performed to characterize the population structure and genetic variation using Arlequin v.3.0GST  and NST were estimated using the program PERMUTGST and NST were compared using a permutation test with 1000 permutations. A higher NST than GST indicates the presence of phylogeographical structure with closely related haplotypes being found more often in the same area than less closely related haplotypesFCT) was retained as the best grouping of populations.Two parameters of differentiation, NASPNASP using Fu’s Fs testD testFs values indicate recent population expansion following a severe reduction in population sizeD values generally suggest rapid demographic expansionsStuckenia species. If the estimate of growth rate is positive and big, but the confidence intervals include zero, it’s quite likely that there is in fact little or no growthS. filiformis (East and South) and the three distribution regions of S. pectinata , we also performed the mismatch distribution analyses, neutrality tests and coalescent simulations.To detect historical population dynamics, mismatch distribution was estimated. The observed number of differences between pairs of haplotypes was compared to the theoretical distribution using the population growth-decline model with DHow to cite this article: Du, Z.-Y. and Wang, Q.-F. Allopatric divergence of Stuckenia filiformis (Potamogetonaceae) on the Qinghai-Tibet Plateau and its comparative phylogeography with S. pectinata in China. Sci. Rep.6, 20883; doi: 10.1038/srep20883 (2016).


Oncorhynchus tshawytscha), was surveyed across three generations using genome-wide approaches. Genetic divergence from the source population was minimal in an integrated line, which implemented managed gene flow by using only naturally-born adults as captive broodstock, but significant in a segregated line, which bred only captive-origin individuals. Estimates of effective number of breeders revealed that the rapid divergence observed in the latter was largely attributable to genetic drift. Three independent tests for signatures of adaptive divergence also identified temporal change within the segregated line, possibly indicating domestication selection. The results empirically demonstrate that using managed gene flow for propagating a captive-reared population reduces genetic divergence over the short term compared to one that relies solely on captive-origin parents. These findings complement existing studies of captive breeding, which typically focus on a single management strategy and examine the fitness of one or two generations.Captive breeding has the potential to rebuild depressed populations. However, associated genetic changes may decrease restoration success and negatively affect the adaptive potential of the entire population. Thus, approaches that minimize genetic risks should be tested in a comparative framework over multiple generations. Genetic diversity in two captive-reared lines of a species of conservation interest, Chinook salmon ( The genetic risks associated with captive breeding are widely recognized. Rearing conditions may result in the relaxation of natural selective pressures and impose artificial selection favoring individuals suited for captivity instead of the natural environment Frankham . Additioin situ population recovery if captive offspring have high reintroduction success. Supportive breeding using hatcheries has become an important component of many recovery plans for anadromous Pacific salmon on the West Coast of North America, where wild populations have steadily declined due to anthropogenic disturbances  because matings that produce no offspring are typically unobserved  was initiated in 1997 in response to declining anadromous spring Chinook salmon returning to the Yakima River, a tributary of the Columbia River, USA . Wild adThe aim of our study was to determine whether managed gene flow between natural and captive environments was effective at reducing genetic divergence relative to a wild founding population over three generations in a Chinook salmon supportive breeding program. We used a population genomic approach to survey genetic variation at 9410 polymorphic restriction site-associated DNA (RAD) markers, including 4405 markers that were anchored to a dense linkage map representing all 34 chromosomes. These markers were first used to test whether genetic divergence occurred in the integrated and segregated hatchery lines over each generation since founding, and to determine whether managed gene flow was effective at reducing differentiation. We also investigated potential mechanisms underlying observed changes. Changes due to genetic drift were examined using estimates of effective number of breeders. Molecular signatures of adaptive divergence, possibly indicative of domestication selection, were detected using multiple outlier analyses. By combining these results, we empirically validated an approach aimed at minimizing potential deleterious genetic effects of supportive breeding, and identified possible causes that might lead to reduced fitness of captive-reared individuals.Oncorhynchus tshawytscha) in the upper Yakima River, the Naches River, and the American River  and integrated (INT) hatchery lines . The segn = 681) from the 1998 wild founders  and hatchery brood years 2002 (F1 Wild and F1 Hatchery), 2006 (F2 INT and F2 SEG), and 2010  were collected from all fish during spawning at CESRF by facility staff or the Washington Department of Fish and Wildlife and stored in 100% ethanol in Olympia, WA. We subsampled tissues  following the animal tissue protocol. Restriction site-associated (RAD) libraries  and expected (He) heterozygosity for each population, and tests for Hardy–Weinberg equilibrium (HWE) at each locus, were computed in the R-package adegenet  for each locus was computed to correct for multiple testing using the R-package qvalue  was used to visualize temporal changes in genetic relationships between the lines and to identify loci that contribute most to population separation. Principal components analysis (PCA) is not optimal for the analysis of population structure because it maximizes total genetic variation but does not account for underlying genetic structure  and temporal methods. To reduce potential bias due to selection, loci that were identified as outliers by FTEMP and Bayescan, as well as DAPC discriminatory loci, were omitted. For each year sampled, we used only four-year-old adults, which represented a single cohort of individuals and thus provided the most appropriate measure of Nb.Effective number of breeders, Nb and 95% parametric confidence intervals were subsequently calculated for all populations , as low return years within a generation have a disproportionate effect on Nb, and multiplied the mean by four to obtain total number of spawners per generation (Ngen). We then calculated the weighted harmonic mean of total spawners for the previous three generations (Wt. Ngen), as past demographic history influences LD estimates of Nb  in each hatchery line. Drift was simulated at 1 × 106 loci for four generations. The model was first run using Nb estimates obtained by the temporal method using all loci . Yet, Nb estimates can themselves be biased by loci under selection. Therefore, FTEMP outlier loci identified in the first run, as well as Bayescan outlier and DAPC discriminatory loci, were removed, and second estimates of Nb were calculated using only non-outlier loci. Estimates of Nb using nonoutlier loci were 466 and 69 for the integrated and segregated hatchery lines, respectively, and final FTEMP simulations incorporated these Nb estimates. However, to test the sensitivity of our results to Nb, we also ran FTEMP simulations using the following estimates: (i) Nb estimates obtained from individuals of all ages  instead of those calculated using only 4-year-olds; (ii) estimates of Ne instead of Nb .n) per generation were also parameterized in the FTEMP simulations, where sample sizes equaled the harmonic means of the number of individuals sequenced in each generation (61 and 57 for the integrated and segregated lines), but randomly varied between 0.8*n and n to account for variation in the number of individuals genotyped per locus per generation. One thousand final FTEMP simulations were performed to account for variability between runs. To correct for multiple testing and control the false discovery rate , low sample sizes (<30), and low neutral FST values (<0.01)  terms, GO Slim terms, and functions associated with each gene were subsequently identified.All loci located within the genomic regions that exhibited overlap among the three tests of adaptive divergence  and expected (He) heterozygosity were similar between the integrated and segregated hatchery lines within each generation, although a small decrease was observed over time . In contrast, the integrated line did not exhibit the same temporal trend of increasing genetic divergence. However, FST compared to the P1 founders was significant for the F2 and F3 INT populations . FST between the F3 SEG and F3 INT populations was also significant , indicating divergence in the two hatchery lines.Population genetic differentiation, o 0.0108 . The F1 F1 hatchery fish and segregated line from the P1 founders  identified 58 PCs