Cotesia vestalis, and its host diamondback moth (DBM), Plutella xylostella, provide opportunities to test whether the specialist natural enemy migrates seasonally with its host or occurs as resident population. We genotyped 17 microsatellite loci and two mitochondrial genes for 158 female adults of C. vestalis collected from 12 geographical populations, as well as nine microsatellite loci for 127 DBM larvae from six separate sites. The samplings covered both the likely source (southern) and immigrant (northern) areas of DBM from China. Populations of C. vestalis fell into three groups, pointing to isolation in northwestern and southwestern China and strong genetic differentiation of these populations from others in central and eastern China. In contrast, DBM showed much weaker genetic differentiation and high rates of gene flow. TESS analysis identified the immigrant populations of DBM as showing admixture in northern China. Genetic disconnect between C. vestalis and its host suggests that the parasitoid did not migrate yearly with its host but likely consisted of resident populations in places where its host could not survive in winter.Genetic comparisons of parasitoids and their hosts are expected to reflect ecological and evolutionary processes that influence the interactions between species. The parasitoid wasp,  Each run started with a burn‐in of 100,000 followed by 1,000,000 iterations employing the admixture model. The final true value of K was determined using the web software STRUCTURE HARVESTER version 0.6.94  with 120,000 MCMC sweeps after a burn‐in of 2,000. The optimal K value was chosen by inspecting bar plots of individual membership probabilities and the K value at which the plots of Deviance Information Criterion (DIC) values against K were stable across replicates and began to plateau. The optimal K value was replicated 100 times, and 20% of the replicates with the lowest DIC were processed with CLUMPP version 1.1.2  and the mitochondrial cytochrome b gene  were sequenced. All PCRs were conducted using the Mastercycler pro system  following standard PCR amplifications conditions and an annealing temperature of 55°C. Amplified products were purified and sequenced directly from both strands using an ABI 3730 xl DNA Analyzer .In order to validate morphological identification of The sequencing results determined from both strands were assembled. Each gene was aligned independently with CLUSTALW .For the microsatellite loci characterized in both species, null allele frequencies are low. No locus was significantly linked across all populations. No locus departed from HWE in all populations and no population departed from HWE across all loci. Both species showed high allelic richness, but allelic richness of DBM (7.4–8.9) was somewhat greater than that of 3.2C. vestalis, but weak in DBM (Table p = .005) for C. vestalis and an r value of 0.579 (p = .046) for DBM.The level of pairwise population differentiation is strong in BM Table . The ManC. vestalis showed that northwestern populations from Xinjiang formed one cluster, while the southwestern population from Yunnan formed another distinct cluster ; while in DBM, an admixture zone was identified in northern populations. The northwestern population was similar to another northern population and the southwestern population, while the other two northern populations were found to be an admixture of southern populations, respectively  estimates obtained from the microsatellite data of C. vestalis show low levels of effective migrants per generation between each pair of the three groups (0.01–52.43) and between the individual population pairs (0.65–17.77). In contrast, estimates of 29.2–249.72 were obtained for the clusters of DBM and estimates of 4.88–91.93 for population pairs of DBM. These estimates indicate higher movement rates for the host in comparison with its parasitoid.The migration , and a major one, which consisted of other haplotypes of all populations Figure . No sign44.1C. vestalis and its host DBM. We collected specimens from southern regions of China extending to the northern regions, covering both emigrant (southern) and immigrant (northern) areas of DBM . Although C. vestalis may show low levels of genetic differentiation across fields as has been found within Taiwan, indicative of high gene flow in C. vestalis at a local scale  system, in which homozygosity at CSD loci lead to the dead end of diploid males  (Ackerly, Ackerly, , while aAckerly, .5C. vestalis in biological control of DBM.We have demonstrated that the parasitoid and its host show contrasting patterns of genetic differentiation. As far as we know, this is the first comparison of genetic structure in populations of a migratory host and its specialist parasitoid in a shared landscape. Strong genetic structure revealed resident populations in the parasitoid in regions where DBM could not survive in winter. Processes of stepping‐stone dispersal followed by adaptation to local conditions may explain the current distribution of the parasitoid out the annual occurring areas of the host. Our study also provides information on conservation and usage of the parasitoid There is no conflict of interest.http://dx.doi.org/10.5061/dryad.45fm6.Data for this study are available at the Dryad Digital Repository: Shu‐Jun Wei and Zai‐Fu Xu conceived and designed the experiments; Shu‐Jun Wei and Zai‐Fu Xu organized the collection of specimens; Yuan Zhou and Xu‐Lei Fan performed the molecular analyses; Yuan Zhou, Shu‐Jun Wei, and Li‐Jun Cao analyzed the data; Shu‐Jun Wei, Yuan Zhou, Ary Hoffmann, Zai‐Fu Xu, Xue‐Xin Chen, and Li‐Jun Cao discussed the results; Shu‐Jun Wei, Yuan Zhou, Ary Hoffmann, and Zai‐Fu Xu wrote the manuscript. Click here for additional data file.


