Nymphon australe, with an apparently large population size compared with other sea spider species, is an ideal target to look for molecular signatures of past climatic events. We analysed mitochondrial DNA of specimens collected from the Antarctic continent and two Antarctic islands (AI) to infer past population processes and understand current genetic structure. Demographic history analyses suggest populations survived in refugia during the Last Glacial Maximum. The high genetic diversity found in the Antarctic Peninsula and East Antarctic (EA) seems related to multiple demographic contraction–expansion events associated with deep-sea refugia, while the low genetic diversity in the Weddell Sea points to a more recent expansion from a shelf refugium. We suggest the genetic structure of N. australe from AI reflects recent colonization from the continent. At a local level, EA populations reveal generally low genetic differentiation, geographically and bathymetrically, suggesting limited restrictions to dispersal. Results highlight regional differences in demographic histories and how these relate to the variation in intensity of glaciation–deglaciation events around Antarctica, critical for the study of local evolutionary processes. These are valuable data for understanding the remarkable success of Antarctic pycnogonids, and how environmental changes have shaped the evolution and diversification of Southern Ocean benthic biodiversity.The evolutionary history of Antarctic organisms is becoming increasingly important to understand and manage population trajectories under rapid environmental change. The Antarctic sea spider  This cosis spp. ,24, Austs, 1902) , Colosseeis spp. ,26, and sing COI ,27.Nymphon australe Hodgson 1902 is the most abundant and frequently collected SO species with wide bathymetric (8–4136 m depth) and geographical ranges. It has been recorded from around the Antarctic continent, the southern tips of South America, South Africa and New Zealand, although New Zealand representatives are considered a subspecies N. australe caecum Gordon, 1944 [N. australe is a single widespread species with intraspecific variation of some characters sometimes overlapping interspecific differentiation [N. australe, based on 57 specimens from the Antarctic Peninsula (AP) [N. australe as a true circumpolar species with distinct genetic differentiation among individuals from different localities and regions.on, 1944 ,28; in getric 8–46 m depthula (AP)  and lateetric 8–46 m depthN. australe populations around Antarctica to understand the wide geographical distribution proposed for the species, and (ii) relate the genetic structure observed with historic events including refugia survival during LGM and population expansion during deglaciations. We examined the genetic structure of the species at a range of spatial (1000s km to 100s km) and bathymetric scales taking advantage of: (i) large geographical coverage of the dataset, one of the largest for benthic SO invertebrates; (ii) wide bathymetric range with individuals collected from 25 to 1260 m depth; and (iii) the unusual and relatively large number of individuals collected at several locations in the EA.The present study aimed to: (i) investigate the genetic differentiation and demographic histories of 2.2.1.N. australe specimens collected by a variety of sampling means from diving to trawling at 69 sites in the SO between 2007 and 2011  aiming to collect, determine and curate well-preserved SO benthic fauna . Expeditn et al.  and Aran [et al. . Sequenc2.2.et al. [p-distance ≤ 1%), discouraging the use of this ITS fragment for within-species level analyses in N. australe. The use of microsatellites to complement this COI dataset was also attempted; however, the developed markers were too conserved. Four markers were monomorphic for all analysed samples with half of them showing no polymorphism even across species boundaries [Most of the samples were submitted to the Canadian Centre for DNA Barcoding as part of the barcoding agreement between CAML  and the et al. . Other get al.  and the et al.  showed vundaries .Nymphon mendosum , an Antarctic species closely related to N. australe [p-distance mean among N. australe sequences was 0.01 while between N. australe and N. mendosum was 0.08. A haplotype parsimony network was constructed using TCS 1.21 [rBayes 3.1.2 [rModelTest v.2 [Uncorrected pairwise distances among the COI sequences were calculated with 1000 bootstrap replicates under a J–K model in MEGA v. 7 . Nymphonaustrale , was incTCS 1.21  with 95%es 3.1.2  using MrTest v.2 . Based oTest v.2 , the HKY2.3.N. australe was measured for each SO region and for the subregions within the EA using DNASP v. 5.00.7 [FST = 0) among SO regions and within the EA, and the corrected p-values for population differentiation among pairs of populations were tested with Arlequin v. 3.0.1 [Based on the sampling localities, COI sequences were grouped in one of four main SO geographical regions: Antarctic Peninsula (AP), East Antarctic (EA), Weddell Sea (WS) and Antarctic Islands (AI). Furthermore, the EA was divided into four subregions: Terre Adélie (TA), Ross Sea (RS), Davis Station (DS) and Bruce Rise (BR). Within the AI, there were two subregions: Bouvet Island (BI) and South Sandwich Islands (SSI) .To infer the spatial genetic structure of v. 2.0.0  in the Rv. 2.0.0 ). The so2.4.N. australe at various spatial and temporal scales related to glacial refugia [D and Fu's SF) were run in DnaSP to estimate deviations from the mutation-drift equilibrium and infer past population processes. The observed distribution of pairwise differences between sequences  was examined in Arlequin under a model of population expansion and the resulting shape of the distribution was compared to a simulated dataset under a spatial expansion model [To detect demographic changes such as population expansion or contraction in  refugia , the numon model ,44 and aon model . Signifiluctuate 1.4 [g) and the θ parameter , the initial value of θ was estimated using the approach of Watterson [We used the coalescent-based method implemented in Fuate 1.4  to estimatterson . All runT) for the analysed COI fragment based on T = τ/(2µk), where τ is the expansion time estimator, µ the mutation rate and k the sequence length [θ0 = 2N0u (before population growth/decline) and θ1 = 2N1u (after the population growth/decline). Here, we apply a 10-fold correction when estimating N. australe population expansion times, as it has been suggested that the short-term mutation rate is 10-fold higher than long-term substitution rates [Molecular clock estimates were used to approximately date the timing of population expansions as in Rogers , calculae length . The demon rates ,51.3.3.1.N. australe had a length of 554 bp and represented 85 unique haplotypes  of which 10 were common haplotypes , while the majority were uncommon haplotypes with only one or two representatives .The final COI alignment of 364 sequences of ntatives . We detek) was lowest in AI (k = 6) and highest in EA (k = 50), although this was probably related to the different sampling effort in each locality given that haplotype diversity was similar in these two locations . Genetic variation was low in the WS, which had around half the haplotypic diversity of the other localities, as well as the lowest average number of nucleotide differences (Π) and mean nucleotide diversity (π). The number and the proportion of private haplotypes were lowest in AI, although this was the region with the smallest sample size . COI pairwise FST relationships and significance comparisons supported the structuring of SO regions , while FST values between EA and AI samples were the lowest, but still significantly greater than zero (FST = 0.10).There was significant genetic differentiation among the four regions sampled around Antarctica . Most haplotypes were found only within a single region except for the AI where a large proportion of haplotypes were found elsewhere. Furthermore, the haplotype network had a predominant star-like pattern at the regional level, particularly for EA (b). Bayesian models computed with Geneland detected three main clusters , cluster B grouped all EA individuals with the SSI (b), and cluster C included all AP individuals (c). Values of cluster membership were approximately P = 0.65 for each cluster locality.A Bayesian phylogenetic analysis did not show segregated, well-supported clades related to region; however, the COI haplotype network coupled with the Bayesian consensus while supporting plotypes a. Most hy for EA b. Bayesis K = 3, : clusters K = 3, a, cluste the SSI b, and clividuals c. ValuesFST results within the AP and WS indicated low differentiation within regions, over 90% of the variation was owing to the differences within populations. Within the EA region, there was significant genetic differentiation among the five subregions sampled , although this was largely a result of the large and significant differences between DS samples and those from all other subregions (FST < 0.025). Bayesian cluster analysis revealed two clusters . Samples from DS were all collected in about 25 m depth, whereas most other EA samples were collected deeper than 200 m. A subset of the TA samples collected by the REVOLTA program from approximately 40 m depth showed no evidence of significant genetic differentiation from deeper TA samples . The AI populations (BI and SSI) were also significantly different , while the WS did not show significant differences  between the easternmost and westernmost populations. Genetic differentiation within AP was significant owing to differences between northeastern and southernmost populations of the AP  seem to indicate that N. australe populations have gone through relatively recent expansions. We obtained a similar pattern in separate analyses of the WS, EA and AP data. Either an L-shaped or a unimodal distribution of pairwise differences was obtained for each region, supporting the existence of a demographic expansion in each region; this pattern was corroborated by the negative and significant Fu's SF test values and the non-significant goodness-of-fit tests. Tajima's D was not significant (p > 0.05) for AP and EA, suggesting there has not been historical reduction in effective population size in these regions  showed non-significant neutrality tests, rejecting the population expansion model  (g = 2187 (s.d. = 170.970) in EA to g = 8151 (s.d. = 403.155) in WS (The on model . Althougctively) . However5) in WS .Sudden growth model analyses detected a population expansion at about 15 thousand years before present (kyr BP) at EA  and an older expansion at the AP (21.2 kyr BP). These estimates (obtained after applying the 10-fold correction) are in agreement with the high genetic diversity calculated for each of the two regions. On the other hand, WS and AI populations with lower values of haplotype and nucleotide diversity seem to have gone through more recent population expansions around 4.3 and 5.9 kyr BP, respectively .Figure 4.N. australe is comprised of regionally distinct populations that appear to have undergone recent population expansion. Here, we show: (i) further evidence to confirm that N. australe, a benthic brooder, is indeed circum-Antarctic; (ii) that the AP and EA populations are genetically more diverse suggesting multiple demographic