to retain, which explained 29.3% of the observed genetic variation among individuals. A plot of the kernel density estimates of individuals along the first discriminant function, which explained 63.7% of the retained variation, showed separation of the founders . The segNb, obtained by the LD method for each sample year largely reflected the effective number of parents that produced the samples  genome. Twenty-two loci, including five outliers, were located within or near annotated RBT genes. While the RBT genes had an array of functions, commonalities among genes were observed. Three genes, including one associated with an outlier locus, were related to the protein ubiquitin, which is important for protein degradation and immune response. Two other genes were also related to the function and response of the immune system. Three genes, with two linked to outlier loci, were involved in the utilization, breakdown, and reception of lipids, while another two genes, including one near an outlier locus, were essential for the production of ribose, a sugar that can be used for energy or the synthesis of nucleotides. Three genes were linked to neurotransmission, including one gene that coded for receptors of gamma-aminobutyric acid (GABA), a major inhibitory transmitter in the central nervous system, and a second gene that has been shown to induce clustering of GABA receptors. Lastly, two of the RBT genes, including one from an outlier locus, were linked to the development of photoreceptors and eye pigmentation. The shared functions of many genes provided insight into the potential targets of domestication selection. Alignment results, as well as gene functions with references, are summarized in The aim of this study was to evaluate the effectiveness of managed gene flow to reduce genetic divergence in supportive breeding programs. This goal was achieved by comparing genome-wide diversity in three generations of integrated and segregated Chinook salmon hatchery lines to their founding wild population. Genetic distance measures at 9410 loci showed that divergence in the segregated line was significant by the second generation. Much of this change can be ascribed to genetic drift, as suggested by the small numbers of effective breeders. However, we also found evidence for a temporal trend in divergence at specific genomic regions, consistent with domestication selection. In contrast, genetic divergence in the corresponding integrated line was marginal over three generations, suggesting that the use of natural-origin broodstock was effective at reducing genetic change in the short term. By comparing contrasting regimes, the results illustrate the range of possible outcomes that may occur when using deliberate gene flow to mitigate genetic impacts in captive breeding programs. This information is therefore highly relevant when weighing the relative risks of different captive rearing strategies.Like many such studies on supportive breeding programs  for the two lines can be compared to the ratio of Nb estimates (Nb INT/Nb SEG) in each generation  estimates reflect the Nb of fish that spawned in 1998 (P1 period). In the P1 period, approximately equal numbers of wild fish spawned in the river and in the hatchery, yet the Nb of fish in the river was over two times greater than the Nb of fish in the hatchery. The result suggests that in this generation, the hatchery environment caused higher variance in reproductive success and decreased Nb, which is consistent with other studies of effective size in hatcheries , were connected to the processing of proteins, lipids, and sugars and may be affected by hatchery feeding regimes. For example, in rainbow trout, the ubiquitin–proteasome pathway, which facilitates protein degradation, was affected by starvation (Martin et al. The two GABA-related genes, one that coded for GABA receptors and a second that induces clustering of GABA receptors, suggest another potential target of domestication selection. GABA has been shown to stimulate the secretion of two gonadotropins, GTH-1 and GTH-2, in rainbow trout (Mañanos et al. FST could equal or exceed values typically observed between natural Chinook salmon populations (e.g., Brieuc et al. The results reported here add to the growing number of studies that have, to date, typically focused on the relative performance of one or two generations of captive individuals released into the wild, and only under a single broodstock management regime. In salmon species, a few such studies documented minimal differences between hatchery and wild fish, including comparable reproductive success (Anderson et al. Determining the impact of different captive rearing approaches on population productivity in species of conservation interest is central to measuring their success, but empirical studies on such species are usually constrained by the natural systems in which they operate. An ideal experiment would demonstrate that integrated fish have greater fitness than segregated fish in the natural environment. However, permitting spawning of the segregated fish in the same environment as the integrated fish would promote gene flow between the lines, and negate both the experiment and the concurrent restoration efforts in the Chinook salmon system we have studied. Therefore, it was necessary to use proxies for fitness by identifying signatures of selection and measuring effective population size, consistent with best practices in genetic monitoring (Schwartz et al. This study is the first to empirically compare multigenerational consequences of alternative management approaches in a nonmodel species of conservation interest using genome-wide surveys, and adds to the range of available tools for assessing risks associated with supportive breeding. Multigenerational observations are advantageous because processes occurring in the wild, including natural selection, could mitigate or exacerbate the effects of captive rearing over time. Our findings provide the first empirical demonstration that using natural-born parents in captive breeding programs is more effective at reducing genetic divergence than using only captive-born individuals. Interpreting these findings in a management context can be challenging on one hand, because determining ‘acceptable’ levels of divergence depends in part on a clear link between the molecular measures we report and population productivity. On the other hand, the study is comparative in nature and considers a range of possible outcomes, an approach that is highly relevant to risk assessment in realistic scenarios Waples . The exp


Populus nigra L., European black poplar, collected from 13 sites across western Europe and grown in a common garden in Belgium. Significant genetic differentiation was observed, with populations from France displaying greater admixture than the distinct Spanish and central European gene pools, consistent with previously described glacial refugia (IBC). Many quantitative traits displayed a bimodal distribution, approximately corresponding to small-leaf and large-leaf ecotypes. Examination of nine climatic variables revealed the sampling locations to have diverse climates, and although the correlation between morphological and climatic differences was significant, the pattern was not consistent with strict local adaptation. Partial Mantel tests based on multivariate summary statistics identified significant residual correlation in comparisons of small-leaf to large-leaf ecotypes, and within the small-leaf samples, but not within large-leaf ecotypes, indicating that variation within the small-leaf morphotype in particular may be adaptive. Some small-leaf populations experience climates very similar to those in large-leaf sites. We conclude that adaptive differentiation and persistent IBC acted in combination to produce the genetic and morphological patterns observed in P. nigra.Identifying processes underlying the genetic and morphological differences among populations is a central question of evolutionary biology. Forest trees typically contain high levels of neutral genetic variation, and genetic differences are often correlated with geographic distance between populations [isolation by distance (IBD)] or are due to historic vicariance events [isolation by colonization (IBC)]. In contrast, morphological differences are largely due to local adaptation. Here, we examined genetic (microsatellite) and morphological (from a common garden experiment) variation in  Identifying the evolutionary processes controlling genetic structure and morphological diversity is a central aim of evolutionary biology. The genetic structures of species have been examined using putatively neutral genetic markers for decades Brown . Geneticet al. et al. et al. et al. et al. Plant morphology, in contrast, is widely considered adaptive Turesson . Phenotyet al. et al. et al. et al. et al. et al. et al. et al. The consequences of these adaptive and neutral processes are not mutually exclusive, and natural populations are expected to experience them in combination  results in genetic differentiation increasing as a function of the geographic distance between populations Slatkin . That iset al. et al. et al. et al. et al. et al. et al. et al. For many species, an extreme geographic