Petrolisthes armatus has a wide native range, which has expanded in the last three decades. We sequenced 1718 bp of mitochondrial DNA from native and recently established populations to determine the population structure of the former and the origin of the latter. There was phylogenetic separation between Atlantic and eastern Pacific populations, and between east and west Atlantic ones. Haplotypes on the coast of Florida and newly established populations in Georgia and South Carolina belong to a different clade from those from Yucatán to Brazil, though a few haplotypes are shared. In the Pacific, populations from Colombia and Ecuador are highly divergent from those from Panamá and the Sea of Cortez. In general, populations were separated hundreds to million years ago with little subsequent gene flow. High genetic diversity in the newly established populations shows that they were founded by many individuals. Range expansion appears to have been limited by low dispersal rather than lack of ability of marginal populations to adapt to extreme conditions.Theoretically, species with high population structure are likely to expand their range, because marginal populations are free to adapt to local conditions; however, meta-analyses have found a negative relation between structure and invasiveness. The crab  The population-genetic constitution of marine invasive species in their native range is increasingly being studied in efforts to determine the source of invasions into new areas  because of the influx of genes from the species centre. Kirkpatrick and Barton7 constructed models that supported this proposal. Their reasoning would lead to the conclusion that species with high levels of genetic structure are more likely to expand their ranges. Existing data from invasive species, however, have indicated the opposite, i.e. that species with high rates of gene flow in their native range are more likely to expand to new areas. Bohning-Gaese, et al.8 found that in warblers of the genus Sylvia ability to disperse –as measured by capacity for long-distance flight– overwhelmingly explained the size of species ranges. In the marine realm, a meta-analysis by Gaither, et al.9 found that the best predictive variable of whether a species would become invasive is the inverse of genetic structure in its native range. Species with low pairwise  FST values between their native populations were more likely to be invasive. Thus, intraspecific phylogeography, of interest in any species, takes an additional dimension when the species is invasive, because it also addresses the basic biogeographical question of the factors that determine range expansion.MayrPetrolisthes armatus (Gibbes), a marine, crab-like anomuran decapod. This species has the largest geographical distribution of any neotropical porcellanid. In the Atlantic its range extends from central Florida and Bermuda to southern Brazil on the American coast, and from Senegal to Angola on the West African coast12. It is also present at Ascensión14. In the eastern Pacific it ranges from the northern part of the Gulf of California to southern Perú, but it seems to be absent from the eastern Pacific offshore islands12. Until the early 1990s, the range of this species in the western Atlantic did not extent any farther north than Cape Canaveral12. Rathbun15 listed a few specimens from Connecticut, but Chace14 expressed doubts regarding this report. Starting in the 1990s, P. armatus has expanded its populations to the shores of Georgia and South Carolina and has become abundant on rocky rubble, oyster reefs, and other intertidal habitats16, where it suppresses recruitment of other crabs and growth of small oysters17. Thus, the species is considered “invasive”20, although its recent spread is probably best classified as a range shift18, rather than an invasion.An invasive species with a wide native geographic range is the porcelain crab Petrolisthes armatus is not due to an exceptionally long planktonic life. The planktonic phase, consisting of two zoea stages, in both eastern Pacific and western Atlantic populations ends in a settling megalopa in 12–19 days in the laboratory22, a length of planktonic stage typical of other porcellanid species28. The wide distribution of the species and its potential to become invasive may be due to its wide habitat preference and physiological tolerance. The species occurs in the lower intertidal zone, under stones, in oyster and mussel beds, around mangrove roots, in corals, sponges and on dock pilings. It has also been dredged from rock, sand, and shell bottoms down to 18 m on the American coasts12 and down to 30 m in the eastern Atlantic14. P. armatus is by far the most abundant porcellanid in the rocky intertidal of the Panamanian Pacific coast, with densities up to 20 individuals per m2. It tolerates salinities as low as 10‰ inside the Panamá Canal Locks, where it is common11. According to a 16S rDNA phylogeny by Hiller, et al.29, P. armatus from both American coasts forms a sister clade to one comprised of P. robsonae and P. zacae, the former adapted to withstand great changes in salinity30, and the later adapted to inhabit spaces formed by entangled roots of the mangrove Rhizophora mangle31.The wide geographic distribution of Petrolisthes armatus reproduces throughout the year both in eastern Pacific32 and western Atlantic34. Females of Pacific P. armatus release larvae synchronously near the times of nocturnal high tides35, probably as result of predation pressure by visual predators36. The larvae may use vertical migrations in order to be retained in estuaries39. Sexual maturity is reached in about a month after settlement.18. It is not known whether the northward range shift of P. armatus along the coasts of Georgia and South Carolina was aided by warming waters, or whether larvae were not capable of spreading northward of Cape Canaveral until adults were transported by humans. Canning-Clode et al.20 studied the possible impact of cold water temperatures on the survivorship of P. armatus. In temperatures similar to the ones experienced by the southern and mid-Atlantic coast of the United States during abnormally cold-spells between January and March 2010, 39% of specimens survived.Recent warming of coastal subtropical waters has facilitated a poleward range expansion of many organisms with tropical distributionPetrolisthes armatus collected from localities spanning a large part of its geographic range to address the following questions: (1) What does the intraspecific phylogeography of this species reveal about genetic connectivity of its populations? (2) Have there been barriers to gene flow within the species range? (3) How long ago were populations separated, and what has the rate of genetic exchange been after their separation? (4) Is the genetic structure of this invasive species consistent with the view that genetic structure is related to range expansions? (5) What is the origin of founders of newly established populations, and what have been the population genetic consequences of invasion of new areas?Here we use DNA sequence data from the mitochondrial cytochrome oxidase I (COI) and 16S rDNA genes of 40, the split in Petrolisthes armatus from the eastern Pacific and the Atlantic cannot be more recent than the completion of the central American Isthmus, and thus their common ancestor could not be younger than 3 MY. Our purpose of reconstructing a phylogeny was to identify and date major clades within the species. For this reason, we employed a conservative approach of collapsing every node that did not receive adequate support in both our Maximum Likelihood and the two Bayesian analyses. This mostly meant that we preserved all nodes supported by >70% of the bootstrap iterations in RAxML, because this program produced much lower clade support than either MrBayes or BEAST. The mtDNA phylogeny of P. armatus from concatenated 16S and COI sequences  comparisons between clades  illustrates the population genetic effect of population subdivision and few migrant haplotypes Table . In the ST values between populations in the western Atlantic were large and highly significant  analysis found a significant relationship between genetic dissimilarity and geographic distance, whether the two variables were measured in a linear , or in a logarithmic  scale. In contrast to the western Atlantic, genetic discontinuities between the populations in the eastern Pacific were mostly governed by the suggested barrier to gene flow between the coast of Colombia and the coast of Panamá. FST values between populations between these two areas were large and significant, whereas within each area –despite long geographic distances between Panamá and the Sea of Cortez– they were indicative of gene flow between distant populations. Correlations between genetic and geographic distance in the eastern Pacific were not significant in either a linear  or a logarithmic  scale. Very small and non-significant FST values between the Gulf of Guinea populations  indicated that P. armatus in this area is genetically uniform between Nigeria, Cameroon and São Tomé  between their concatenated haplotypes is 4.04%, we have calibrated estimated times of divergence and migration between populations on the assumption that the 1718 bp fragment experiences a substitution rate per lineage of 7.5–15 mutations per MY. In comparisons between the population at Georgia and the rest of the samples (for which only a 499 bp fragment of COI-HL was available), we used the divergence in this DNA fragment between West Atlantic and Pacific populations of 5.02% to set the substitution rate prior for this fragment as 2–5 mutations per lineage per MY. Posterior