contraction–expansion events possibly associated with deep-sea refugia, while the low genetic diversity at the WS points to a more recent expansion possibly of shelf ice-free refugia; (iii) N. australe seems to have undergone demographic expansions during the mid-Pleistocene (15–21.2 kyr BP), suggesting multiple LGM refugia; (iv) support for population expansion in the AI associated with colonization from the continent, and (v) no genetic boundaries detected in East Antarctica, except for the clear segregation of samples from the vicinity of DS, an area particularly isolated from the more open habitats sampled off TA.The circum-Antarctic sea spider, 4.1.N. australe as a circum-Antarctic species [N. australe individuals from across Antarctica that together formed a single haplotype network precluding any suggestion of cryptic speciation  within the species. Although the use of a single mitochondrial marker is often considered limiting in the context of detecting recent speciation and inferring spatial genetic structure [N. australe.Our study supports previous work proposing  species . Here, wtructure ,52, our tructure  and ITS tructure ,31. ThusColossendeis megalonyx Hoek, 1881 [Pallenopsis patagonica  [N. australe is thus surprising, and raises questions on how to reconcile a wide distribution, more characteristic of a species with pelagic dispersal, with the life-history traits of the species  ,24); simk, 1881) –57; Amphk, 1881) ,59; Ostrk, 1881) , Nudibrak, 1881) ; Crinoidk, 1881) ,62). By k, 1881) ,64. The the male ). The roale FST, . Unfortu4.2.N. australe  is higher than that for other invertebrates distributed throughout Antarctica including echinoderms [et al. [A. cornigera, in which distant populations on the Antarctic continental shelf (WS and TA) clustered together while the islands segregated as distinct clusters [et al. argue that A. cornigera dispersal is limited by the ACC acting as major dispersal barrier, and by a relatively restricted depth range (max of 1180 m) limiting access to the deep sea. Here, the results could be seen as support for the hypothesis of connectivity between the SO islands and the continent [N. australe (known max depth 4136 m); however, the large and significant FST values found indicate regions are not connected. An alternate explanation for the grouping of samples from the islands with those from the continent is that these geographical populations are isolated and are in the process of diverging, but owing to post-LGM colonization, ancestral genetic signatures might remain.The genetic diversity estimated for inoderms  and arthinoderms  in whichinoderms . The obs [et al.  for the  [et al.  and Antaclusters . Dömel eontinent ,26 being4.3.N. australe populations, as corrected estimates (instead of 40–220 kyr BP with no correction applied) fit well with accepted hypotheses of benthic invertebrate refugia survival and population expansions associated with deglaciation events after the LGM about 20 kyr BP [N. australe populations expansion , as welturqueti ).ex situ, on the shelf of neighbouring continents or sub-AI [in situ on the continental slope and deeper waters of the SO [in situ in shelf refugia [in situ. Our N. australe findings agree with those views, but, different hypotheses apply (2 and 3 above) depending on the region.There is strong evidence that, during the last glaciations, ice sheets extended to cover the continental shelves of Antarctica ; howeverr sub-AI ,77; (ii)f the SO ; and . These time estimates of expansion, 15.5 kyr BP at EA (13.6 excluding the differentiated DS samples), and 21.1 kyr BP at the AP, fit with the dating of post-LGM deglaciation events in these regions . It BP in AP .N. australe populations from EA and AP are genetically diverse possibly owing to repeated colonization events from shallow to deep and deep to shallow and changing landscape owing to ice expansion and retraction, supporting the hypothesis of multiple independent glacial refugia [N. australe could be expected to retain high levels of genetic diversity [It is plausible to assume that  refugia . Also, eiversity ,27,63, diversity .N. australe from the WS are rather low. The strongly reduced diversity, lower estimates of theta and higher growth rates  detected in WS populations compared to EA and AP  belong to a single haplotype that is also the most common in the SSI [N. australe results support the notion of a recent, ‘founder effect’ type of colonization of BI from the WS, rather than a potential insular refugium, a hypothesis already proposed for a crinoid species [The genetic signal from samples collected from BI and SSI AI in  shows a  the SSI . The N.  species . This sc species ,16,27,77N. australe. Effect of homoplasy in an expanding population is a possibility, but also animals being transported from EA by oceanic hitchhiking or rafting, or by drifting on the ACC has been proposed [N. australe, this event seems relatively recent as haplotypes from SSI are mostly shared with EA haplotypes  reveal a lack of geographical structure within the EA. Low FST estimates among localities and Bayesian clustering corroborated a single group , except for the DS samples. In contrast to the AP with its complex topography seemingly facilitating the heterogeneity of populations within the region [N. australe. On the contrary, species restricted to shallow waters might form distinct faunistic communities on rocks and algae [Orchomenella [The iversity . The mosiversity b reveal e region ,27, the e region . It is le region  as is thnd algae  and may omenella . The sliomenella  or movem2 situated in the Vestfold Hills (see www.antarctica.gov.au). Segregation may be attributed either to a depth constriction (a maximum depth of 25 m) or to geographical isolation. This genetic differentiation of the DS samples from the remaining EA individuals is also found in other studies [b), probably because all samples were taken from a relatively small area by SCUBA.Pycnogonids from DS seem to represent the most genetically distinct population. DS is enclosed in a well-sheltered ice-free bay of 400 km studies ,58 and mN. australe genetic structure within EA suggest that the eurybathic condition of the species has allowed gene flow over extensive areas of EA, as there is no evidence of bathymetric constraint for the migration of this species. Individuals collected by the REVOLTA program from TA at 40 m depth clustered with deeper waters samples from EA collected between 200 and 1230 m.Clearly, N. australe is one of the Antarctic brooding invertebrates with the widest distribution. The species shows different demographic histories depending on the Antarctic region, possibly shaped by the characteristics of the deglaciation events post-LGM. The study reinforces the notion of the strong effect of climatic events and environmental conditions on the patterns of diversity and structure in Antarctic benthic fauna. Nymphon australe is a key species for the understanding of microevolutionary forces that could reveal connectivity and dispersal mechanisms and demographic processes in the SO.In conclusion, our results confirm that


Hyalella amphipods. It was found that geographic isolation plays a key role in the evolution of reproductive isolation and divergent morphology and that divergence cannot be explained by molecular genetic variation.Geographic isolation is known to contribute to divergent evolution, resulting in unique phenotypes. Oftentimes morphologically distinct populations are found to be interfertile while reproductive isolation is found to exist within nominal morphological species revealing the existence of cryptic species. These disparities can be difficult to predict or explain especially when they do not reflect an inferred history of common ancestry which suggests that environmental factors affect the nature of ecological divergence. A series of laboratory experiments and observational studies were used to address what role biogeographic factors may play in the ecological divergence of  The nominal species H. azteca  “What role does geographic isolation play in determining the patterns of molecular and morphological differentiation?”, (ii) “How are levels of reproductive isolation related to morphological and molecular genetic differentiation?”, and (iii) “Can variation in reproductive isolation be explained by biogeography?” In order to answer these questions, we surveyed morphological and molecular genetic variation, and experimentally quantified the strength of reproductive isolation among lineages, and the answers to these questions contribute to a foundation for understanding the multifarious processes that shape the evolution of reproductive isolation.The objectives of this study were to address three questions regarding the patterns of reproductive isolation among 2Hyalella—a widespread and abundant taxon of freshwater amphipods distributed across North America. Two of the populations studied herein fit the expectations of the H. cf. azteca morphotype . SMS Hyalella sp. and H. texana are both documented to be endemic to physicochemically stable springs separated by hundreds of kilometers of ambient surface water, suggesting that physiological limitations are responsible for the geographic isolation of these populations. We compared morphological and genetic variation, in combination with attempted mating experiments and study of biogeographic distributions, in an attempt to explain factors contributing to reproductive isolation.This study used five populations Table  belongin2.1Hyalella were established to provide a continuous source of live animals for experimentation and to control for the possibility of morphological differentiation due to phenotypic plasticity in situ. Cultures of Hyalella spp. were collected from the four localities listed in Table Amblystegium sp. and organic detritus ad libitum on a daily basis. Great care was taken when handling cultures to ensure that organisms did not get moved between cultures.Stock cultures of 2.2www.digimizer.com). We also counted the number of dorsal mucronations and calculated the ratio of the length of the longest mucronation to total length.To quantify morphological variation while controlling for potential effects of phenotypic plasticity, cultures from all five populations were reared in common‐garden replicates. As conditions in all stock cultures (described above) were maintained under the same conditions, we anticipated that five generations would be sufficient to control for environmental or maternal effects; a lack of variation in neonate size across all the populations and generations in captivity suggests that maternal effect was not a factor Glazier, . TherefoPrincipal components analysis (PCA) was used to examine morphological characteristics of wild‐caught populations. The PCA analysis included three independent variables: head length/total length ratio and longest spine as continuous variables, and spine count as a meristic variable. PCA was conducted in R using the “princomp” function.Using the same morphological variables, the degree that common‐garden populations morphologically differentiated from wild‐caught populations, if at all, was assessed. Pairwise permutational multivariate analysis of variance (perMANOVA) was used to test for differences across wild‐caught populations, across cultured populations, and between wild‐caught and cultured