isolation took place at the most recent glacial maximum (ending 15 000 years before present), which was sufficient to reduce gene flow among refugia across the Northern Hemisphere. This historic vicariance resulting from subsequent postglacial migration, called isolation by colonization (IBC), is distinct from contemporary IBD and may produce different patterns of genetic differentiation  and served as a proxy for latitude.r programing language (version 3.0.2) using the package stats. The first two climate components were compared to morphological (discriminant factors) and genetic  differences among populations. Correlation between climatic differences and geographic distances between populations was examined using Mantel tests as implemented in genalex v. 6  was conducted as a means of data reduction as implemented in the N = 308 in total) were analysed for neutral nuclear markers (Table Populus Molecular Genetics Cooperative (http://www.ornl.gov/sci/ipgc/ssr_resource.htm); and WPMS_14, WPMS_18 and WPMS_20 , mean alleles per locus (A), effective number of alleles (Ae), observed (Ho) and expected (He) heterozygosity and the fixation index over loci (F), as implemented by GenAlEx v6 , and as individual locus FST, as implemented by genalex v. 6 . Plantings were established on a grid with 0.75 × 2.0 m spacing, surrounded by a double row of the P. nigra in the common garden was examined for 12 morphological characteristics: five leaf traits, two biomass traits and five cell traits. Leaf measures included leaf area, leaf length, leaf width, leaf length:width ratio (LL_LW) and specific leaf area (SLA). Biomass traits were measured as stem height and circumference at the start of the 2006 growing season. Cellular traits were measured from epidermal imprints and included epidermal cell area (mm2), stomatal number, stomatal density (SD), stomatal index (SI) and cell number per leaf (CN). Detailed methods are provided in the In the third year of growth (2006), each ramet of y) using the model y = ax + by + ɛ, where ax is the population mean, by is the genotype nested within each population, and ε is the error. Scores for LL_LW were arcsine-transformed, and SD were log-transformed prior to analyses. This linear mixed model was implemented over genotype means using the lme function provided in the nlme package in r  In addition, phenotypic correlations were estimated for each pair of traits using spss .To explore the variation in individual morphological traits, differences among population means were tested for each trait .To assess overall morphological differentiation of P. nigra: (i) genetic differences calculated as the Euclidian distance between the population means for the first two coordinates of the PCoA, (ii) morphological differences assessed as the Euclidian distance between the population means for the first two factors of the discriminant classification analysis, (iii) climate differences calculated as the Euclidian distance between the population means for the two climatic variables from the PCA and (iv) geographic distance from the latitude and longitude of the site of sample collection. To assess IBD, IBA and infer IBC, each response matrix  were compared to the two predictor matrices (climate differences and geographic distance) using simple Mantel tests .To test for IBA directly, correspondence between pairwise genetic, morphological and geographic distances was assessed using partial Mantel tests The first tested for IBD as the correlation between genetic and geographic differences when controlling for morphological differences . The second tested for IBA as the correlation between morphological and genetic differences when controlling for geographic distances . Partial Mantel tests were implemented using the Isolation by Distance Web Service v. 3.15 , NE3 and SP1 (P < 0.05), indicating these populations have undergone a recent population bottleneck. Results for populations NE3 and SP1 may be influenced by the small sample size in this analysis. The bottleneck in population IT1 may have contributed to increased inbreeding and homozygosity identified as possible null alleles (above).Sign tests for heterozygosity excess at Hardy–Weinberg equilibrium compared to that expected at mutation–drift equilibrium was observed for populations IT1 , indicating gene flow among populations of P. nigra is restricted across western Europe. Per-locus estimates ranged from 0.044 to 0.118  (Supporting Information).Overall genetic differentiation was significant (K) identified by the admixture analyses varied with the method of inference used. The structure harvester method of Evanno et al. . The discriminant factor analysis (DFA) was required to reduce the variation of the highly correlated phenotypic measures to a smaller number of orthogonal factors Table . The firr = 0.582, P < 0.001), genetic and geographic matrices  and morphological and genetic matrices . Weaker but significant correlations were observed between the morphological and climate matrices  and the genetic and climate matrices .Simple Mantel test identified significant correlations between the genetic, morphological, geographic and climatic matrices distance matrices Table . CorrelaP. nigra in western Europe , indicates that IBD has influenced the genetic structure of pe Table . Genetic Rousset  measuresIsolation by adaptation also contributed to the genetic structure of populations, as genetic similarity increased with phenotypic similarity, even when controlling for geographic structure among populations . Analyses of log-transformed data were consistent (data not presented). This result indicates that genetic differentiation at microsatellite loci may be influenced by reduced gene flow between morphologically distinct populations or reflect historic vicariance due to IBC.F1,76 = 28.1, P < 0.001; slope = 0.038, 95% CI 0.023–0.052). Categorical comparisons of small-leaf and large-leaf pairs revealed the trend to be driven by differences among the small-leaf populations. Significant positive relationships were observed in small-leaf to small-leaf comparisons , and small-leaf to large-leaf comparisons , but not large-leaf to large-leaf comparisons . Thus, the genetic and morphological variation observed in the large-leaf populations was fully explained by IBD, with gene flow decreasing as a function of distance. However, when comparing small-leaf to small-leaf, or small-leaf to large-leaf populations, the genetic differentiation between populations resulted from IBA as well, and populations are more genetically dissimilar than their geographic distance would predict.Examination of partial regression plots of the genetic and morphological differences between populations further revealed the putative source of IBA Fig. . IBA waset al. et al. P. nigra revealed distinct morphological and genetic variation in trees from across western Europe and related these patterns to differences in local climate at each site. We conclude that adaptive differentiation and persistent IBC acted in combination to produce the observed genetic and morphological patterns.In the strictest terms, evidence of local adaptation requires that a species displays multiple morphotypes, each having higher fitness in its native habitat than the others, as confirmed through reciprocal transplant experiments , and no evidence was observed for IBD , current genetic structure may reflect historic rather than extant demographic patterns . Attempting to identify biologically meaningful differences, we considered the k of the highest probability maintaining a small variance, K = 5, in which samples clustered according to country of origin, except those from Germany and the Netherlands clustered together. Samples from France were admixed between two genetic clusters. This pattern of admixture and differentiation was more consistent with previous AFLP and microsatellite data , as estimated by the number of alleles in each sample. Human actions may have particularly affected population IT1, sampled along the Ticino River. A previous study reported the depletion of the natural poplar stands in the Ticino River region in northern Italy during the Second World War  experience a climate more similar to the site in Germany than Spain, but display morphology more similar to the Iberian collections (Fig.et al. If morphological differentiation strictly tracks differences in environment, then comparing the current distribution of morphotypes and predicted distribution of environments may provide insight for conservation efforts (Sork ions Fig.. If the Together, these patterns of morphological, genetic, geographic and climate differences indicate that IBC due to serial colonization events likely continues to influence the morphological and genetic structure in a forest tree. Genetic structure was significantly correlated with geographic distance but not climatic differences, consistent with models of IBD or isolation by serial colonization. Morphological variation was correlated with both geographic distance and climatic differences. These patterns confirm that multiple evolutionary processes influence the morphological and genetic structure of long-lived species and indicate that the response to climate change may be influenced by historic factors.