probabilities of successful runs in IMa2 are supposed to rise to a peak at particular values of each parameter, then drop back down. In some pairwise comparisons, despite many chains, wide priors and long runs, posterior probabilities rose to a plateau and remained there, so that no unique value could be assigned to the relevant parameters. In the western Atlantic, times of initial separation estimated by IMa2 between populations . The most recent separation is between the Colombian and the Isla Puná populations. Rates of migration were generally not significantly different from 0, except for asymmetrical gene flow, from the population at Isla Puná to Salinas and from the coast of Colombia into Isla Puná  was >1.5 × 10rs Table . Despitená Table .ST values and recent separation  and large divergence between populations within the same clade. Clearly, this is a species with low ability to disperse, either within the western Atlantic or within the eastern Pacific, although our limited sample from the eastern Atlantic indicates higher genetic connectivity between the more closely spaced samples in this region. The degree of structuring within the West Atlantic and East Pacific is evident in the mtDNA phylogeny, in FST values and in the coalescent analysis. Yet a few errant haplotypes of one clade found in the region mainly inhabited by the other, along with the coalescence estimates of gene flow subsequent to initial separation, attest to infrequent long distance genetic exchange. What barriers and mechanisms of gene flow could account for the observed patterns?Genetic structure of Petrolisthes armatus22 is conducive to high population structure, but it is not so limiting, as to be the entire explanation for the deep divisions seen in this species. Other tropical species in the same regions, which in the laboratory complete their planktonic cycle in approximately the same period of time, such as the sea urchin Echinometra lucunter with a larval life of 19 days43, and the sponge Cliona delitrix with a larval life of about 10 days44, show much less genetic structure within the Caribbean. Only the sea anemone Nematostella vectensis45, the gorgonian Gorgonia ventalina46 and the barnacle Chthamalus proteus47 show evidence of such high population differentiation between local populations. Shulman and Bermingham48 calculated that if larvae were transported as passive particles, it would take only 13 generations for genes of a species with a larva that stays in the plankton for two weeks to traverse through stepping stones the entire Caribbean following the main Caribbean Current, given average current vectors52. It is unlikely that P. armatus is lacking stepping stones, as, in addition to our sampled localities, it is also found in the Greater and the Lesser Antilles10. The lack of long-distance dispersal indicated by the high population structure, therefore, is more likely due to local retention of larvae, which appears to be a characteristic of this species38. The tendency of larval retention, however, cannot be absolute in a species of such a wide range, so barriers to gene flow must also be present to account for the phylogeographic divisions.Patterns of spread of genes between sedentary marine populations are obviously a function of the dispersal abilities of the species relative to potential barriers to planktonic transfer. The 12 to 19 day planktonic larva of 53. Some authors55 have agreed that there were water connections until this time, but they presented their conclusions so as to imply that there may have been previous interruptions of gene flow between the eastern Pacific and the western Atlantic57. Whether the water connections had narrowed at instances prior to 3 MYA bears little relevance to the phylogenetic reconstruction of P. armatus, because among five sister clades in the genus Petrolisthes with representatives on either side of the Isthmus of Panamá, eastern Pacific and Atlantic populations of P. armatus have diverged the least59, and are thus most likely to have been the most recently separated. This, however, does not necessarily mean that the trans-isthmian split was contemporaneous with the final isthmus closure, because it could have pre-dated it by any length of time. Our use of wide priors in the split between the Pacific and the Atlantic clades in the BEAST reconstruction has resulted in an estimated date of 3.5 MYA, which seems reasonable for a species with wide environmental tolerances, such as P. armatus. Based on this, we have estimated the split between western and eastern Atlantic clades at 3.1 MYA. The barrier that separated populations at these two regions was undoubtedly the long distance between American and African coasts. Scheltema60 estimated that, based on the speed of currents, larvae of marine organisms would require between 9 and 28 weeks to cross the contemporary tropical Atlantic. This distance barrier also accounts for distinct amphi-Atlantic clades in other porcellanids29, sea urchins62 and fishes66. Given the short length of larval life of Petrolisthes, the puzzle is not how the separation happened, but rather how the original colonization was effected. The only explanation for the expansion of P. armatus to the coast of Africa is that this species can occasionally raft, possibly on mangrove roots, on which adults and juveniles are frequently found. We can only speculate about ocean circulation in the Pliocene, but modern-day currents between the coast of Brazil and the coast of Africa68 are conducive to rafting transport; Ascensión may have been a stepping stone.An obvious such barrier is the Isthmus of Panamá, the timing of which we have used to date all other separations between populations. A multitude of lines of evidence has indicated that the final closure of the Isthmus, after a 12 MY process of narrowing water connections, occurred at approximately 3 MYAet al.69 found that a population of the brittle star Ophiothrix suensoni in the Gulf of Honduras was genetically isolated from populations in the rest of the Caribbean, which they attributed to larval entrapment arising from the Mesoamerican gyre71. A similar explanation was offered by Jackson, et al.72 for the isolation of populations of the Nassau Grouper, Epinephelus striatus in this area. However, our sample from Yucatán was from the northern side of the peninsula and, given that in Petrolisthes armatus there is significant isolation by distance in the western Atlantic, the geographic separation of the two clades may be due to distance alone. Although larval entrapment in the Yucatán area could be the reason haplotypes in this area are so similar to each other, the FST values between the Yucatán population and populations on the coast of Colombia and Panamá are significant but relatively small . Isolation of Brazilian marine populations of other species from their conspecifics in the Caribbean is usually attributed to the fresh-water plume of the Orinoco and the Amazon79; however, given the wide salinity tolerance of P. armatus, the isolation of its Brazilian populations may be due to the extensive mud habitat off the coast of Guyana and Surinam80.The barrier that could account for the presence of two clades in the Caribbean, one predominantly on the coast of Florida, the other from Yucatán to south Brazil, is less obvious. The population at Yucatán shows the lowest nucleotide diversity among all sampled localities Table , suggest81. There is ample rocky habitat between Ecuador and Panamá, and there is little in patterns of ocean circulation to suggest that larvae could not spread through stepping stones84. It will be interesting to find out whether other eastern Pacific species of Petrolisthes show a similar genetic discontinuity.In the eastern Pacific, the monophyletic clade composed of haplotypes at the coasts of Ecuador and Colombia, highly differentiated from those at Panamá and the Sea of Cortez, is unusual. No other organism sampled genetically to date displays this genetic break between populations at Colombia and PanamáPetrolisthes armatus in South Carolina and in Georgia are of particular interest, because they have earned the title of invasive species for this crustacean20. Our data clearly show that these populations were initiated by the movement of individuals from Florida. Their haplotype and molecular diversity is as high as that of native populations, a frequent characteristic of invasive populations when they are established by a large number of propagules87. Transfer of large numbers of the oyster Crassostrea virginica from the Gulf of México to the Chesapeake Bay during the early 1960s to replace oyster stocks infected with “MSX” disease has been associated with invasions by crustaceans inhabiting oyster beds from the Gulf to the Atlantic seaboard of the United States88; the same may have been true for introductions into Georgia and South Carolina. A large number of recently introduced founders, however, confounds the detection of evolutionary processes within the populations with the mark left by the genetic constitution of haplotypes that founded them. This is particularly true when there are multiple invasions from different sources90. At the time that our samples were taken at Georgia and South Carolina, a maximum of 22 years had elapsed since P. armatus was noticed in these areas. Despite the obvious tendency of this species to form deep genetic divisions between its populations over time, ~250 generations have apparently not been enough for haplotypes to sort themselves to the degree that coalescence can correctly infer the history of isolation and migration. Comparisons of the Georgia population with those from the native range