populations using Bray–Curtis distance and the “Adonis” function using the statistical package “vegan” in R. For each perMANOVA analysis, a sequential Bonferroni was applied to the results. The statistical package vegan in R was used for the perMANOVA analyses  by placing all or part of individuals in microtubes containing a chelating resin , heated to 60°C for 20 min, then 100°C for 20 min. A fragment of the COI gene was PCR‐amplified using the primer TrpPar1 (5′—GTTATATAAACTATTAGCCTTCCAA—3′) paired with either COIaV9 (5′—ACTGCCACAACAGAYAARTAMGACCC—3′) or COIaV10 (5′—ACAGCAACAACAGATAARTARGACC—3′). PCR was carried out with TopTaq (Qiagen) kits in 50‐μl reactions containing 2.0 μl of template DNA. Cycling conditions were 5 min at 94°C, followed by 35 cycles of 45 s at 94°C, 45s at 51°C, and 60 s at 72°C, followed by 5 min at 72°C. PCR products were gel purified and sequenced with TrpPar1 (COI) using an ABI 3730 automated sequencer.A molecular phylogeny based on the mitochondrial cytochrome C oxidase subunit I (COI) locus was constructed in order to analyze the relationship between morphological similarity, geographic factors, and a history of shared common ancestry. During collection of organisms for the establishment of stock cultures, some specimens were preserved in 95% EtOH and stored at room temperature. DNA was extracted from these individuals (blast.ncbi.nlm.nih.gov) which returned 332 Hyalella sequences (including the 15 sequences generated herein) as well as 242 sequences belonging to ten other families of amphipods (a subset of one sequence per amphipod family was randomly selected to serve as outgroups). Of these 332 Hyalella sequences, geographic data were available for 269; therefore, only these 269 sequences were retained for further analysis. Additional sequences belonging to amphipods in the families Chiltoniidae, Gammaridae, Gammarellidae, Ischyroceridae, Lysianassidae, Metacrangonyctidae, Niphargidae, and Talitridae were compiled into an alignment with Hyalella sequences to serve as outgroups and to provide context for the depth of divergence within Hyalella. The resulting alignment was trimmed to 500‐bp to remove missing data using Geneious R9 .All sequences generated by this study were queried in GenBank using a BLAST search  to annotate codon positions. PARTITIONFINDER  and between‐population  mating experiment where we attempted to achieve all possible combinations with respect to both population source and sex Table . PairingAmblystegium sp. and organic detritus ad libitum. Cultures were maintained at a constant 22°C, and water in containers was refreshed weekly.An experimental replicate consisted of one male and female pair. Replicates were placed individually in sealed 150‐ml containers. Each container was given the same sand substrate and fed a diet consisting of A = L–1, where A = age in weeks and L = length in mm . However, geography was found to be an important factor  H. cf. azteca is a paraphyletic complex of populations with appreciable molecular divergence between several distinct lineages despite fitting the usual expectations of a morphological species . However, this could be due to a generation time effect leading to different rates of molecular divergence (Chao & Carr, Hyalella has roughly four generations per year—a much faster rate of reproduction than observed in the other amphipod families discussed herein (Crawford & Tarter, It is remarkable that We also only used a single mitochondrial locus because of the abundance of archived COI sequences for amphipods; however, the rate of divergence may be too rapid at the COI locus to properly estimate relationships with such deep divergence Figure . It is pIt is important to point out that the present study recovered fewer haplotypes than previous authors despite sequencing the same locus and using the same sequences published by other authors on GenBank. This is likely due to the trimming of sequences to much fewer base pairs in order to have a complete alignment as different authors amplified different regions of the COI locus. Therefore, it is likely that variable sites were eliminated that other authors used to identify haplotypes.Divergence in isolation may lead to scenarios that allow for trait deterioration due to genetic drift Bromham, , includiM. L. D. Worsham involved in research design, field collections, laboratory experiments, molecular phylogeny, statistical analysis, manuscript preparation, and maps. E. P. Julius involved in field collections and laboratory experiments. C. C. Nice involved in molecular phylogeny, statistical analysis, and manuscript preparation. P. H. Diaz involved in multivariate statistics and maps. D. G. Huffman involved in field collections, material support and manuscript preparation.Genbank accessions: see Table  Click here for additional data file.


GBS) to investigate genetic patterns in the threatened coral, Acropora cervicornis, across the Florida Reef Tract (FRT) and the western Caribbean. Results show extensive population structure at regional scales and resolve previously unknown structure within the FRT. Different regions also exhibit up to threefold differences in genetic diversity (He), suggesting targeted management based on the goals and resources of each population is needed. Patterns of genetic diversity have a strong spatial component, and our results show Broward and the Lower Keys are among the most diverse populations in Florida. The genetic diversity of Caribbean staghorn coral is concentrated within populations and within individual reefs (AMOVA), highlighting the complex mosaic of population structure. This variance structure is similar over regional and local scales, which suggests that in situ nurseries are adequately capturing natural patterns of diversity, representing a resource that can replicate the average diversity of wild assemblages, serving to increase intraspecific diversity and potentially leading to improved biodiversity and ecosystem function. Results presented here can be translated into specific goals for the recovery of A. cervicornis, including active focus on low diversity areas, protection of high diversity and connectivity, and practical thresholds for responsible restoration.Threatened Caribbean coral communities can benefit from high‐resolution genetic data used to inform management and conservation action. We use Genotyping by Sequencing ( These individual wild reef samples represent a much higher‐density sampling method. For each colony, approximately 0.5 cm apical tips were collected with a razor blade, transferred to 250 μL of chaotropic salt preservative  and stored at 4°C until processing. Overall, samples were collected from 177 individuals across entire Florida Reef Tract , Dominican Republic (n = 29), and Belize (n = 6). In addition, 67 samples from four wild reefs in Florida and two wild reefs in the Dominican Republic (Table Samples for this study were collected between June 2012 and August 2015 from (1) nurseries representing regional populations and (2) individual wild reefs. Nursery stocks are composed of single colonies originally collected from individual reefs spanning the study area  with the goal of minimizing collection of clonemates and maximizing genotypic and genetic diversity. Rarely, several samples in a nursery were collected from the same individual reef, but were presumed to represent nonclonemates due to distance separating colonies and sequencing results of this study. These nurseries contained 2.2Samples (skeleton and preservative) were homogenized using silica beads in original collection tubes, and DNA was extracted using a silica column protocol and a vacuum manifold modified from Ivanova, Dewaard, and Hebert . DNA deg2.3Acropora digitifera genome  were identified using Arlequin 3.5.1 . LOSITAN , which were removed. Finally, clonality was assessed using pairwise genetic distance of eight samples collected from a single colony, calculated in TASSEL using the formula of Endelman and Jannink (n = 8).Called SNPs were quality‐filtered following Drury et al.  to produ Jannink . Samples2.4FST was analyzed in Arlequin (3.5.1), with 10,000 permutations for significance values. Discriminant Analysis of Principal Components (DAPC) was completed using the adegenet package in R  were taken from the population 50 times with replacement, and adegenet  of comparisons  were significantly different from each other but show variable relationships with populations in Florida. FST values ranged from 0.012 to 0.171, including four comparisons between Belize and other populations with FST values greater than 0.1.In total, 265 samples from regional populations and 67 samples from wild reefs were analyzed at 3,136 and 3,165 loci, respectively  although patterns of structure in the FST comparisons between Broward and other Florida populations are not apparent. Among Florida populations, the same outliers were evident, along with strong overlap between Upper Keys, Miami Dade, and Dry Tortugas  vary nearly tenfold from about 0.004 (Dry Tortugas and Belize) to 0.036 (Lower Keys). These alleles may be disproportionately influential and explain some of the spread in DAPC, where higher values correspond to some outlying clusters  . The Lower Keys, Middle Keys, and Broward regions show the highest diversity values, while the Upper Keys and the ecotonal habitats between Broward and Miami‐Dade counties show the lowest diversity , the Upper Keys and Miami‐Dade, and Palm Beach County have not been extensively sampled.There is significant Isolation by Distance within Florida . Although percentage of variation for Among Group and Among Population was larger in the Wild AMOVA than Regional, similar patterns of variation were maintained. Approximately 84% Table b of vari4A. cervicornis   is comparable to other Florida populations. This relationship is like due to the small sample size, but may also relate to the upstream location of this population in the Florida Current , where nonreference alleles are assumed to be errors unless sufficient evidence in the dataset suggests otherwise. Due to multiplexing in the laboratory protocol, reads per individual were limited, which could lead to inflated homozygote calls as the secondary allele was missed, contributing to an underestimation of heterozygotes during read processing. Individuals with low heterozygosity did not disproportionately influence the results; however, this is an important potential bias in most restriction‐digest sequencing that would affect all populations within the study.Observed heterozygosity is much lower than expected for all populations, which may be an effect of biological or technical influences. Potential inbreeding within isolated Symbiodinium  would be homogenized over small scales by gene flow  and protect unique or highly diverse areas  may allow for more efficient and effective conservation of an important foundation species, with cascading effects on individual reefs and eventually throughout the species range.Genetic patterns of Caribbean CD and DL designed and coordinated the study. CD completed library preparation and analysis and wrote the manuscript. SS, EG, EB, KM, KN, CM, MJ, and VG managed nursery resources, contributed samples, and contributed to manuscript review.Acropora cervicornis unfiltered VCF and sampling locations are available on Dryad.The  Click here for additional data file.