Salvelinus alpinus) populations in northern Fennoscandia, where this species is considered near threatened. We discovered that all populations were genetically highly (mean FST ≈ 0.26) differentiated and isolated from each other. Evidence was found for historical, but not for recent population size bottlenecks. Estimates of contemporary effective population size (Ne) ranged from seven to 228 and were significantly correlated with those of historical Ne but not with lake size. A census size (NC) was estimated to be approximately 300 individuals in a pond (0.14 ha), which exhibited the smallest Ne (i.e. Ne/NC = 0.02). Genetic variability in this pond and a connected lake is severely reduced, and both genetic and empirical estimates of migration rates indicate a lack of gene flow between them. Hence, albeit currently thriving, some northern Fennoscandian populations appear to be vulnerable to further loss of genetic variability and are likely to have limited capacity to adapt if selection pressures change.Variation in presumably neutral genetic markers can inform us about evolvability, historical effective population sizes and phylogeographic history of contemporary populations. We studied genetic variability in 15 microsatellite loci in six native landlocked Arctic charr ( Evolutionary adaptation to abiotic and biotic selection pressures is fuelled by genetic variability. Low genetic variability, or lack thereof, can reduce the rate or even prevent adaptation. Reduced genetic variability is common in small and isolated populations, such as many freshwater fishes landlocked in discrete lakes and ponds e.g. ,2). The . The 2])Although there has been considerable debate around the informativeness of (presumably) neutral marker genes–such as microsatellites–as indicators of populations’ adaptive potential  is the northernmost freshwater fish in the world, with a circumpolar distribution – 1, where n1 and n2 are the numbers of charr in catches of day 1 and 2 respectively, and m is the number of charr recaptured. In addition, immigration and emigration between Galggojavri and Leenanlampi were investigated in the 4.3 km long creek connecting them. The creek is about 3 m wide and 20 cm deep when it drains from Leenanlampi, but it becomes narrower (20–100 cm) and shallower (2–10 cm) soon after. The average channel slope is 3.5 cm/m . The creek was completely blocked with a net fence  near the pond entrance (about 400 m from the pond) for the whole summers in 2011–2013. The width of the creek at the gate site was about 1 m. Fish traps  were located on both sides of the mesh gate, and nets  were set to guide fish into the traps. The fence and traps were checked at least three times a week, and the number of Arctic charr on either side of the mesh gate was counted. Immigrants and emigrants were evaluated based on the fish found at the Galggojavri and Leenanlampi sides, respectively. The fish collected at the Galggojavri side (i.e. immigrants) were photographed and released to the other side to see if they reach to Leenanlampi. They were identified based on size and shape and were distinguishable from emigrants from Leenanlampi as their body lengths were much bigger than those of Leenanlampi fish. We were particularly interested in examining if they can pass a stony stretch near Leenanlampi where they need to climb 1.3 m against a water flow of 1–2 m/s. The fish caught from the Leenanlampi side were released on the Galggojavri side. Since their adipose fins were clipped earlier in the sampling and mark-recapture study, they were distinguished from “true immigrants” from Galggojavri based on adipose fins.To estimate adult population size in Leenanlampi, a mark-recapture study was performed using two 40 m long and 5 m high gill nets  in 2010 (two days in August). Adult population size was estimated according to Chapman's low-bias modification of the Petersen’s estimator . In shorDNA was extracted from fin clips or muscle tissue with silica-based methods ,33 or a FIS and FST /4 /4  with theCoregonus lavaretus) [Thymallus thymallus) [Pungitius pungitius) [Perca fluviatilis) [Although the study populations reside at a watershed divide, three lakes currently discharging into the Baltic Sea used to discharge into the Atlantic Ocean . Out of varetus) , graylinymallus) , nine-spngitius) ,71 and piatilis) . Howeveriatilis) ,74. Giveiatilis) ,73,75–77The clustering analyses identified possible admixtures in a small proportion of individuals in some of the study localities, although none of them are known to be subject to stocking or farming activities. The possible introgressed individuals in Galggojavri and Kilpisjärvi were indicated to have been admixed with the ancestors of Leenanlampi and Somasjärvi, respectively. Since these admixtures were found within the same watercourses, migration might have taken place via the watercourses. Similarly, a possible admixture of Somasjärvi into Urtas-Riimmajärvi was observed within the same watercourse. However, other possible introgressed individuals in Urtas-Riimmajärvi were indicated to have admixed with the ancestors of Galggojavri and Gallajavri currently belonging to a different watercourse. Since it is unlikely that migration could take place from these lakes to Urtas-Riimmajärvi, the possible admixtures might result from artificial transfers by indigenous fishermen in the past, although no such records are available. It should be also noted that the accuracy of clustering and admixture analyses largely depends on the number of markers utilized ,79. GiveNe) with precision and without bias, it can provide a useful proxy for predicting population viability and fitness (e.g. [Ne were below 600. In most cases the estimates suggested effective sizes around 100 or less (average = 84). While these values are very low, especially in the case of the Leenanlampi population with Ne = 7 (95% C.I. = 4–11), they are well within the range of previously reported values from a wide range of taxa [Ne < 500). In addition, two of the populations with Ne < 50 may also suffer from adverse effects of inbreeding depression.Despite the difficulty of estimating effective population size . In . In Ne)ss e.g. –85). Whi. WhiNe) of taxa . Applyin of taxa ,87, all NC). By conducting an exhaustive mark-recapture study, we estimated the size of the census population to be around 300 individuals. This translates to Ne/NC-ratio of 0.02, which is much lower than the median value (0.15) of 66 studies reviewed in Palstra and Ruzzante [Ne = 12–228, we obtain NC = 519–9754 individuals. However, as discussed in Palstra and Ruzzante [Ne/NC-ratios can be considered temporally constant, and hence, caution should be exercised when estimating Ne from NC (or vice versa).Leenanlampi population provides an interesting benchmarking case because of the availability of data on the census population size  and average number (55) of genotyped individuals per population utilized in this study certainly contributed to this. These figures are amongst the highest used in population genetic studies of Arctic charr: typically ≤10 microsatellite markers have been utilized. Nevertheless, an even larger panel of markers–as obtainable for instance by using high-throughput sequencing –and samOur study uncovered a high degree of genetic differentiation among the northern Fennoscandian Arctic charr populations on a very small geographical scale. This differentiation is understandable in light of very limited gene flow and strong genetic drift. Although some of the study populations are connected by pristine rivers and brooks, there is little or no evidence for ongoing gene flow between them neither from genetic or field data. In addition, our results suggest a strong impact of historical factors on the contemporary genetic diversity and effective population size. Given the low genetic variability and small effective size of the Leenanlampi population, it appears to be vulnerable to further loss of genetic variability and may have limited capacity to adapt on changing selection pressures.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.