are further complicated by their being based on a short fragment of mtDNA, which is less likely to conform to the infinite sites mutation model assumed by the IMa2 algorithm. Population density of P. armatus in Georgia and South Carolina fluctuates widely91, as it also does in its native range93, but the species appears well-established in areas occupied after its range shift91. P. armatus was present–though not as abundant–in South Carolina, even after the 2010 cold snap20, which suggests that larvae as well as adults are able to tolerate temperate temperatures. It is apparently too early to tell whether local adaptation in this marginal environment will permit the species to expand its range farther north.The newly established populations of 7. Local adaptation is expected to promote range expansion95. Petrolisthes armatus, according to our mtDNA data, is a species characterized by high structure, although this structure may need thousands or millions of generations before it becomes established so that local adaptation can ensue. The signature of natural obstacles to migration remains in the genetics of both the newly established and the old populations. Two major barriers are identified by the phylogeny, the separation of Atlantic and Pacific populations by the completion of the Isthmus of Panamá, and the almost contemporaneous separation of two clades on the opposite shores of the tropical Atlantic. The subsequent separation of the North and South clades in the western Atlantic and a similar break in the eastern Pacific do not lend themselves to obvious explanations as to their causes, but further support the conclusion that dispersal in P. armatus is limited. The recent range expansion from Florida to the northern Atlantic seaboard of the United States is most likely due to human transportation of oysters, but the ability of the species to survive in the new habitats shows that it possesses the necessary plasticity to tolerate new physical and biotic environments. Thus, the range limits of this species are more likely set by species characteristics related to low ability to disperse, rather than by narrow physiological tolerance. Given these characteristics, any subsequent spread of this invasive species is likely to be caused by human mediation.With sufficient variation, pronounced genetic structure is expected to allow local adaptationPetrolisthes armatus from both sides of the Atlantic and from the eastern Pacific , except for 15 samples from Fort Pierce (collected in July 2010) and 11 samples from South Carolina (collected in November 2012). We additionally downloaded from GenBank 139 sequences of partial Cytochrome Oxidase I (COI) (Accession numbers FJ693377–693515) from Sapelo Island, Georgia, obtained by J.D. Robinson, E. Díaz-Ferguson, S. Pennings, T. Dale Bishop and J. Wares. These sequences were obtained from specimens collected in 2006–7 .We collected, or obtained from museums, 276 individuals of ® Blood & Tissue Kit (Qiagen), following the manufacturer’s protocol for animal tissues. A 530 bp fragment of the ribosomal 16S rDNA was amplified using primers 16Sar-5′ (CGCCTGTTTATCAAAAACAT) and 16Sbr-3′ (CCGGTCTGAACTCAGATCACGT)96, and was trimmed to 508 bp in the alignment. A 640 bp of Cytochrome Oxidase I (COI) was amplified using primers COIf-5′ (CCTGCAGGAGGAGGAGAYCC) and COIa-3′ (AGTATAAGCGTCTGGGTAGTC)96, and was trimmed to 532 bp in the alignment. A second 680 bp COI fragment was amplified using primers LCO1490 (GGTCAACAAATCATAAAGATATTGG) and HCO2198 (TAAACTTCAGGGTGACCAAAAAATCA)97, and was trimmed to 678 bp in the alignment. We refer to the first COI fragment as COI-af and to the second as COI-HL. Double-stranded amplifications were performed in 25 ml volume reactions containing 5 µl of Taq buffer (5×), 2.5 µl of dNTP mix (8 mM), 1.2 µl of each primer (10 µM), 2.5 µl of MgCl2 (25 mM), 0.2 µl of GoTaq® Flexi DNA Taq Polymerase (Promega), 1 µl of DNA template, and 11.4 µl of ddH2O. Thermal cycling conditions consisted of an initial denaturation step at 96 °C for 3 min, followed by 30 cycles of 95 °C for 1 min, 50 °C for 1 min, and 72 °C for 1 min. An extension step at 72 °C for 5 min followed the last cycle. Amplifications that resulted in unique PCR products were cleaned using the ExoSap-IT® kit (USB Corporation) following the manufacturer’s protocol. Samples that amplified multiple products were purified by cutting the band of the appropriate molecular weight out of a 2% low-melt agarose gel after electrophoresis in 1× TAE buffer. PCR products were recovered by incubating the sample at 70 °C for 10 min, and then at 45 °C for 60 min after adding 1 µl of GELase™ (Epicentre Biotechnologies). Clean PCR products were cycle-sequenced in both directions using the BigDye® Terminator v. 3.1 Cycle Sequencing Kit, and electrophoresed in an Applied Biosystems® 3130 Genetic Analyzer. DNA from one individual of Petrolisthes robsonae was also sequenced to be used as outgroup in the phylogenetic analyses. Sequences were aligned by eye using BioEdit98. Multiple attempts to identify an informative nuclear sequence failed.DNA was extracted from chelipeds or walking legs using the DNeasy100, was used to select the model of nucleotide substitution that best fit each DNA region. These models were TIM3+I (I = 0.92) for 16S, GTR+I+G  for COI-af, and TIM+I+G  for COI-HL. The three fragments of each individual were concatenated and subjected to partitioned phylogenetic analysis after removal of redundant haplotypes, with the appropriate model applied to each partition.The Akaike Information Criterion [AIC; ref. 101, applying a GTR model with different values of α for each partition, as indicated by jModelTest2, and using the options for rapid bootstraps and automatic halting. Support values for the nodes were estimated from 456 bootstraps. Bayesian reconstruction was conducted in two Bayesian algorithms, MrBayes v.3.2.2102 and BEAST v. 1.8.2103, using as priors the models indicated by jModeltest2, but letting the programs estimate the parameters. Mr. Bayes was run in 4 chains and for 6 × 107 steps, which allowed the average standard deviation of split frequencies to fall below 0.01, and the potential scale reduction factor to be equal to 1.00. Convergence was also determined in multiple runs, which produced the same topology. Node credibility values were determined by sampling every 600th tree after discarding 25,000 trees as a burnin. BEAST results came from eight separate runs of different numbers of steps, combined in Logcombiner v. 1.8.2 taking every 10th tree and a burnin of 10% per run, for a total of 26,085 trees. Tracer v. 1.6 verified that Effective Sample Size (ESS) of all estimates of the combined runs exceeded 200.Maximum Likelihood (ML) phylogenetic reconstruction was conducted in RAxML v. 8.2.6104. However, as there are claims that only “narrow, shallow, and transient channels” connected the Caribbean with the Tropical Pacific as of 13 MYA (million years ago)54, the priors for this calibration point were set as uniform, ranging from 0.2 to 10100 MY. RAxML and BEAST analyses were run on the CIPRES Science Gateway105. A median-Joining network106 of haplotypes in the same geographic region was constructed in PopArt v.1 (http://popart.otago.ac.nz) to visualize relationships between haplotypes and geography within subclades in which the ancestral sequences may still be present in the populations40.In addition to reconstructing the phylogeny, BEAST was used to estimate date of divergence between major clades. For this purpose, the separation between Atlantic and Pacific haplotypes was given an offset of 3 million years (MY) in a Lognormal Uncorrelated Relaxed clock. This is the generally accepted date of the completion of the Central American Isthmus107 and sequence-based pairwise FST (ΦST) values were calculated in Arlequin 3.5.1.2108 using Tamura and Nei109 distances. Analysis of Molecular Variance (AMOVA)110 was also carried out with this program, with significance determined in 10,000 reshufflings of haplotypes between populations. For a coalescent analysis, intended to determine times of separation of populations and subsequent direction of gene flow, we used IMa2 v. 2.0111. In the MCMC mode of this program we used geometric heating with 60 chains and a burnin of 5 hours. The runs were continued until an ESS of >200 was obtained for each parameter. L mode, intended to determine whether gene flow was significantly different from 0 and significantly different in each direction, was run for 3 × 107 steps. The uniform prior for mutation rate per lineage was estimated from assuming that divergence between Atlantic and Pacific populations of P. armatus reflects separation of 3 MY. The prior of the substitution rate was given a range equal to the mean and distributed symmetrically on either side of the mean. Comparisons between populations were made in pairwise fashion in separate runs. For Isolation by Distance analysis, Mantel112 correlations between ΦST and geographic distance by sea we employed IBDWS113 with 104 iterations.For the purposes of population genetic analyses, 4 samples from the Pacific shore of Costa Rica were pooled with those from the adjacent coast of Panamá, and 3 samples from Belize were pooled with those from Yucatán. All analyses were based on the full concatenated sequences of 16S, COI-af and COI-HL except for those involving the invasive Georgia population, for which only sequences from COI-HL were available. Haplotype and molecular diversityDatasets generated during the current study are available in GenBank under accession numbers KY856997–857273 (for 16S rDNA) and KY857274–857550 (for COI) and in Dryad (https://doi:10.5061/dryad.pm1r2) .