Small and peripheral populations often contain low levels of genetic variation. This may limit their ability to adapt to environmental change, including climate warming. In a recent study published in AoB PLANTS, Birkeland, Skjetne and colleagues show that many rare and threatened plant species in the High Arctic archipelago Svalbard harbour low levels of genetic variation. Most of them are probably relicts from the early Holocene warmer period. They have likely experienced strong genetic founder/bottleneck effects due to climatic limitations. Even though temperatures now are rising, it is highly uncertain whether this will be beneficial for these warmth-demanding species. Botrychium lunaria, (2) Carex capillaris ssp. fuscidula, (3) Comastoma tenellum, (4) Kobresia simpliciuscula ssp. subholarctica, (5) Ranunculus wilanderi, (6) Sibbaldia procumbens and (7) Tofieldia pusilla. In addition, we gathered population size data in Svalbard. The Svalbard populations had low genetic diversity and distinctiveness and few or no private markers compared to populations outside the archipelago. This is similar to observations in other rare species in Svalbard and the genetic depletion may be due to an initial founder effect and/or a genetic bottleneck caused by late Holocene cooling. There seems to be limited gene flow from other areas and the Svalbard populations should therefore be considered as demographically independent management units. Overall, these management units have small and/or few populations and are therefore prone to stochastic events which may further increase vulnerability to inbreeding depression, loss of genetic variation, and reduced evolutionary potential. Our results support theory predicting lower levels of genetic diversity in small, isolated and/or peripheral populations and may be of importance for management of other rare plant species in the Arctic.Small, isolated and/or peripheral populations are expected to harbour low levels of genetic variation and may therefore have reduced adaptability to environmental change, including climate warming. In the Arctic, global warming has already caused vegetation change across the region and is acting as a significant stressor on Arctic biodiversity. Many of the rare plants in the Arctic are relicts from early Holocene warm periods, but their ability to benefit from the current warming is dependent on the viability of their populations. We therefore examined Amplified Fragment Length Polymorphism (AFLP) data from regional red listed vascular plant species in the High Arctic archipelago of Svalbard and reference populations from the main distribution area of: (1)  Small, isolated and/or peripheral populations may harbour low levels of genetic variation due to genetic drift, inbreeding, bottlenecks and founder effects . SimilarThe Arctic has been warming at approximately twice the global rate since the 1980s , and speThe remote High Arctic archipelago Svalbard (74–81°N and 10–35°E) is among the best studied regions in the Arctic, with detailed knowledge of the local distribution of species , and mansee Supporting Information—Table S1]), seven study species were chosen as they all were in need of more data to ensure informed conservation decisions. Our aim is to (i) evaluate their vulnerability in terms of population size and genetic diversity in Svalbard, (ii) examine their genetic relationships to populations outside Svalbard and (iii) determine if the Svalbard populations constitute management units with special conservation value.In this study, we gather population size data and examine Amplified Fragment Length Polymorphism (AFLP) data from several red listed vascular plant species in Svalbard. Based on the regional red list from 2006  Carex capillaris ssp. fuscidula, (3) Comastoma tenellum, (4) Kobresia simpliciuscula ssp. subholarctica, (5) Ranunculus wilanderi, (6) Sibbaldia procumbens and (7) Tofieldia pusilla (see ). All species are seed plants, except B. lunaria, which is a pteridophyte. Furthermore, all are herbaceous plants with larger distributions outside Svalbard. However, R. wilanderi is considered an endemic microspecies for the archipelago  c plants .Figure To estimate population sizes in Svalbard, we either counted all visible individuals, or extrapolated the total population size from the number of individuals counted in a smaller area. Tussocks or clusters of clonal plants were treated as single individuals if they were separated by more than five centimetres, although we cannot be entirely sure that they were not connected belowground. All previously recorded localities for the seven species were revisited . In addiKobresia simpliciuscula ssp. subholarctica we were only able to obtain material from a different subspecies, the European ssp. simpliciuscula (Carex capillaris: ssp. fuscidula and ssp. capillaris (fuscidula (Tofieldia pusilla) was also sampled for all study species to serve as outgroup in the neighbour-joining analysis , following the manufacturer’s protocol. DNA from Tofieldia pusilla individuals was isolated using the E.Z.N.A.™ SP Plant DNA Mini Kit, following the protocol for dry specimens . The protocol was modified by adding a freezing step (at −80 °C for 10 min) prior to cell lysis. To increase the final DNA concentration of C. tenellum and T. pusilla samples, the amount of AE buffer was reduced to 30–50 µl, the first eluate (i.e. DNA dissolved in AE buffer) was re-eluted in a second elution step, and incubation was done at 65 °C. The DNA concentration of the samples was measured using a spectrophotometer . Samples isolated with the Ziegenhagen protocol were diluted so that the final DNA concentrations were approximately the same within each species (60 ng/µl). The Qiagen DNeasy Plant Mini Kit and the E.Z.N.A. Plant DNA Mini Kit gave concentrations of approximately 20 ng/µl, which were kept undiluted.Approximately 20 mg of silica dried leaves were placed in 2 ml tubes with two tungsten carbon beads and crushed at 20 Hz for 2–8 min on a mixer mill . To obtain optimal purity and concentration of DNA, two to three different extraction protocols were tested on a few individuals of each species, and the best protocol was used further. DNA from the individuals of see Supporting Information—Table S3]. Finally, 3–4 primer pairs were chosen for each species [see Supporting Information—Table S3]. The 6-FAM EcoRI-primer and all non-labelled primers and adaptors were ordered from MWG  or IDT , while the other fluorescent-labelled primers were ordered from Applied Biosystems/Life Technologies. A set of negatives, replicates and duplicates was included in all final AFLP runs to check for contamination and replicability  was used to generate dominant molecular markers from the sampled individuals . Unambiguously scorable fragments (peaks) in the size range of 50–500 bp were scored as absence/presence, following the approach of see Supporting Information—Table S3] and at population level. Monomorphic markers at species level were excluded from further analyses. Within-population genetic diversity was estimated as the average proportion of pairwise differences between individuals, D  using STRUCTURE ver. 2.3.3 , contradicting the results from STRUCTURE. Due to its uncertain affiliation, the Greenlandic population was omitted from further analyses .Genetic groups were delineated for each species  were run in Arlequin ver. 3.5 (Ranunculus wilanderi and Kobresia simpliciuscula) was inferred by performing multi-locus assignment tests in AFLPOP ver. 1.1 (The source area(s) of the Svalbard populations (except for ver. 1.1 . Geograpsee ). To quantify the adaptation to the current climatic conditions in Svalbard, we used two measures of temperature requirement and rated their rarity (see ). The measures of genetic founder/bottleneck effects and climatic adaptation were summarised in two separate principal component analyses (PCA), using R ver. 3.2.1 .As in r. 3.2.1 . The firBotrychium lunaria, Carex capillaris ssp. fuscidula, Ranunculus wilanderi and Sibbaldia procumbens) to ten (Tofieldia pusilla); all populations were situated within the warmest bioclimatic subzone in Svalbard (the Middle Arctic Tundra Zone). Two populations of T. pusilla and one of Comastoma tenellum were previously unknown. The population sizes ranged from less than five individuals  to more than 2000 . These new population size data led to a downgrading of C. capillaris ssp. fuscidula, T. pusilla, S. procumbens, C. tenellum and R. wilanderi in the 2010 Red List [see Supporting Information—Table S1]. However, T. pusilla was upgraded from ‘Least Concern’ to ‘Near Threatened’ in the 2015 Red List due to a higher weighting of fragmentation of its range. The same year, an adjustment to the IUCN criteria also led to a further downgrading of C. tenellum (now ‘Vulnerable’) and C. capillaris (now ‘Near Threatened’). At present, five of the seven study species are considered threatened in Svalbard [see Supporting Information—Table S1], mostly due to restricted extent of occurrence (criterion B1), limited area of occupancy (criterion B2) and/or a low number of reproducing individuals (criterion D1)  .Botrychium lunaria, Sibbaldia procumbens and probably also in Ranunculus wilanderi  and three genetic groups in B. lunaria (K = 3). However, T. pusilla . Two main STRUCTURE groups (K = 2) were also delineated in C. capillaris , the Svalbard population was separated as its own group. The assignment test confirmed Northwest Europe and Northern Norway as the source areas for the Svalbard individuals of S. procumbens and C. capillaris ssp. fuscidula, respectively. However, source area was only confirmed in half of the C. capillaris ssp. fuscidula individuals [see Supporting Information—Table S6]. In both C. capillaris ssp. fuscidula and S. procumbens, the Svalbard population scored below average on the rarity index . Furthermore, two private Svalbard markers were also found  possessed one possible private marker each . Second, all examined Svalbard populations are likely demographically independent as there seems to be little current gene flow between these populations and populations outside Svalbard. This is clearly demonstrated by the strong founder/bottleneck effects. Finally, conservation of edge populations may be important for maintaining evolutionary potential as e.g. stress tolerance alleles may be more common here than in more optimal habitats , and only shares its unusual habitat preference (damp moss tundra) with one other member from the complex; the diploid, and probably sexually reproducing, Ranunculus boecheri from eastern Greenland  is beyond the scope of this study due to the lack of adaptive markers, information on  complex . MembersCarex capillaris ssp. fuscidula, Sibbaldia procumbens and Comastoma tenellum. Although Svalbard is known to be predominantly colonized from Northern Russia and only occasionally from Northern Norway and Greenland  as source area for S. procumbens but S. procumbens to belong to the North-American/North-Atlantic group using plastid markers—the opposite group of what is reported here. One explanation for these contradictory results might be that the current population of S. procumbens in Svalbard was established through multiple introductions from different sources, followed by hybridization and subsequent decline in genetic variation .IGA conceived the idea, designed the project and served as project leader together with AKB. RE was the main taxonomic advisor. SB and IEBS contributed equally to this work and performed all laboratory and data analyses, as well as the drafting of the article. All authors conducted field collections and commented on the manuscript.None declared.Supplementary DataClick here for additional data file.