Trypanosoma cruzi  infection is an ancient and widespread zoonosis distributed throughout the Americas. Ecologically, Brazil comprises several distinct biomes: Amazonia, Cerrado, Caatinga, Pantanal and the Atlantic Forest. Sylvatic T. cruzi transmission is known to occur throughout these biomes, with multiple hosts and vectors involved. Parasite species-level genetic diversity can be a useful marker for ecosystem health. Our aims were to: investigate sylvatic T. cruzi genetic diversity across different biomes, detect instances of genetic exchange, and explore the possible impact of ecological disturbance on parasite diversity at an intra-species level.T. cruzi I  from different major Brazilian biomes with twenty-seven nuclear microsatellite loci. A representative subset of biologically cloned isolates was further characterised using ten mitochondrial gene loci. We compared these data generated from Brazilian TcI isolates from around America.We characterised 107 isolates of Genetic diversity was remarkably high, including one divergent cluster that branched outside the known genetic diversity of TcI in the Americas. We detected evidence for mitochondrial introgression and genetic exchange between the eastern Amazon and Caatinga. Finally, we found strong signatures of admixture among isolates from the Atlantic Forest region by comparison to parasites from other study sites.T. cruzi as a sentinel for ecosystem health, and the impact disrupted sylvatic transmission cycles might have on accurate source attribution in oral outbreaks.Atlantic Forest sylvatic TcI populations are highly fragmented and admixed by comparison to others around Brazil. We speculate on: the possible causes of Atlantic Forest admixture; the role of  Trypanosoma cruzi  infection is an ancient and widespread zoonosis distributed throughout the Americas south of 33′ latitude, where it infects approximately 8 million people  calculated in MICROSAT [K-means clustering algorithm, implemented in adegenet [For the 19 locus dataset, individual level sample clustering was defined via a neighbour-joining tree based on pairwise distances between multilocus genotypes MLGs [evaluated using MICROSAT  . Ar is highest among PARANORTH and PARASOUTH samples in the Eastern Amazon (Ar = 2.027 & 2.134), as well as in the Atlantic Forest (Ar = 2.010) and Tocantins, in the Cerrado (Ar 1.959). While Ar is a useful measure of overall sample size corrected genetic diversity, structured diversity within a population may be overlooked. We thus also calculated mean pairwise allele sharing (DAS) between multilocus genotypes (MLGs) in each population – Table AS values are particularly informative. Diverse populations with elevated standard deviations  are likely to possess intra-population sub-clusters. By contrast genetic diversity is uniformly distributed among samples within populations with low standard deviations about the mean DAS . Observed heterozygosity varied considerably across populations. However, where population sizes (N > 10) are likely to facilitate meaningful interpretation, positive values for FIS prevailed, and by inference heterozygous deficit compared to Hardy-Weinberg expectations  is detailed in the map inset.Given the intense degree of admixture and substructure in several populations we decided not to calculate population specific linkage disequilibrium indices. Substructure is known to inflate such measures and increase the likelihood of a type 1 error . InsteadGPI sequences for this group nonetheless confirmed this group as TcI and no affinities with Tcbat were apparent based on the same target (data not shown).The inclusion of nuclear reference microsatellite profiles from throughout the Americas in Figure GPI gene rejected this hypothesis (data not shown). In contrast, all remaining TcI isolates from Brazil fall alongside their congeners, including isolates from Bolivia and Argentina, but distinct from isolates north of the Amazon basin .TcI diversity in Brazil is clearly considerable by comparison to that in the rest of South, Central and North America. Figure Didelphis albiventris). Given that this pattern of introgression fits precisely with that observed in hybrids in the laboratory [The available data suggest that genetic exchange is a fairly common phenomenon among TcI isolates ,42, whicboratory , and betboratory , recombiT. cruzi genetic diversity within the Atlantic Forest  of the Atlantic Forest region [There is a circumstantial link between Atlantic Forest loss (88% of its former extent ), human t region . It is tT. cruzi diversity may be now relatively rare in South America, especially where human population densities and infections rates have been historically high. The presence of disturbed and admixed sylvatic T. cruzi populations in populous areas has major implications for the effective source attribution and thus future prevention of oral outbreaks [T. cruzi as a sentinel species for ecosystem health.Rather like primary rainforest, ‘pristine’ sylvatic utbreaks . Many suutbreaks . As suchThe authors declare they have no competing interests. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.VL and ML generated the data. VL, ML and LM analysed the data. VL, MM and ML wrote manuscript. AJ and ML designed the study. All authors read and approved the final version of the manuscript.Trypanosoma cruzi I isolates evaluated in this study.Click here for file


Avicenniagerminans and A. schaueriana, using microsatellites markers. As reported for other sea-dispersed species, there was a strong differentiation between A. germinans and A. schaueriana populations sampled north and south of the northeastern extremity of South America, likely due to the influence of marine superficial currents. Moreover, we observed fine-scale genetic structures even when no obvious physical barriers were present, indicating pollen and propagule dispersal limitation, which could be explained by isolation-by-distance coupled with mating system differences. We report the first evidence of ongoing hybridization between Avicennia species and that these hybrids are fertile, although this interspecific crossing has not contributed to an increase in the genetic diversity the populations where A. germinans and A. schaueriana hybridize. These findings highlight the complex interplay between intrinsic and extrinsic factors that shape the distribution of the genetic diversity in these sea-dispersed colonizer species.Mangrove plants comprise a unique group of organisms that grow within the intertidal zones of tropical and subtropical regions and whose distributions are influenced by both biotic and abiotic factors. To understand how these extrinsic and intrinsic processes influence a more fundamental level of the biological hierarchy of mangroves, we studied the genetic diversity of two Neotropical mangrove trees,  These plants establish discrete communities, known as mangrove forests , which aorldwide . Moreoveorldwide ,4.These patterns of species diversity are influenced by abiotic factors, such as oceanography, climate, topography and soil conditions . AdditioSince the 1980s, the genetic variation of mangrove plants at the molecular level has been evaluated using a variety of methods in both biogeographic regions . More reAvicennia L. (Acanthaceae) as models, we evaluated the extrinsic and intrinsic factors that shape the genetic variation of different species of mangrove plants. Avicennia, the most widely distributed genus of mangrove plants [Avicennia species with the largest distribution is A. germinans L., which is distributed across the entire region except for southern Brazil, where A. schaueriana Stapf and Leechman ex Moldenke are dominant , regional  and continental geographic scales   , Table 1A. germinans and A. schaueriana, respectively; localities from which the samples were obtained are indicated with three letter abbreviations. For A. germinans, we collected individuals from two nearby localities in