Protoreaster nodosus in Singapore has similarly high levels of genetic diversity as comparable Indonesian populations from the Coral Triangle. The high genetic diversity of this population is remarkable because it is maintained despite decades of continued anthropogenic disturbance. We postulate that it is probably due to broadcast spawning which is likely to maintain high levels of population connectivity. To test this, we analysed 6140 genome-wide single nucleotide polymorphism (SNP) loci for Singapore's populations and demonstrate a pattern of near panmixia. We here document a second case of high genetic diversity and low genetic structure for a broadcast spawner in Singapore, which suggests that such species have high resilience against anthropogenic disturbances. The study demonstrates the feasibility and power of using genome-wide SNPs for connectivity studies of marine invertebrates without a sequenced genome.The Coral Triangle is widely considered the most important centre of marine biodiversity in Asia while areas on its periphery such as the South China Sea, have received much less interest. Here, we demonstrate that a small population of the knobbly sea star  Sequence chromatograms were checked for quality, assembled and translated to check for stop codons in Sequencher™ v. 4.5 (Gene Codes Corporation).Genotyping was performed using the tRNAasn42F and ValvaCOI-770R primers  for the P. nodosus populations from the Coral Triangle [rlequin v. 3.5 [ΦST values) were calculated with 10 000 pairwise permutations for which the p-values were subject to the Benjamini–Hochberg procedure to correct for multiple comparisons [D [FS [COI sequence data were also obtained for Triangle  (figure Triangle . After rTriangle . Other pn v. 3.5 : (i) paiparisons , (ii) a isons [D ,30 and Fs [D [FS , and  buffer . Briefly, 18 µl per sample was subjected to size-selection with sequential bead : DNA ratios of 0.78× and 0.95× to extract 250–600 bp DNA-adapter fragments, washed with 85% ethanol and resuspended in 20 µl molecular grade water. Triplicate PCRs were used to amplify 2 µl of the size-selected, adapter-ligated DNA fragments in a 10 µl reaction containing 1× Q5 reaction buffer, 200 µM each dNTP, 0.2 µM each PCR primer and 0.2 U Q5® High-Fidelity DNA polymerase. PCR cycling conditions were as follows: 98°C for 30 s, two cycles of 98°C for 10 s, 55°C for 30 s and 72°C for 1 min, 18 cycles of 98°C for 10 s and 68°C for 1 min, followed by a final extension at 72°C for 2 min. Size-selection was repeated using the same bead : DNA ratios as before, to ensure clean products of the desired fragment size. Purified DNA libraries for 36 P. nodosus samples were quantified using the Qubit® 2.0 Fluorometer, pooled in equal proportions, and sequenced on a single Illumina HiSeq lane .DNA extracts were prepared from two to four tube feet following the manufacturer's protocol of the Biospin tissue genomic DNA extraction kit. Extracted DNA samples were quantified using NanoDrop 1000 and assessed for quality on a 1% agarose gel. In total, 36 samples were selected from across the three main sampling localities, Pulau Sekudu, Pulau Semakau and Cyrene reefs, based on DNA quality. A double-digest restriction enzyme associated DNA sequencing (ddRADseq) library was then prepared from these extracts for genome-wide SNP analyses, using the adapters and PCR primer pairs in Peterson et al.   was excluded from subsequent steps due to low amounts of data retrieved. (ii) For each sample, reads were sorted into ‘stacks’ of at least five identical reads using the ustacks module. A sensitivity analysis of the mismatch thresholds (M) for merging of stacks was conducted with 1, 2, 4, 7 and 14 bp mismatches between stacks. The removal and deleveraging algorithms were enabled to remove highly repetitive stacks and resolve over-merged stacks. (iii) A catalogue of radtag loci was created with loci from across all samples using cstacks with the same number of mismatches allowed between stacks during step (ii) as the number of mismatches between loci to generate the catalogue. (iv) Stacks from each sample were matched against the radtag locus catalogue to determine the allelic state in the sstacks module. (v) The populations module was then used to filter and retain the first SNP per radtag locus with a stack depth of at least 30 and not more than 20% missing data, and calculate basic population statistics such as nucleotide diversities and heterozygosities. Several trial runs were conducted under the exclusion of one to several samples with little data. Two samples, SE20 and TP14, were eventually excluded from further analyses because the number of SNPs called dropped below 100 when these samples were included. The final dataset consisted of 33 samples. A minor allele filter was also applied to remove alleles present in only one (MAF > 0.06) of these 33 individuals. Preliminary sample clustering analyses suggested little differences in the final population structuring patterns across different parameter setting combinations . We chose to focus on a mismatch threshold of four as the number of SNP loci mined increased to almost a plateau for most datasets from four to seven . Effects of minimum stack depths on population genetic structure were then explored (mismatch thresholds fixed at 4) using initial stack depths of 3 and 5, and final stack depths of 20 and 10 . Because the version of STACKS used here did not consider indels which may lead to over-splitting of loci, SNP calls were also made using pyRAD v. 3.0.65 [Read quality of the raw sequencing data was assessed using FastQC v. 0.11.2 . Data pr. 3.0.65  at severrlequin was used to screen for loci that were found to deviate from Hardy–Weinberg equilibrium (HWE). Identification of loci possibly under selection was performed using Bayescan v. 2.1 [4 followed by 1 × 105 iterations, and all other parameters left as default. At false discovery rate (FDR) levels of 0.05, 0.1 and 0.2, loci were determined as under directional (diversifying) selection when positive alpha values were encountered, and under balancing or purifying selection (background) when zero or negative α values were encountered [K) considering correlated allele frequencies in the admixture model and without using sampling locations as priors [α. StrAuto [5 burn-in followed by 1 × 105 MCMC iterations for data collection. The optimal K was determined using STRUCTURE HARVESTER [Greedy algorithm in CLUMPP v. 1.1 [G′ST [DJost [FST [fastDivPart function in the package diveRsity [Input files for downstream analyses were formatted using PGDSpider v. 2.0.5.1 . Arlequin v. 2.1 , with a ountered ,40. Locis priors –43. Prel StrAuto , which iARVESTER ,46. To aP v. 1.1  was usedP v. 1.1 . Pairwis.1 [G′ST , DJost [T [DJost , FST [51iveRsity  that is iveRsity . The disiveRsity , was periveRsity .3.P. nodosus populations as assessed by COI haplotype diversity was high; rarefied haplotype diversity of the three main populations at Pulau Sekudu, Cyrene reefs and Pulau Semakau in Singapore (0.798–0.869) fell in the upper end of the spectrum of the Coral Triangle (KU896219–KU896296)—most of which were found at Cyrene reefs . Inspection of the raw sequence files revealed that this high drop-out rate was largely due to the presence of an N base call at the second position of the barcode region. Of these, 93.0 ± s.d. 1.7% were retained per sample after filtering for potential sites of chimera formation. The sensitivity analysis of mismatch thresholds for calling SNPs showed that varying the mismatch threshold had little effect on results. Log-likelihood score profiles of the assembled stacks  although the distributions for M = 1 and M = 2 were similarly close to zero . FIS distributions across the mismatch thresholds also displayed a similar pattern of minimal effect . Applying a minor allele frequency filter of requiring alleles to be present in at least two or more individuals (MAF > 0.06), however, resulted in a shift in peak away from FIS = 0 and increase in the number of loci at FIS = 1 , which was similar to