Procambarus clarkii) is one of the most economically important farmed aquatic species in China. However, it is also a famous invasive species in the world. This invasive species was dispersed most via human activities including intentional or unintentional carry in China. Thus, P. clarkii naturally distributed in China provides us a desirable mode to investigate the genetic structure of an invasive species dispersed mainly by human-mediated factors. To reveal the impact of human-mediated dispersal on genetic structure of P. clarkii in China, a total of 22,043 genome-wide SNPs were obtained from approximately 7.4 billion raw reads using 2b-RAD technique in this study. An evident pattern of population genetic structure and the asymmetrical migrational rates between different regions were observed with 22 populations based on these SNPs. This study provide a better understanding of the population genetic structure and demographic history of P. clarkii populations in China, inferring that anthropogenic factors (aquaculture or by accident) and ecological factors , as well as its special biological traits could account for the current population structure pattern and dispersal history of P. clarkii.The red swamp crayfish ( Procambarus clarkii (Girard 1985), native to north-eastern Mexico and south-central United States, is one of the world’s most invasive species2, and its aggressive burrowing leads to the damages of levee, dam and paddy field3. In 1920s, P. clarkii invaded China from Japan and now widely distributed in almost all types of freshwater habitats, e.g., swamps, sloughs, ditches and paddy fields, in China. Several traits of its life history, e.g., polytrophism, rapid growth, high fecundity and disease resistance, make its invasion of the wild successful4. Interestingly, it is widely favored and consumed in China, and is one of the most economically important farmed aquatic species rather than a devastating invasive species. In the cities located in the middle and lower reaches of the Yangtze River, annual consumption of P. clarkii reaches more than 600,000 metric tons during the peak summer season (www.yyj.moa.gov.cn). Thus, recently, much attention has been paid to the cultivation technique5, artificial reproduction6, and antibacterial or antiviral mechanisms of P. clarkii8. The dispersal rate of P. clarkii appeared to greatly exceed the estimated rates, and now it has invaded into most areas in China except Tibet plateau. Study on the population genetic structure of this alien species would contribute to understanding its biological invasions and establishing possible methodologies for its prevention and control9. Human-mediated jump dispersal of P. clarkii including intentional or unintentional carry, combined with its natural expansion influenced the population structure and genetic diversity11. Especially, with P. clarkii consumption demand increasing in recent years, the crayfish was carried into many areas by commercial transportation. The wild crayfish populations were mixed with the crayfish escaped from cultured population due to the natural disaster or human negligence. Therefore, the genetic structure of P. clarkii populations in China is complicated. Information on genetic structure and diversities of P. clarkii populations, which are mainly dispersed by human-mediated factors, would be more helpful to find a way to control its invasion and scientifically utilize the germplasm resources. Undoubtedly, P. clarkii could provide a desirable mode for investigating the genetic structure of an invasive species mainly dispersed by human-mediated factors. Previous studies13 proposed that the populations of P. clarkii in China exhibited a high level of genetic diversity after successfully invading a new environment. Meanwhile, in the past years, the human-mediated dispersal in aquaculture accelerated the invasion of P. clarkii in China and might result in the differnet  levels of gene flow among different populations11. However, the impacts of anthropogenic factors on the population structure of P. clarkii need to be comprehensively assessed, and the population genetic features of P. clarkii have changed during the past decades after the invasion. Therefore, the dynamics of population genetics in P. clarkii should be evaluated with a powerful method. The population structure and genetic diversity of this alien species help us revealing the historical dispersal pattern of P. clarkii in China, and also provide new insights into invasion prevention and utilization of genetic resource. Previously, population genetic studies of P. clarkii have generally used mitochondrial genes12, microsatellites15 and AFLP13, and that the genomic data of P. clarkii is quite limited16. The growing accessibility to high-throughput sequencing technologies allows the production of massive data and the discovery of genome-wide resources at relatively modest and decreasing costs. As a simple and flexible technique for genome-wide genotyping, 2b-RAD method can provide the rapid discovery of thousands of SNPs that are to some extent evenly distributed across the genome18. To date, to our knowledge, there is no document about investigating the population structure and genetic differentiation of P. clarkii at genome level after its successful invasion.The red swamp crayfish, 20. Theoretical considerations indicate that invading populations should be prime candidates for both adaptive and non-adaptive evolutionary change21. It is clear that invasion can drive evolutionary changes in the phenotypic traits of invasive organisms within periods of years to decades. To adapt the environmental conditions of its introduced area, invasive species rapidly evolved adaptive clines in key morphological and life-history characteristics after its invasion. Presently, most researches in this area tended to concentrate on comparing genetic diversity between native and invasive populations. Few studies have investigated the phenotypic divergence of the invasive animal species after its invasion. Zhang et al.22 firstly reported that the morphological differentiation of 12 different populations in P. clarkii and proposed the Chinese populations exhibited phenotypic variations  compared to American populations. Based on the basis of that previous study, the investigation of morphological characters between different populations of P. clarkii could help us understanding the rapid evolution of phenotypic traits of P. clarkii after its invasion.Recently, rapid adaptive evolution of invasive species has been a hot topicP. clarkii using 2b-RAD technique and then used into clarifying the genetic structure of P. clarkii in the main distribution areas in China. The genetic dynamics and genetic structure impacted by human-mediated dispersal, and phenotypic divergence of P. clarkii in China, would be presented in the study. We investigated the population genetic signatures and conducted morphological analyses to test the hypotheses that (1) the P. clarkii populations impacted by anthropogenic factors show persistent asymmetrical gene flow and exhibit an evident pattern of genetic structure; (2) rapid evolution or selection affects the phenotypic divergence of different P. clarkii populations. These results would contribute to exhibiting the genetic signatures of P. clarkii populations after the successful invasion, providing management strategies for the invasion of P. clarkii in China and utilization of germplasm resource for the hatchery breeding in aquaculture.In this study, amounts of genome-wide SNPs were identified in P. clarkii genome survey sequences (not published) with the average coverage was 26.08 ± 6.29 reads per locus. The sequences were discarded due to alternative alignments and insufficient depth of coverage  reads with restriction site per individual were retained. Of the retained sequences, an average of 10.89 million (40.98%) aligned to the FST values between populations ranged from 0.0400 to 0.2521. Also, we calculated the FST based on the SNP sites  had noticeably high FST values (>0.15), of which 2284 even exceeded 0.25. Analysis of molecular variance (AMOVA) revealed that a significant fraction of the total variance was due to between-populations variance  with evidence of significant genetic differentiation (P < 0.05); most of the variance was due to variation within populations (87.31%). Notably, the pairwise comparison of FST values from seven populations in the middle reaches of Yangtze River basin revealed the significant lower level of genetic differentiation between these populations than others with the average of 0.085, indicating the high level of genetic communication among the populations in the middle reaches of Yangtze River basin. The Mantel test for matrix correlation between genetic similarity and geographical distances were not significant , indicating the pattern of isolation by distance for the P. clarkii didn’t exist in the main distribution areas of China. Additionally, the mismatch distribution displayed a unimodal curve for P. clarkii  across all individuals ranged from 0.1335 to 0.1964 among 22 populations. The K value from the STRUCTURE output showed the model value of parameter K was 13, thus indicating that the uppermost hierarchical level detected by STRUCTURE was 13 distinct genetic clusters  also recovered these groupings  tree of these crayfish populations Fig.  featuresθ) were consistently low and ranged from 0.0487 for lower reaches region  to 0.0702 for the UM region  in P. clarkii (P < 0.05). Path analysis showed that carapace length (LC), carapace width (WC) and abdominal segment length (LA) had strong positive direct effects (Table LC) and carapace width (WC), are indicative of determining the body weight of P. clarkii. In total, 90.4% of the variation in weight could be explained by the variation of two independent variables. The unexplained variation, 9.6% of the total, may be due to variation in the other components under consideration. To investigate the phenotypic divergence of the individuals from different populations, we compared the five external characters among the populations, which were standardized by total length (LT). The results showed that carapace widths of P. clarkii were differed among these populations . The lowest value of carapace width/total length ratio was observed in NJ population, where P. clarkii was firstly introduced into China. These populations were divided into three groups based on the geographic distribution. Further, the phenotypic divergence of carapace width between the populations in the lower reaches  and Huai River region (SD and PZ) was not significant (P = 0.38).Since the level of genetic differentiation is relatively low between populations and is higher within populations. Investigation of morphological characters between different populations of ts Table , indicati P < 0.0. Path anP. clarkii, one of the most famous invasive species in China, dramatically became an important cultured species in China. Since it invaded into China in 1920s, it has widely dispersed in Yangtze River basin and Huai River basin. Barbaresi et al.3 proposed that the wide dispersal of P. clarkii throughout the world is attributed to human activities. It is evident that aquaculture and aquatic product transportation promote the spread of this invasive species in China. Also, the dispersal capabilities  of P. clarkii contribute to the rapid and widespread diffusion. Therefore, population genetic structure, genetic diversity and evolutionary history, and phenotypic divergence of this alien