Bragança, Pará, Brazil that experience different tidal influences: one area in which inundation frequency was reduced due to changes in the hydrographic regime caused by highway construction (AgPAa) [A. germinans individuals by their ovate leaves that usually present a blunt apex; by their long, exserted stamens; and by their conspicuously hairy petals. We identified A. schaueriana by their obovate leaves; by their inserted stamens; and by their glabrous inner-face corolla [A. germinans presenting flowers at AgPRC (Each species sample is presented in  (AgPAa) , and ano corolla . Voucherat AgPRC .A. schaueriana mating system analyses , which were isolated to achieve more reliable results using a larger number of loci for both species. The A. schaueriana microsatellites were developed using a method previously used to isolate A. germinans markers [A. germinans [A. germinans and A. schaueriana, respectively. Markers showing intra- or interspecific polymorphisms were considered in subsequent examinations  were performed as previously described  or with A. schaueriana markers, we tested the occurrence of linkage disequilibrium (LD) for all pairs of markers per sample using the FSTAT 2.9.3 software program [A. germinans and A. schaueriana, we considered 22 and 17 polymorphic markers for further analyses, respectively , the expected (HE) and observed heterozygosities (HO), and the fixation index (f) for each sample using GenAlEx 6.5 software [ta) was determined as (1 - f)/(1 + f) assuming Wright equilibrium [Using the MICRO-CHECKER software program , we obsesoftware   by performing a Mantel test in the ade4 software program [ST [A. germinans and 15 for A. schaueriana, with 105 permutations performed to verify the significant correlations.Considering the different sets of microsatellite markers for each species, we used different approaches to evaluate how genetic diversity within each species is organized. We assessed population structure using the summary statistics D  and GST ST  using thST . The rel program , considegram [ST  and D [3gram [ST . The Mangram [ST , consideA. germinans and A. schaueriana samples, respectively, with the number of clusters (k) varying from 1 to 50. The number of PCs was chosen using the optim.a.score function to avoid over-fitting during discrimination. The choice of an optimal k value was based on the Bayesian information criterion provided for each k tested. We also used the model-based clustering method implemented in the Structure 2.3.4 software program, assuming correlated allele frequencies and admixture [a priori information. We performed 50 independent Markov chain Monte Carlo (MCMC) runs for each k, which ranged from 1 to 10 for A. germinans and from 1 to 15 for A. schaueriana, with 5×105 iterations following a burn-in period of 5×105 iterations. The k value that best explained our data was determined using both maximization of the logarithm likelihood of the data, lnL [ad hoc statistic ΔK [Greedy algorithm, with 106 random input orders. For A. schaueriana, to determine fine-scale population structure revealed throughout the analyses, we used the same strategy described above for each of the two clusters we obtained (see below), with k ranging from 1 to 10 for each group. We used an extended model-based approach implemented in the InStruct software [6 MCMC repetitions with a 5×105 burn-in period for each. We also performed hierarchical analyses of molecular variance (AMOVA) using permutation procedures (105 iterations) in the Arlequin 3.5 program [ST analog estimator under the infinite alleles mutation model (IAM) [To further analyze how genetic diversity is structured within each species, we used a multivariate method known as discriminant analysis of principal components (DAPC)  implemendmixture ,39, and ata, lnL , and theistic ΔK . We usedistic ΔK  to addresoftware  to consi program , consideel (IAM) .Avicennia species, we used a different set of markers as only microsatellites that yielded PCR products for both species were considered to reduce marker development bias. We evaluated 20 microsatellites in these analyses, including intraspecific monomorphic markers that showed variation between species  of eventual two-generation hybrids between each “pure species,” employing a different model-based method implemented in the NewHybrids 1.1 beta software [6 MCMC following 5×105 burn-in steps without prior allele frequency information, considering Jeffrey-type and uniform distribution priors for θ and π. We also evaluated the groups identified with these approaches using hierarchical AMOVA to evaluate how genetic diversity is organized between and within A. germinans and A. schaueriana.The same methods used to study genetic diversity and population structure described above, with the exception of InStruct, were used in this investigation. For DAPC analysis, we used seven PCs, which explained 67.7% of the variance using every species sample as ed above ,39, withsoftware . For thiA. schaueriana under the mixed mating model for unlinked markers [tm) and single-locus (ts) outcrossing rates, the outcrossing rate between related individuals (tm-ts), the average single locus inbreeding coefficient of maternal parents (Fm) and the multilocus paternity correlation (rp(m)). Standard error was determined based on 104 bootstraps among families.Using 24 open-pollinated progeny arrays composed of 288 individuals, we evaluated the mating system of  markers  using th markers . AccordiA. germinans and A. schaueriana from the Brazilian Institute of the Environment and Natural Renewable Resources—IBAMA (currently Chico Mendes Institute for Biodiversity Conservation—ICMBio). We confirm that Avicennia germinans and A. schaueriana are not endangered or protected species.We confirm that we obtained two licenses (Nos. 17159 and 17130) to collect the leaves and propagules of A. germinans and A. schaueriana, eight were polymorphic for A. germinans, and nine were informative for A. schaueriana  for this progeny array, which was in accordance with the ta (0.529) estimated for AsAJU, which was composed of only reproductive individuals, with Fm = 0.232 ± 0.065 assuming Wright’s equilibrium. We also observed that biparental inbreeding (tm—ts = 0.151 ± 0.026) contributed to the apparent selfing rate of the progeny, which showed a fraction composed of full-sibs (rp(m) = 0.178 ± 0.057).Regardless of the method used , the results were similar and differed only when thousandths were considered; therefore, only the EM results disregarding null allele outcomes are shown. We observed a predominantly outcrossing mixed-mating system . The observed IBD was even more evident when we evaluated the correlograms of rM and classes of geographic distance , as the most likely number of populations according to the ΔK approach, and k = 8, as an alternative scenario of finer genetic structure based on lnL and a smaller ΔK value peak , based only on ΔK . Therefore, both species clearly exhibit variations in genetic structure on different geographic scales, which is in partial agreement with the IBD results as we found two inferred groups (AgPAa and AgPAb) that were clearly distinguished at the genetic level despite their geographic proximity. In all cases, a correspondence was observed between samples from each locality and the inferred clusters, which also allowed for the inference of admixed individuals and migrants  . For this species, the among-group fixation indexes were significant, although they were lower in terms of the proportion of variation  when fin(15.37%) .ST and D values of 0.583 and 0.549, respectively, which were considerably higher than the values observed for individual species. Pairwise comparisons between samples, both within and between species, also indicated that the interspecific differences were much more pronounced than the intraspecific differences   . No sign process .A. schaueriana to complement the molecular markers developed for A. germinans [We characterized