the effect of using the rxstacks catalogue correction module in STACKS . A similar effect was also reported in [Excluding three data-deficient samples, 82.0 ± s.d. 3.5% of the barcoded reads from the Illumina HiSeq sequencer remained after quality filtering (1.7 to 7.1 × 10g errors ) displayorted in . All in b). Considering only loci that were polymorphic, genetic diversities across the three main sampling sites in Singapore were similar with minimal variation , P. nodosus populations in both the mitochondrial COI and ddRADseq SNP data. Of the 22 COI haplotypes found in Singapore waters, the three most common haplotypes were detected at three of the five locations  was detected among Singapore's ocations . Pairwis, et al. , a signi4.P. nodosus population in relation to those within the Coral Triangle, despite the many anthropogenic impacts on Singapore's marine environment [P. nodosus, we show that Singapore's P. nodosus populations can be important contributors to the global gene pool, with 18 COI haplotypes unique to a small area.The most surprising finding is the remarkably high level of genetic diversity in Singapore's ironment  and the ironment ). This cironment , but it ironment ,2), our ironment  and highironment . DespiteP. nodosus populations  such as nodosus . This isingapore ,68, and ingapore . High leingapore –72 obseringapore –78). Alsingapore  and can ingapore ). Prelime waters  suggests [et al.  previous [et al. –22), whi [et al.  that red [et al.  across m [et al. , and/or  [et al.  for larvFST values in the SNP dataset, tables FST values that reflect relationships or clustering patterns have also been shown despite changes in absolute values with different mismatch parameter settings during SNP loci clustering [FST values obtained in this study should be cautiously interpreted because the inclusion of only a subset of 10–12 samples per population in SNP analyses may have resulted in an over-estimation of pairwise FST values . Howevehan 4–6;  should shan 4–6; . A false islands , was alsPlatygyra sinensis [et al. [P. nodosus ‘populations’ in Singapore, which demonstrate their ability to quickly recover from disturbances. In December 2006 and January 2007, an unusually heavy rainfall event over southern Johor in Peninsular Malaysia caused an excessive discharge of freshwater and hence sudden drop in salinity into the area around Pulau Sekudu [P. nodosus, but the populations appear to have restored their genetic diversity levels within 6 years of the devastation. At Cyrene reefs, only few individuals of P. nodosus were encountered in 2007 , but in 2013 while sampling for this study, they were extremely abundant and genetically diverse . The reiewed in ).Our study of genome-wide SNPs supports previous research using fewer genetic markers, showing patterns of high genetic diversity and likely high connectivity consistent among several broadcast-spawning marine organisms in Singapore, and supports the concept of unified conservation strategies for marine biodiversity. With the advent of next-generation sequencing technology, more population studies can be performed rapidly using genome-wide SNPs for non-model organisms (e.g. –22,59,61


Arbutus unedo in the western Iberia Peninsula, using plastid markers with conservation and provenance regions design purposes. This species is currently undergoing an intense domestication process in the region, and, like other species, is increasingly under the threat from climate change, habitat fragmentation and wildfires. We sampled 451 trees from 15 natural populations from different ecological conditions spanning the whole species’ distribution range in the region. We applied Bayesian analysis and identified four clusters . Hierarchical AMOVA showed higher differentiation among clusters than among populations within clusters. The relatively low within-clusters differentiation can be explained by a common postglacial history of nearby populations. The genetic structure found, supported by the few available palaeobotanical records, cannot exclude the hypothesis of two independent A. unedo refugia in western Iberia Peninsula during the Last Glacial Maximum. Based on the results we recommend a conservation strategy by selecting populations for conservation based on their allelic richness and diversity and careful seed transfer consistent with current species’ genetic structure.Mediterranean forests are fragile ecosystems vulnerable to recent global warming and reduction of precipitation, and a long-term negative effect is expected on vegetation with increasing drought and in areas burnt by fires. We investigated the spatial distribution of genetic variation of  The contemporary genetic structure of a species offers important information about its responses to past geological and climatic events, which had often played a crucial role in shaping the current distribution range. The spatial distribution of genetic variation is strongly influenced by neutral and adaptive processes and, at the population level, the amount of extant genetic variation plays a key role in maintaining the adaptive potential to face future environmental changes . Among oArbutus unedo L. (strawberry tree) is a small tree native to the Mediterranean region found in western, central, and southern Europe, north-eastern Africa, Canary Islands and western Asia. The species distribution is mostly typical of Mediterranean sclerophyllous and laurel-like vegetation, mainly within coastal and inland areas, where frost or summer dryness are not too intense [Arbutus unedo genetic structure has been investigated at both local and biogeographical scale using different types of markers [Arbutus unedo fruit has strong antioxidant properties and it can be consumed fresh or processed, and it is used in Portugal to produce the liquor ‘aguardente’, an important source of income for land users, which has led to an on-going process of domestication of the species through clonal propagation of genotypes selected for fruit quality [The evergreen  intense . Arbutus markers ,11,12,13 markers . Arbutus quality  and to t quality , 17. Nev quality  developeAnthropogenic impacts on forest genetic resources include practices and processes that may affect the genetic layout of natural populations, including: (i) breeding and selection, (ii) seed transfer and (iii) environment alterations . The effA. unedo genetic diversity at the regional scale to build dynamic conservation programs in Portugal [A. unedo in Portugal, and to ii) provide recommendations for species’ conservation and help in provenance region design.The ongoing domestication process, together with the increasing frequency of wildfires due to higher temperatures and lower precipitations, demands further knowledge of the Portugal . Our speArbutus unedo is an evergreen species, a shrub or a small tree up to 8–10 m. The fruit is a round berry (ca. 20 mm wide) and bright red when ripe in autumn. The berries with a fleshy pulp are mainly eaten by seed-disperser birds, which represent the main A. unedo seed dispersal vector [A. unedo plants can easily resprout and survive if damaged  [Arbutus unedo prefers siliceous or decarbonated substrata and can grow on alkaline and relatively acidic soils, but grows satisfactorily under a very wide range of soil conditions, with pH varying from 4 to 7, excluding waterlogged soils [l vector ,27. The grazing) . This spgrazing) . Arbutused soils .The leaves for DNA extraction were collected in ca. 30 individuals from 15 natural populations sampled across the species’ natural range in Portugal , S2 Fig.A. unedo adult trees was non-destructive. No specific permission was required to sample the species in Portugal. Field studies did not involve endangered or protected species.Leaf sampling of We extracted total DNA from frozen leaves with a Dneasy® Plant Mini kit , following the manufacturer’s instructions. Six chloroplast microsatellite primers  were desTwo multiplex PCR were performed using the Type-it® Microsatellite PCR Kit (Qiagen). Forward and reverse primers for each locus were