species deserve a special mention in response to challenges in biodiversity conservation and invasion control.23, and P. clarkii is no exception24. Therefore, the genome assembly of P. clarkii becomes much more difficult even though the NGS technology rapidly developed in the past few years. As an efficient and flexible method for genome-wide genotyping, 2b-RAD technique17 provides an excellent fractional representation of the targeted genome and has been widely applied in the previous studies27. In this study, to reveal the characteristic of genetic structure of P. clarkii, a total of 22,043 SNP sites were identified from the P. clarkii genome based on 2b-RAD technique, which were adequate for the study of population genetics.It is generally known that crustaceans were considered to be problematic for karyological and cytogenetic studies, as the majority of species have relatively high diploid chromosome numbers (>150) and relatively small chromosomesP. clarkii populations was detected in this study, which is consistent with the previous studies12 and could contribute to invasion success28. Meanwhile, the high level of variance within populations rather than among populations was revealed in this study, which might help to the invasion success of P. clarkii12, and also it appears to be an typical genetic feature of invasive species after its successful invasion, which is also reported in other invasive species31. Meanwhile, the high FST values revealed less genetic exchange between most P. clarkii populations, which may be related to its migrational ability in the wild and high fecundity. In addition, the low rate of migration between natural populations, which is affected by the human activities (extensive fishing of this economic species), to some extent reduced the level of genetic exchange between the populations in China. Furthermore, other methods should be adopted to further confirm this conclusion in the future studies, such as investigation with slow evolving sites or haplotype distribution. Additionally, no pattern of isolation by distance (IBD) detected in the P. clarkii populations to some extent further verified that human-mediated jump dispersal was the main dispersal pattern for P. clarkii.The high level of genetic diversity in et al.11 proposed that the P. clarkii was most likely originally introduced into China from Japan, and then expanded its range primarily into the middle and lower reaches. Here, we infer that the populations in upper reaches may originate from the populations in middle reaches of Yangtze River basin. Notably, two populations  from Huai River region and three populations  from lower reaches region obviously separated from UM region, which coincided with the phenotypic divergence of carapace width. These three regions revealed asymmetrical migration and low migration rates. Meanwhile, the mismatch distribution was adopted to investigate the demographic history of P. clarkii, and the typical unimodal distribution indicated that populations had undergone demographic expansions. To estimating population expansion through time, Bayesian skyline plots revealed that the population sizes of three regions were fluctuant and experienced one or two period of rapid increase. Interestingly, the population size (θ) of UM region remained at high levels in the longtime, which experienced two periods of rapid increase. These findings confirmed our hypothesis that persistent asymmetrical gene flow occurred among these three regions, and UM region was the main distribution area. The population sizes of lower reaches and Huai River regions were increasing, which were consistent with the conclusion reported in Li et al.12. In summary, this successfully invasive species that was mainly influenced by human-mediated dispersal revealed some characteristics of genetic landscape including asymmetrical migration, population size rapid increasing, high genetic differentiation within population, etc.The populations from upper reaches of Yangtze River basin exhibited similar population structure with the populations from middle reaches. Li 33 might account for the phenomenon that the low genetic differentiation among populations and high differentiation within population were detected in this study. Unfortunately, it was difficult to carry out the related analysis in this species because it seemed impossible to identify the slow evolving sites based on the limited SNPs information in this study. The bottleneck was that the reads outputted from 2b-RAD technique was only 33 bp in length. A comprehensive conclusion could not be given for the results mentioned above due to the limitations of the SNPs with limited information used in this study. The identification of more informative SNPs in P. clarkii must be carried out using other better methods  in the future studies. In addition, the crayfish genomic information is still very limited although we had sequenced the genomic sequences. The SNPs related with phenotypic divergence could not be identified even though the morphological characters were investigated in the subsequent section. Thus we could only exhibit the trend of divergence of morphological traits for P. clarkii as a whole.As the characteristics for an successfully invasive species mentioned above, the notion of fast evolving DNA markers reaching saturation levels of genetic distance between populations and maximum genetic diversity (MGD) theory34, and high level of phenotypic plasticity should enable a colonizing species to cope with, and become established under new environments. Many studies have focused on the phenotypic differences between populations in invasive plants38 , and fewer studies have proposed the phenotypic divergence of invasive animals40. As a famous invasive species, P. clarkii provide a desirable opportunity to investigate rapid adaptation of its phenotype. Given that the genome complexity of P. clarkii, phenotypic divergence is a better breach for predicting future invasion scenarios and understanding how populations can evolve rapidly in response to novel and changing environments. In the present study, the morphological differentiation of carapace width in P. clarkii was observed after invaded into China, and the lowest value of carapace width was observed in NJ population. Since it was firstly introduced into Nanjing City and invaded other areas10, the hypothesis that the carapace has been gradually widening for the rapid evolution of P. clarkii after its invasion was supported by the comprehensive comparisons of morphological characters. Therefore, it could be inferred that rapid evolution or selection affects the phenotypic change of the different populations in P. clarkii. Path analysis revealed that higher carapace width indicates the higher weight of individuals in P. clarkii, which are exclusively preferred in aquaculture at present.Generally, invasive species show higher phenotypic plasticity than native speciesP. clarkii plays two roles in China, a famous invasive species and a popular aquaculture species. The middle reaches of Yangtze River dominates the crayfish industry of China in both aquaculture and wild capture fisheries, where the industry contributes well in excess of 56.4 billion RMB annually in 2016 (www.cnfm.gov.cn/). Although the burrowing activities of P. clarkii can lead to damage to water courses and to crops, particularly rice, and its feeding can disrupt native ecosystems41, Chinese farmers are adept at avoiding the ecological damage  and utilization of its biological characteristics . For example, the rice-fish culture system, a complex ecological aquaculture mode, is widely developed in southern China42. P. clarkii is better adapted to growing in a rice field, and the concurrent culture of rice and crayfish makes good use of land, resources, equipment, and infrastructure already being used for rice production. Also, the wild capture of P. clarkii is still in a certain proportion in Chinese crayfish industry presently, while the wild crayfish’s burrows result in dam damages and a huge loss of irrigation water, causing significant economic loss43.P. clarkii populations and low level of genetic exchange between most crayfish populations were observed in our study. High level of genetic diversity promotes availability of natural variations for use in breeding germplasm. The P. clarkii populations with high level of genetic variation might be used as a source for selection of desirable germplasm and development of new varieties. In addition, the carapace of P. clarkii has been widening since it was firstly introduced into China in 1920s. Path analysis revealed that the characters in carapace play major role in body weight of P. clarkii. It provides novel insights into future selective breeding of P. clarkii, for improving the economic value of crayfish culture. The results of the present study, together with those of previous population genetic studies in P. clarkii12 investigated the comprehensive genetic information of P. clarkii populations in China, could provide a significant guidance into utilization of the valuable genetic resources and more efficient management of its invasion.The high level of genetic diversity in P. clarkii populations, and uncovered the population genetic landscape of this famous aquatic animal as well as the phenotypic differentiation between different areas in China that could provide us some fresh ideas to conduct selective breeding program in future. However, imperfectly, this study has two weaknesses that should be considered when interpreting these results. First, we used only a small number of populations from the Huai River region to draw conclusions as a whole. A larger population size would be an important way to make more powerful conclusions about migration rate, population structure, and genetic diversity. Also, the gene flow from unsampled populations in Huai River region could be influencing the poor estimates because Migrate-n assumes that all populations exchanging migrants are sampled. Second, the sequencing method used in this study had few limitations that the SNPs identified based on this method without high-quality reference genome were not informative due to the generation of short reads (33 bp in length). However, such an ideal assumption cannot meet for most studies, yet this method has been widely used to infer migration rates in natural populations. Given the above mentioned, our future work will collect more samples from more areas especially the Huai River region if possible, and focus on evaluating the evolving rates of genomic variants that contribute to understanding the genetic diversity and phylogeny of P. clarkii and the potential connectivity between environmental conditions and genetic variations.Overall, this study provided some suggestive clues for comprehensively revealing the impact of human-mediated dispersal on the genetic structure and diversity of P. clarkii, a total of 22 natural populations, which selected from the major cultivation areas and river basins where P. clarkii distributed in China, were collected . DNA integrity was assessed by 1.0% agarose gel electrophoresis. The 2b-RAD libraries were constructed for each individual following the methods from Wang et al.17 Different from original 2b-RAD technique, our procedure produced single-tag constructs using modified adaptors and biotin-labeled primers, and then digested by the Bsa XI enzyme to generate distinct cohesive ends and then ligated in a predefined order to produce five concatenated tags for Illumina paired-end