the first set of microsatellites for erminans –24. We oA. schaueriana, we found an intermediate outcrossing rate, which significantly departed from both 0 and 1, indicating that this population has a mixed mating system [a values estimated for A. schaueriana from AsAJU (0.529), we observed that this variable closely approximated the tm estimated via the progeny array (0.542 ± 0.062), indicating that inbreeding is a consistent feature of this population across generations. These results are consistent with the reproductive biology of these species as this tree is self-compatible with a pollinator-dependent generalist pollination system [A. marina [A. germinans progeny arrays from the Pacific coast, we found comparable tm values  and proportions of progeny that shared the same male parent for two of the three progeny, indicating that this species may also exhibit moderate levels of self-fertilization. Considering the evaluations of the A. germinans mating system, substantially lower biparental inbreeding was apparent [m and ts is sensitive to the number of loci, with more markers resulting in values closer to the true difference between these parameters [A. germinans mating system, indicating that inbreeding in these species could be affected by both selfing and mating among relatives. Taken together, this evidence suggests that A. germinans, whose flowers are visited by a wide range of insects [A. schaueriana generally exhibit similar mixed mating systems.Considering the progeny array of g system . Comparin system , similar. marina , which iapparent . Unfortuapparent  (six) anrameters . Howeverrameters  suggeste insects , and A. a of 0.704 for A. germinans and 0.610 for A. schaueriana) and varying degrees of inbreeding for most of the evaluated samples  [Ceriops tagal (Perr.) (Rhizophoraceae) [Kandelia candel (L.) Druce (Rhizophoraceae) [Lumnitzera racemosa Willd (Combretaceae) [As has been previously discussed ,9,60, th= 2 or 6 . This re. mangle . Regardioraceae) , Ceriopsoraceae)  Kandeliaoraceae)  and Lumnetaceae) . ConsideA. germinans and A. bicolor, a species with a limited distribution along the Pacific coast of Central America [A. germinans and A. schaueriana in the Atlantic basin [A. germinans and A. schaueriana sympatry zone. To the best of our knowledge, this is the first report of ongoing hybridization in Avicennia.There is evidence of an ancient introgression between  America , as wellic basin . AlthougST and D values between samples or using the DAPC approach, in which the genetic diversity between each species is much greater than the variation within each species  and extrinsic factors  shape the genetic diversity of A. germinans and A. schaueriana, leading to genetic diversity structured at the micro-, meso- and macro-scales for both of these Avicennia species.Using the first set of microsatellites developed for Rhizophora mangle [Avicennia species. Moreover, we are aware that these different spatial scales imply distinct temporal scales of evolutionary response. Considering this, future efforts will be focused on elucidating the processes that generate and maintain these patterns.Similar patterns of neutral genetic variation organization have been observed in different taxa of mangrove species, which is likely related to their shared colonization ability ,55. For a mangle ,9; howeva mangle . This mua mangle . The gena mangle . HoweverS1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S1 File(XLSX)Click here for additional data file.S1 FigPhotographs of two geographically close environments under different tide regimes (photos by Gustavo Maruyama Mori).(PNG)Click here for additional data file.S2 Fig(PNG)Click here for additional data file.S3 Fig(PNG)Click here for additional data file.S4 Fig(PNG)Click here for additional data file.S5 Fig(PNG)Click here for additional data file.


Ochlerotatus scapularis is a potential vector of filarias and arboviruses in the Neotropics. This species was once typically associated with sylvatic environments; however, cases of synanthropy and urbanization of this species have been increasingly reported in southeast Brazil. Despite the medical relevance of Oc. scapularis, its populational variability is not yet known. To our knowledge, this is the first report describing the morphological and genetic variabilities of this species.Population samples were characterized using the cytochrome oxidase subunit I (COI) mitochondrial gene and wing geometrics. Adult mosquitoes were collected from five sampling sites from remnants of the Atlantic forest embedded in the urban or rural areas of southeast Brazil.st = −0.009 to 0.060) and morphological distances (Qst = 0.47) indicated that populations were not identical. No correlations were noted for phenetic and genetic diversities (p = 0.19) or for genetic or phenetic distances with geographical distances .In the 130 individuals analyzed, 46 COI haplotypes were detected. Haplotype diversity was high and ranged from 0.66 to 0.97. Six haplotypes were present in 61% of the individuals, whereas the remaining haplotypes were less frequent (39%). Wing shape was also highly polymorphic. Differentiation of populations across sampling sites according to genetic distances  contains supplementary material, which is available to authorized users. Ochlerotatus scapularis (Rondani 1848) is a Neotropical culicid widely distributed in the southeastern region of Brazil [ Rondani 848 is a f Brazil .Oc. scapularis can be synanthropic and enter human dwellings [Oc. scapularis preferentially develop in natural temporary pools of water in the soil, they might also exploit permanent natural or artificial water containers [Nonetheless, in southeast Brazil, this species has been reported to occur in sylvan rural and urban ecotones ,4 that awellings , and hasntainers .Oc. scapularis was possibly involved in the transmission of Rocio encephalitis virus in the State of São Paulo (southeast Brazil). This human encephalitis epidemic [The concurrence of insects and humans in this case is particularly problematic, because this mosquito has vectorial competence for several human and animal pathogens such as Rocio virus . Oc. scaepidemic  occurredDirofilaria immitis [Wuchereria bancrofti [Outside southeast Brazil, this mosquito might also be a vector of the worms  immitis  and Wuchancrofti , and theancrofti , Ilheus ancrofti , and Melancrofti . The epiancrofti .Oc. scapularis had not yet been performed. Even the remarkable population polymorphisms regarding chaetotaxy and genitalia, which were empirically reported by Arnell [Heretofore, some key investigations such as biological variability of y Arnell  and Foray Arnell , have noOc. scapularis, we, for the first time, investigated the populational variability of this species. We then compared population samples collected from some southeast Brazil municipalities with epidemiologically relevant conditions by using largely used genetic  and morphological (wing geometry) markers.To better understand the biology of Sampling sites were located adjacent to the remnants of the southeastern Brazilian Atlantic Forest. Between 2007 and 2011, adult mosquitoes were collected using a portable insect aspirator from five locations  of the five populations was comparatively analyzed. Extraction of DNA was performed according to the Vidal & Suesdek . The COITaq DNA Polymerase was used.PCR amplicons of COI were electrophoresed in 1.0% agarose gel in TAE buffer . We used 2 μl of the PCR amplified, add 1 μl of 6× DNA Loading Dye (0.1% bromophenol and 30% glycerol) and 1 μl gel red. The primers 5′-LCO1490 GGTCAACAAATCATAAAGATATTGG-3′ and HC02198 5′-TAAACTTCAGGGTGACCAAAAAATCA-3′ were used for sequencing. For the final reaction 10 μl were added: 2 μl of 5× sequencing buffer, 3.6 pmol of primer (Forward or reverse), 0.25 of the Big Dye, 10–50 ng of DNA and ultrapure water. Reaction conditions were as follows: 96°C (15 s), 50°C (15 s) and 60°C (4 min) for 25 cycles . For thest) test