mixed at equal proportion and used in the multiplex PCR reactions. The first multiplex mix (with AU2 and AU7) was prepared in a 6 μL final reaction volume containing 1 μL of DNA (~10 ng), 3 μL of 2× Type-it mix, 1.4 μL dH2O and 0.6 μL of primer pre-mix 1 (0.1 μM each primer). The second mix (with AU1 and AU4) contained the same amount of total volume, DNA and master mix and 2 μL of primer pre-mix 2 (0.1 μM each AU1 primer and 0.2 μM each AU4 primer). The two multiplexes were amplified with the same PCR profile: an initial activation step of 5 min at 95°C, followed by 30 cycles of 30 s denaturation at 95°C, 90 s annealing at 57°C and 30 s extension at 72°C, concluding with a final extension for 30 min at 60°C. The amplified products were mixed with Hi-Di™ Formamide (Applied Biosystems) and GeneScan™ 500 LIZ™ dye size standard (Applied Biosystems) according to standard protocols and separated on an AB 3500 DNA genetic analyzer (Applied Biosystems). The molecular sizes of the fragments were binned using the GeneMarker® v2.4.0 software.mst function and visualized through the plot.mst function in the pegas package in R [h), the unbiased haplotypic diversity based on haplotype frequencies (He) [R , were computed for each population using the program CONTRIB v. 1.4 [e) and the number of private haplotypes (Ph) were calculated in GenAlEx v. 6.501 [2sh, [2sh was estimated on the original data and on a transformed dataset (ng = no gaps), where the gap between alleles were eliminated to mitigate the possible impact of indels, by in-house R scripts. The relationship between the estimates of diversity (Nh and He) and the genetic distances among individuals within populations D2sh and D2shng was tested with Pearson's product-moment correlation in R.The four cpSSR fragments analysed were combined in order to derive the chloroplast haplotype of each individual. An haplotype network, a minimum spanning tree based on the differences in number of repeats between haplotypes, was created using the age in R ,36. The ies (He)  and the B v. 1.4 . The effv. 6.501 . CONTRIBv. 6.501 . Additio01 [2sh, , as modi01 [2sh, . D2sh waK (from one to 15) to obtain the optimal number of clusters. The best partition of populations into K clusters was identified as the one with the highest log marginal likelihood.Bayesian analysis using a spatial clustering model implemented in BAPS 5.4  was usedet al. [The extent of the genetic structure was further explored using a non-hierarchical and hierarchical analysis of molecular variance (AMOVA) implemented in the Arlequin 3.5 software . We usedet al. . OverallST) and ordered alleles (NST) with the PermutCpSSR 1.2.1 software [ST is defined as a measure of genetic differentiation among populations when the distance between haplotypes is taken into account, whereas GST is a measure of genetic differentiation that makes use only of haplotypic frequencies. The significance of the two differentiation statistics was tested with 10,000 random permutations of individuals among populations. To test if the observed NST value is larger than the GST, we counted how many permutated NST values were larger than the observed NST. Statistical significance may indicate the presence of a phylogeographic structure [ST/(1-FST) as genetic distance. The statistical significance of the Mantel test correlation coefficient was obtained from 1,000 permutations using Arlequin 3.5.We checked for the presence of a phylogeographic structure by comparing differentiation of unordered alleles  and effective number of haplotypes (Ae). The population EC showed also the highest values for Nh (6). Other populations from the central region , had He values over 0.50 and Ae > 2. The populations PG and M were fixed for the most common haplotype and also the population SM showed a very low value of diversity (0.13).The average haplotypic diversity within populations was 0.40 , with po2sh and genetic diversity estimates . However, if we consider the genetic distance with no gaps (D2shng), a significant correlation was found between the average genetic distance and the estimates of genetic diversity (Nh and He) (We found no correlation between Dstimates . The pop and He) . This di and He) .The population SM showed the highest contribution to the differentiation of populations, while populations G and ON showed the highest contribution to the total diversity. The populations that gave the highest contribution to the population diversity were AV, ON and EC. The populations PG and M contributed negatively to both components of the total diversity (diversity and divergence components), showing only one haplotype CT%, . The popThe Bayesian clustering suggests the presence of four major groups of populations: a northern cluster, a central cluster with populations from the coast and inland, a southern cluster, and, finally, one cluster with the outlier population SM . The hieST = 0.29±0.11 was among population differentiation based on unordered alleles and the NST = 0.28±0.11 was based on ordered alleles. The observed NST was not significantly different from the observed GST, showing absence of a phylogeographic structure.Finally, the Mantel tests showed an absence of correlation between genetic and geographic distances with both mutation models and thus a lack of isolation by distance pattern. Considering all the populations, the GA. unedo in Portugal during the LGM, although it is worth noting that continuous pollen records in the region are missing [A. unedo is a low pollen producer often underrepresented in the pollen diagrams [Quercus coccifera L., including A. unedo and other thermophiles shrubs, during the Middle Holocene in the southern region. Santiso et al. [A. unedo, during the last glacial period [A. unedo in this area, the hypothesis of the presence of different refugia cannot be excluded. Two well-differentiated genetic clusters were found, one in central and another in southern Portugal. In both clusters, there are populations displaying high genetic diversity  and some private haplotypes . Such findings contrast with the hypothesis of a common postglacial origin of A. unedo populations in Portugal.There is evidence about two possible refugial areas for  missing  and thatdiagrams . Neverthdiagrams  found pao et al.  also posl period . Accordil period . In the l period . Thus, cl period  with ourA. unedo at its westernmost distribution limit that need to be clarified, and for which the use of genetic markers characterized by higher mutation rates (e.g. nuclear microsatellites) would be helpful. We observed an absence of phylogeographic structure and IBD patterns in our study that could be explained by not having considered the genetic structure found in the analysis. Nevertheless, when tests were repeated only on the central cluster, the only one with sufficient data to obtain meaningful results, the lack of phylogeographic structure and IBD was confirmed (results not shown). Overall, this points towards low levels of gene flow among populations probably due to limitations in the seed dispersal by birds. Whether limited gene flow may increase the effect of genetic drift in erasing IBD patterns, depends on overall gene exchanges among populations and, therefore, on both pollen and seed gene flow [A. unedo to understand the impact of genetic drift on the spatial distribution of genetic diversity found in the present study.There are several points about the recent evolutionary history of ene flow . This coA. unedo survival and reproduction that prefers open-canopy environments. However if fires are too frequent or intense, the impact on population genetic diversity can be negative, as it is probably the case for the PG population  [Another important aspect to be addressed is the effect of contemporary factors on the distribution of genetic variation, with suitable markers. The occurrence of wildfires in the region potentially drives to abrupt changes in population size. Wildfires help in keeping the canopy open, a key factor for ce 1975) . We shouce 1975) , yet witce 1975)  and evenA. unedo