sequencing. Meanwhile, a draft genome of P. clarkii was assembled with 102.73 Gb clean data, and the maximum contig was 83,480 bp in length and the contig N50 value of 1,138 bp (unpublished). This genome was adopted as a reference which was used in the subsequent analysis. The restriction enzyme recognition sites of Bsa XI were detected on the genome and the recognition sites number was 99,273 in the scaffolds.Total DNA was extracted from muscle tissue using the ammonium acetate methodBsa XI restriction enzyme and ligated to the two library-specific adaptors. To reduce marker density, the adaptors with fully degenerate 5′-NNC-3′ overhangs were chosen. For estimating Bsa XI sites coverage, simulant detections of the Bsa XI sites in P. clarkii reference genome were carried out. PE300 sequencing of 221 libraries was performed on Hiseq X10 platform .To assess the robustness of the method and subsequent data analyses, FH11 library was replicated . The starting DNA was digested with the www.bioinformatics.babraham.ac.uk/projects/fastqc/). The Perl scripts from Wang et al.17 were run for quality filtering and adaptors trimming of the reads. Assembly of the PE reads and extraction the assembled reads containing five tags was performed on PEAR script. The processed five-tag dataset from PEAR output was divided into single-tag data sets using a Perl script, and then the single tags with restriction site were extracted. These tags of each individual were mapped to the reference genome using SOAP2 program (http://soap.genomics.org.cn/) with the parameter −M 4, −v 2 and −r 0 allowing four mismatches. The sequences that aligned with only one location were regarded as unique tags and the expected potential markers. The aligned reads were performed SNP calling using RADtyping45 with default Posterior probability is calculated for two possible genotypes (homozygote and heterozygote) at a given locus using a maximum likelihood approach. To look for the relationship between sequencing data size and Bsa XI sites coverage, a correlation test was performed based on two repeated samples. To improve the accuracy of SNP genotyping, the loci that were present in all samples in at least 80% of the individuals from each sample, with at least three RAD tags per allele at each locus  were included. To avoid linkage bias for the SNP calling, only the loci containing 1~2 SNP and minor allele frequency (MAF) >0.05 were retained in the final analysis. The SNP dataset is publicly available at the website: https://doi.org/10.6084/m9.figshare.5537584.v1.Demultiplexed reads were returned by the sequencing facility in FastQ format and their quality was checked by FastQC (FST) per pair of populations were calculated using Arlequin suite version 3.546 to identify genetically differentiated localities. The potential number of genetic clusters and the membership of each individual were estimated using STRUCTURE Ver. 2.3.447. The software uses Markov chain Monte Carlo (MCMC) simulations to estimate those parameters, with the number of clusters to be tested (K) specified by the user. The MCMC simulation was run for 300,000 repetitions, after a burnin period of 100,000. For each value of K , the genetic ancestry of each individual was estimated based on the admixture model without any prior population assignment; estimations were obtained from the 300,000 iterations that followed a burn-in period of 100,000 iterations. The correlation between genetic distance and geographic distance was assessed using IBDWS version 3.2148. Additionally, the distribution of genetic variation was analyzed by AMOVA analysis using the Arlequin suite version 3.5. Principal components analysis (PCA) was performed using GCTA software49, and the scatter plot of the first and second components was done using R (https://www.r-project.org/). We then used these average pairwise FST values to cluster populations by a neighbor-joining method implemented in the MEGA 6.0 program50. Patterns of migration and past migration rates between populations were determined with MIGRATE-N program following the procedure described by Beerli and Palczewski51. The tested connectivity model was chosen based on the hydrography of the region and the previous genetic structure analysis. Full migration model with all migration paths open  was evaluated in the analysis.Pairwise F-statistics , total length (LT), carapace length (LC), abdominal segment length (LA), first abdominal segment length (LFA), abdominal segment width (WA) and carapace width (WC), were investigated. Simple correlation and stepwise multiple regression analysis were performed using SPSS program . The relative importance of direct and indirect effects of measured traits on weight was determined by path analysis for the morphological data. In the path analysis, weight was the dependent variable and the six characters (mentioned above) were considered as independent variables. Given the variables of the individual size, total length was used to standardize the size effect in the statistical analysis. The statistics were executed in R (https://www.r-project.org/) using the package coin52. Significance was assumed if P < 0.05.To investigate the divergence of morphological traits in Raw sequence data were deposited into the NCBI Short Read Archive (SRA) with the accession number SRP135662.Dataset 1


Aedes aegypti is one of the species most favored by changes in the environment caused by urbanization. Its abundance increases rapidly in the face of such changes, increasing the risk of disease transmission. Previous studies have shown that mosquito species that have adapted to anthropogenic environmental changes benefit from urbanization and undergo population expansion. In light of this, we used microsatellite markers to explore how urbanization processes may be modulating Ae. aegypti populations collected from three areas with different levels of urbanization in the city of São Paulo, Brazil. Specimens were collected at eleven sites in three areas with different degrees of urbanization in the city of São Paulo: conserved, intermediate and urbanized. Ten microsatellite loci were used to characterize the populations from these areas genetically. Our findings suggest that as urbanized areas grow and the human population density in these areas increases, Ae. aegypti populations undergo a major population expansion, which can probably be attributed to the species’ adaptability to anthropogenic environmental changes. Our findings reveal a robust association between, on the one hand, urbanization processes and densification of the human population and, on the other, Ae. aegypti population structure patterns and population expansion. This indicates that this species benefits from anthropogenic effects, which are intensified by migration of the human population from rural to urban areas, increasing the risk of epidemics and disease transmission to an ever-increasing number of people. Aedes aegypti [Urbanization continues to increase as more and more people migrate from rural to urban areas. Indeed, there are now more people living in cities than in rural areas. This phenomenon is responsible for the extinction of species that are not adapted to the urban environment and is especially important for the epidemiology of vector-borne diseases, as there is a clear association between reduced species richness in urban areas and an increased incidence of these diseases ,2. This  aegypti –7.Aedes mosquitoes. It endangers over 2 billion people of the world's population, causing approximately 390 million infections a year [Ae. aegypti [Dengue is a tropical disease caused by a flavivirus transmitted by s a year . Ae. aeg aegypti .Ae. aegypti is one of the most favored species when a rural area is urbanized. In this situation there is a rapid increase in its abundance and a consequent increase in the risk of dengue transmission [For these reasons, smission –13. The smission ,12,14,15Ae. aegypti is considered the primary vector for the Zika and chikungunya viruses, as well as several other arboviruses, and is responsible for numerous outbreaks of insect-borne diseases worldwide [Ae. aegypti, especially on a microgeographic scale, can lead to a better understanding of how human modifications to the physical environment are modulating population structure in this mosquito and the implications of this modulation for disease transmission and vector control.Also, orldwide ,17. IdenAedes fluviatilis populations in São Paulo, Brazil, had undergone population expansion primarily as a result of urban growth, highlighting the epidemiological significance of the association between urbanization processes and population structuring in vector mosquitoes.Microsatellites are highly polymorphic neutral markers often used in genetic population studies . RecentlAe. aegypti in the city of São Paulo and the major role played by this species in the transmission of the dengue, Zika and chikungunya viruses, this study used microsatellite markers to explore how urbanization processes can modulate populations of Ae. aegypti collected in three areas with different levels of urbanization in the city of São Paulo.In light of the high abundance of Ae. aegypti mosquitoes were collected from eleven sites, no more than 30 km apart, in three areas with different levels of urbanization in the city of São Paulo, Brazil.Conserved areas (CON): five municipal parks consisting of large green areas  open to the public from 5:00 am to 8:00 pm. No biological or chemical measures are used to control mosquitoes in the parks, which are home to wild birds, reptiles and mammals. Intermediate areas (INT): four collection sites on the University of São Paulo Armando de Salles Oliveira campus . The campus extends over 3,648,944.40 m2, of which some 800,000 m2 has been built on. Over 100,000 people travel through, visit or work or study on the campus every day. Urbanized areas (URB): two collection sites on the University of São Paulo health sciences campus, which is in a highly urbanized, densely populated area extending over 83,050.82 m2 (with less than 3% of vegetation cover). Much of this area  has been built on  following the manufacturer’s instructions. Polymerase chain reactions (PCRs) were carried out individually with ten microsatellite primers originally designed by Chambers et al.  and SlotTen microsatellite primers were labeled with a fluorescent dye  , and the PCRs were carried out according to the manufacture’s protocol using an E6331000025 Eppendorf Thermocycler . PCR products were visualized on a 1% agarose gel stained with GelRed™ .PCR products were diluted 1:7 by mixing 3 μL of each product labeled with a different dye with 21 μL of Ultra-Pure Water  to a final volume of 30 μL. Next, 2 μL of the diluted PCR products were mixed with 8.925 μL of Hi-Di formamide  and 0.075 μL of the size standard GeneScan-500 ROX  to a final volume of 11 μL. The samples were submitted to the University of São Paulo Center for Human Genome Studies and size-sorted in an ABI 3730 automatic sequencer . Fragments were sized with GeneMarker .O) and expected heterozygosity (HE), deviations from the Hardy-Weinberg equilibrium, inbreeding coefficient (FIS) and pairwise FST with significance values  were calculated with Arlequin (v3.5) [P-values) and number of migrants (Nm) were calculated using Genepop (v4.2 http://genepop.curtin.edu.au/) [Allele frequency, observed heterozygosity (Hn (v3.5) . Linkageedu.au/) . Allelicedu.au/) .ST) and Cavalli-Sforza and Edwards chord distance  were calculated per locus per population with FreeNa [ST values