for short divergence time was performed using Arlequin 3.11 software. The genetic distance among populations of Oc. scapularis was calculated using the Kimura 2-parameter (K2P) by using software Mega version 5.1 [In order to quantify genetic polymorphism and compare across populations, the COI gene sequences were aligned in ChromasPro 2.4 . Next, tsion 5.1 . A haplosion 5.1 , and theOc. scapulars because it is a sensitive and cheap technique, which has been increasingly used to describe inter and intraspecific variability [Wings are good biological markers owing to its bidimensionality and heritability . Geometriability ,29-32.The geometry of (right) wings of females from the five populations was comparatively analyzed. Samples sizes were PET, ITA, TRE, PAR, and BUT Table . GeometrMorphological diversity was estimated using the “amount of dispersion” of individuals in the morphospace of PCs. Such dispersion was calculated as follows: plots in the morphospace of PCs (each corresponding to a single mosquito) were digitized using TpsDig software (as performed for wings) in order to register their positional coordinates in an imaginary Cartesian plane. The centroid size of a set of individuals (a population) was calculated using TpsRelW software. Such centroid size was then considered as an indicator of the morphological diversity of a population. Theoretically, the amount of dispersion of individuals (of a single set) in the morphospace of PCs is proportional to the morphological variability of that set  were also included in one of the comparative morphometric analyses. Those species were collected with a similar procedure of Oc. scapularis in locations mentioned in the Additional file Wing images of other species  in the morphospace overlapped partly Figure . Althougtructure .Figure 3The phenogram of Mahalanobis distances determined on the basis of wing shape Figure  and resuOc. scapularis was also high when compared to those of 6 other culicid species  was noted in the five populations. Such high polymorphism might indicate that these populations have not been recently affected by a bottleneck, despite the dramatic fragmentation of the Atlantic Forest during the past 300 years . Similarmorphism . These aOc. scapularis is naturally infected with Wolbachia , and the presence of this endosymbiotic bacterium could decrease the number of haplotypes, as documented by Morais et al. [Culex quinquefasciatus. However, the COI gene in Oc. scapularis still displays a high rate of polymorphism. Another Neotropical species with high genetic polymorphism is An. cruzii Dyar & Knab, which presented 60 haplotypes in 96 individuals, with a haplotypic diversity = 0.98 [An. darlingi Root (a predominant species in South America), from five locations in Colombia [Ae. albopictus (Skuse), a species evolutionarily close to Oc. scapularis, which has only 16 haplotypes from 377 specimens [Oc. scapularis appears to be a highly genetically diverse species. We find it improbable that such discrepancy is because of experimental errors or sampling biases because amplifications/sequencing were replicated and the high genetic diversity is consistent with the high morphological diversity observed.s et al.  in Culexy = 0.98 . NeverthColombia . Anotherpecimens . Thus, OOc. scapularis was high in all populations. Even other Neotropical sylvatic species  showed lesser diversity than Oc. scapularis.In addition, the morphological diversity of Oc. scapularis, we believe that wing shape may be a morphological indicator of genetic variability. A similar interpretation was given in studies of Culex coronator Dyar & Knab [The removal of both isometric and allometric effects of size permitted us to assess the wing geometry almost purely based on its shape. Considering that wing shape of culicids is mainly determined by quantitative polygenic heritage  and was r & Knab .st scores were below 0.07, and gene flow was estimated to be significant. The absence of correlation between genetic and geographical (p = 0.2) distances is also consistent with a weak population structure.Bayesian phylogenetic inference derived using COI data (data not shown) failed to show reliable clusters of populations, and thus, do not suggest the presence of a population structure. Although the interpretations of the phylogenetic relationships among haplotypes cannot yet be inferred, the major haplotypes H1 and H5 seem to be closer to a common ancestor. All or most of the populations shared H1 and H5, and the interpopulation differentiation was low. In fact, all FAnopheles arabiensis Patton and Anopheles gambiae Giles [As H1 and H5 were the only haplotypes shared by all populations, they might be considered as the remnants of an ancestral polymorphism. This might be an alternative explanation to interpopulation migration, as revealed in studies of the retention of mitochondrial haplotypes in ae Giles .Oc. scapularis  are discontinuous across southeast Brazil. Even in the populational sample obtained from BUT, where collections were made from the smallest forest remnant  surrounded by densely urbanized neighborhood, high genetic and phenetic diversities were noted. The observed pattern of population similarity could be explained by the retention of ancestral polymorphisms or current passive migration, or might merely be a result of the colonization history. Among Culicidae, another species that has maintained interpopulation similarity despite wide geographical distribution and habitat discontinuity, is the malaria vector An. darlingi [Remarkably, suitable habitats for darlingi .Wing shape characteristics provided some evidence of an incipient population structure, coherently supported by canonical variates morphospace, Mahalanobis distances, and Qst. The different Mahalanobis distance scores can be interpreted as different degrees of structuration, but the underlying factors to this heterogeneity are still unknown. The phenogram of morphological distances showed that there was no correlation between phenetic and geographic distances. For instance, ITA and PET clustered together despite being the farthest localities. As this analysis is not phylogenetic we could not study in detail the evolutionary relationships between groups, but we can assume that the evolution of wing shape did not follow an isolation by distance pattern.Results from the analyses of wing shape and COI variability were consistent but not identical. While both markers indicated slight populational differentiation, divergence derived from wing shape was more conspicuous. In fact, no statistical correlation was noted between the phenetic and genetic distances . Apparently, wing shape is less evolutionarily stable than the COI gene, as previously hypothesized by other authors . DespiteOc. scapularis, suggesting that they have vectorial competence [Accordingly, wings and COI revealed high morphogenetic variability . This was unexpected because the Atlantic Forest, the original habitat of this species, has been highly degraded in the past three centuries. This finding led us to pose a new hypothesis: Such biological richness might contribute to plasticity or a broad adaptation capacity of this species. This conjecture is in accordance with the findings from previous studies on mpetence ,11, abilmpetence , tendencmpetence . FurtherOc. scapularis, despite the species being from a fragmented habitat. Wing shape denoted incipient population structure and showed that this species is morphologically more diverse than other sylvatic Neotropical culicids. Unfortunately, studies on Oc. scapularis are still rare despite its possible epidemiological relevance. Future investigations need to be performed to better understand the structure and dynamics of the biological variability of this species as well as the possible implications of this variability on the plasticity, synanthropy, and vectorial capacity of this species.We found many morphogenetic polymorphisms in the studied populations of