using Past (v2.17c) [The probability of null alleles, genetic heterogeneity (Fh FreeNa . A Mante(v2.17c) .ST/(1-FST) and geographic distance (km) was performed using Past (v2.17c).A dendrogram based on the Cavalli-Sforza and Edwards chord distance was constructed using Statistica (v7.0) . A lineaE<HEQ), or heterozygosity excess, a signature of a bottleneck event (HE>HEQ), where HE = the expected heterozygosity based on allele frequencies and HEQ = the expected heterozygosity based on observed alleles.Bayesian analysis was performed with Structure (v2.3.3) , and theP = 0.0001). These results are not considered significant because there were no repetitions of pairs of loci combinations across the populations tested.Linkage disequilibrium for the 495 possible tests, per locus per population, was found only between loci A10 and AC7, AG2 and AC5, AT1 and B07, AT1 and AG2 and AT1 and AG7 after Bonferroni correction  for locus AC1 in five populations, AC5 in three, AC7 in two, AG1 in seven, AG2 in two, AG5 in one, AG7 in one, AT1 in two and B07 in one. Locus A10 had a low probability of null alleles (<0.2) . Allelicwas 0.35 .ST indicated significant genetic structure between populations. Values ranged from 0.00629 to 0.11169, and only 7% of the values (4 out of 55) were not significant. When the potential bias due to null alleles was taken into account, the results ranged from 0.002133 to 0.093649. There were no statistically significant differences between the corrected and uncorrected FST values  (The results for F<0.0001) .2 = 0,03; P = 0,18) between populations.Number of migrants between the populations was 3.94766 per population per generation based on 30 specimens per population, showing a low degree of allelic similarity. There was no correlation between genetic and geographical distances . The CON-3, CON-4 and CON-5 populations segregated close to each other, as did the INT-1, INT-2, INT-3 and INT-4 populations .The method described by Evanno et al.  was usedP<0.05). Although not observed in any of the CON populations, heterozygosity deficiency was detected in all the INT and URB populations (except for INT-4), indicating a recent population expansion (E<HEQ) with a highly significant P-value (0.00015). This result supports the hypothesis that the Ae. aegypti populations in this study have suffered a major recent population expansion.Tests to assess heterozygosity deficiency using the SMM showed that there was a significant degree of deficiency in 5 of the 11 populations tested  did not show signs of population expansion, unlike the INT and URB populations, corroborating the hypothesis that the urban environment benefits this species and that it is highly adapted to human changes to the landscape.The Ae. aegypti populations and is correlated with the level of urbanization, supported by the lack of correlation between genetic and geographic distances. This can be explained by the fact that this species is well adapted to the urban environment and therefore does not have to actively seek new areas in order to find hosts or breeding sites as these are widely available in cities regardless of the level of urbanization and are plentiful even in the more conserved environments, such as urban parks.Bayesian analysis showed that structuring has been occurring in Ae. aegypti was reintroduced in Brazil in the 1970’s [A possible hypothesis to explain this situation is that after e 1970’s , the groAe. aegypti, which resulted from anthropogenic impacts on its evolution may be closely associated with microevolutionary processes [Ae. aegypti were consistent with the results of previous studies of this species [Further, the domiciliation of rocesses , such as species ,39,41, iAe. aegypti populations tends to intensify since this species is favored by urbanization. As a result, there is an increase in diseases transmission indirectly caused by human effects on the environment, such as the development of urban heat islands [Our finding of urbanization-dependent structuring is of great significance for the dynamics of disease transmission, particularly because there appears to be low Nm between the populations collected in areas with different levels of urbanization in this study. Furthermore, with increasing urbanization the demographic expansion of  islands , and a c islands .Ae. aegypti population structure patterns and population expansion. This suggests that Ae. aegypti benefits from human changes to the environment, which increase in intensity as the human population migrates from rural to urban areas, increasing the risk of disease transmission and epidemics for an ever-increasing number of people.The findings of this study indicate a robust association between, on the one hand, urbanization and increasing human population density and, on the other, S1 Fig(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 TableNa) and private allelic richness (Np).Allelic richness ((DOCX)Click here for additional data file.S3 TableP-values in bold.Significant (DOCX)Click here for additional data file.


Oncorhynchus mykiss) populations and show that (i) they are more closely related to native coastal steelhead than to the California Central Valley lineage, with no evidence of introgression by domesticated hatchery rainbow trout, (ii) populations above and below barriers within watersheds are each other's closest relatives, and (iii) adaptive genomic variation associated with migratory life‐history traits in O. mykiss shows substantial evolutionary differences between fish above and below dams. These findings support continued habitat restoration and protection of San Francisco Bay Area O. mykiss populations and demonstrate that ecological conditions in novel habitats above barriers to anadromy influence life‐history evolution. We highlight the importance of considering the adaptive landscape in conservation and restoration programs for species living in highly modified habitats, particularly with respect to key life‐history traits.The streams draining of into San Francisco Bay, California, have been impacted by habitat alteration for over 150 years, and roads, dams, water diversions, and other impediments now block the paths of many aquatic migratory species. These changes can affect the genetic structure of fish populations, as well as driving adaptive evolution to novel environmental conditions. Here, we determine the evolutionary relationships of San Francisco Bay Area steelhead/rainbow trout ( In western North America, much of the landscape has been heavily affected by anthropogenic impacts over the past century , a widespread fish native to western North America that has been introduced all over the world for recreational fishing , that is, strongly associated with life history of populations of steelhead and rainbow trout  loci and test for signals of local adaptation to migratory opportunities. First, we evaluate the evolutionary ancestry of San Francisco Bay Area populations to determine whether they are more closely related to the coastal steelhead or Central Valley evolutionary lineages, and whether the release of large numbers of hatchery rainbow trout of diverse origins in many of the reservoirs in the study area have affected the genetic structure of the species. Introductions and artificial propagation can have many consequences on native populations, and understanding them is important for establishing effective conservation and management strategies  desiccated on blotter paper until DNA extraction. In addition, previously published data , California Central Valley (CCV), Central California Coast (CCC), and South‐Central California Coast (SCCC) DPSs were included in the analysis for comparison with San Francisco Bay Area populations  amplification of 15 microsatellite loci previously used in population genetic studies of O. mykiss , 0.6 μl dNTPs (10 mM), 1 μl Primers (5 μM), 6.95 μl dH2O, and 0.045 μl Taq DNA polymerase. PCR products were mixed with formamide, loading dye, and an internal size standard, then denatured at 95°C for 3 min, and electrophoresed on an ABI 3730 DNA Analyzer (Applied Biosystems). All genotypes were called independently by two people using GeneMapper software (Applied Biosystems). Discrepancies were resolved by reconciling the two calls, by regenotyping, or by deletion of that genotype from the dataset.Genomic DNA was extracted from all tissue samples using Qiagen DNeasy Tissue Kits on a BioRobot 3000 (Qiagen) following the manufacturer's protocols. Extracted DNA was diluted with dHO. mykiss . The assay mix contained 1.3 μl TaqMan Assay, 2.5 μl Assay Loading Reagent, 0.25 μl ROX and 1 μl dH2O. Assays and samples were loaded into the array, with two no‐template negative controls included in each genotyping array. Genotypes were called using Fluidigm SNP Genotyping Analysis Software v3.1.1.All samples were also genotyped at 95 SNP loci previously developed and used for parentage and population genetic analysis in 2.3O. mykiss were combined with data previously collected from a range of California populations, as well as hatchery rainbow trout strains commonly used in California to evaluate their influence on population structure. We first removed the two loci located in the Omy5 MAR , as well as a third locus (Omy127236‐583) that is partially linked to these loci , expected heterozygosity  was calculated to account for sample size effects using the rarefaction method in the program HP‐RARE and a base sample of 16 gene copies  in Genetix  to each K genetic cluster and were visualized using the programs CLUMPP and distruct  was performed with the R package “adegenet”  between the two Omy5 markers for each population using the R package genetics ‐ and anadromous (AD)‐associated haplotypes . These two loci are in near‐perfect linkage disequilibrium (LD) in coastal‐lineage 3A total of 1,419 genotypes from San Francisco Bay Area populations were included in the present analysis (Table 3.1O) and expected (HE) heterozygosity ranged from 0.20–0.42 and 0.20–0.44, respectively (Table R) ranged from 2.12 (Permanente Creek) to 5.95 (Napa River). Mean HO, HE, and AR were not significantly different between populations above and below dams, but were all significantly higher in wild San Francisco Bay populations than in the hatchery trout strains . Pairwise FST values among all populations ranged from 0.030–0.095, and all values were significantly different from zero. As expected, given the known relationship between genetic diversity and FST .Observed  for the San Francisco Bay populations, hatchery strains, and coastal and Central Valley populations showed a similar, clear primary dichotomy between coastal/San Francisco Bay and hatchery/Central Valley fish  between the two loci located in the Omy5 MAR ranged from 1 (perfect linkage) to <0.1 (very low) and was significantly lower in Central Valley populations and hatchery strains than in coastal or San Francisco Bay Area populations, both above and below barriers  structure clustering and PCA analyses showing that fish from San Francisco Bay Area streams align with coastal populations; (ii) the unrooted neighbor‐joining tree, in which San Francisco Bay Area populations branch with coastal populations to the exclusion of Central Valley populations and hatchery rainbow trout strains; and (iii) the strong linkage disequilibrium between two loci located in the Omy5 Migration Associated Region (MAR), which is similar to the LD observed in coastal O. mykiss .All data for this study are permanently archived at the Southwest Fisheries Science Center Fisheries Ecology Division (