is, currently, undergoing an intense domestication process, with the commercialisation of improved genetic material (clones) to produce new plantations and the introduction of genetic material through seed from unknown origin [In the studied region n origin ,17. Domen origin , this laet al. [A. unedo candidate populations for conservation, besides the genetic diversity parameters, we used the contribution of each population to the total diversity partitioned into two components. The first was related to the level of diversity of the population and the second to its ‘uniqueness’, i.e., its divergence from the other populations [Populations are not similar in their capacity to adapt to varying environmental conditions and genetic information can ensure a better use of the available resources by maximizing the evolutionary contribution of the set of populations to conserve . Diversiet al. . To seleulations .A. unedo in Portugal since rare haplotypes will be conserved from a putative remnant population. The southern cluster EC population should be also considered as a potential gene reserve, due to its high genetic diversity and haplotypic richness, and for harbouring a private haplotype as SM. This population has the highest contribution to the total haplotypic richness in both the differentiation and diversity components. Similarly, the ON and AV populations, from the central cluster, should also be considered in conservation programs. The first population show the second highest values of genetic diversity and number of haplotypes, besides the average genetic distance among individuals, and the later has the highest haplotypic richness and number of private haplotypes in this cluster. Conservation should include the i) possible impact of very frequent wildfires in the genetic diversity, and the ii) impact of the species domestication at the region scale. In this region, maritime pine was a paradigmatic example of domestication impact in the species genetic structure: the use of seeds from unknown origin has completely blurred it and affected the species’ forest resources [We found that the SM population, representing a single-population genetic cluster, was the most differentiated, with a high contribution to divergence, but not to diversity levels. We suggest including this population in a conservation programme for esources , and thiConsidering the species’ genetic improvement, the breeding population used in improvement program, including the clones used for deployment, should mirror the diversity and the haplotypic richness of the species in the region, and care should be taken to monitor the breeding generations ahead. This could be done by comparing the haplotypic and genetic diversity of the breeding population with the current study populations’ haplotypic and genetic diversity and by using additional markers, such as nuclear microsatellites and single nucleotide polymorphisms ,58.The information retrieved in the current study will also be helpful to design provenance regions aiming at collecting reproductive material for plantations. Those provenance regions should be built based on: i) genetic clustering results, and ii) ecological attributes, including wildfire information . In ordeS1 Fig(A) Percentage of burned area development. (B) Number of wildfires development. European Commission (2007) Forest Fires in Europe 2006. European Commission, Joint Research Centre. Institute for Environment and Sustainability, Report 7. Luxembourg.(TIF)Click here for additional data file.S2 FigA. unedo stand distribution according to the 2006 Portuguese National Forest Inventory: http://www.icnf.pt/portal/florestas/ifn (orange triangles).Map highlighting the populations’ sampled (black dots) and the (TIF)Click here for additional data file.S1 TableHaplotype refers to four chloroplast microsatellites composition respectively: AU1, AU2, AU4 and AU7.(DOCX)Click here for additional data file.


Calidris maritima) is a medium‐sized shorebird that breeds in the Arctic and winters along northern Atlantic coastlines. Migration routes and affiliations between breeding grounds and wintering grounds are incompletely understood. Some populations appear to be declining, and future management policies for this species will benefit from understanding their migration patterns. This study used two mitochondrial DNA markers and 10 microsatellite loci to analyze current population structure and historical demographic trends. Samples were obtained from breeding locations in Nunavut (Canada), Iceland, and Svalbard (Norway) and from wintering locations along the coast of Maine (USA), Nova Scotia, New Brunswick, and Newfoundland (Canada), and Scotland (UK). Mitochondrial haplotypes displayed low genetic diversity, and a shallow phylogeny indicating recent divergence. With the exception of the two Canadian breeding populations from Nunavut, there was significant genetic differentiation among samples from all breeding locations; however, none of the breeding populations was a monophyletic group. We also found differentiation between both Iceland and Svalbard breeding populations and North American wintering populations. This pattern of divergence is consistent with a previously proposed migratory pathway between Canadian breeding locations and wintering grounds in the United Kingdom, but argues against migration between breeding grounds in Iceland and Svalbard and wintering grounds in North America. Breeding birds from Svalbard also showed a genetic signature intermediate between Canadian breeders and Icelandic breeders. Our results extend current knowledge of Purple Sandpiper population genetic structure and present new information regarding migration routes to wintering grounds in North America.The Purple Sandpiper ( They concluded that although there were notable differences among breeding populations in both morphology and DNA sequence data, these differences were not large enough to warrant distinct subspecific status. Barisas et al. , and Wennerberg, Marthinsen, and Lifjeld  and found evidence for the use of multiple refugia across Beringia that corresponded to different glacial cycles. Rock Sandpipers, which are distributed along the Pacific coast in northwestern North America, Kamchatka, and the Aleutian Islands  and ten newly developed, polymorphic microsatellite markers to extend the genetic analysis conducted by Barisas et al. . We asse22.1Blood, tissue, and pre‐isolated DNA samples were obtained for a total of 279 Purple Sandpipers from a variety of sources, outlined in Figure C. m. maritima based on Engelmoer and Roselaar , spin column protocol. For blood samples, we used 190 μl phosphate buffered saline in step 1b of the animal blood protocol as recommended by Bush, Vinsky, Aldridge, and Paszkowski . For liv® Blue PCR SuperMix, 2 μl forward primer (10 μmol/L), 2 μl reverse primer (10 μmol/L), and 2 μl template (~25 ng/μl) or up to 5 μl for DNA from feathers, which typically had <5 ng/μl DNA concentration. When possible, the control region and cytochrome b regions were amplified in one fragment. However, for some samples, which were presumably partly degraded, the control region was amplified in three fragments and cytochrome b was amplified in two fragments. Control region reactions were heated to 94°C for 3 min for initial denaturing, followed by 40 cycles of 1 min at 94°C for denaturing, 1 min at 50°C for annealing, and 1 min at 72°C for extension. Finally, the temperature was brought to 72°C for 5 min for a final extension. Cytochrome b reactions were heated to 94°C for 3 min for denaturing, followed by 40 cycles of 45 s at 94°C for denaturing, 45 s at 50°C for annealing, 1 min at 72°C for extension, and the final extension step of 72°C for 5 min. Unpurified PCR products were sent to McGill University and Génome Québec Innovation Centre where they were purified and Sanger sequenced on an Applied Biosystem 3730xl DNA Analyzer using the same primers as in the amplification reactions.The mitochondrial DNA control region and cytochrome b mtDNA fragments were amplified separately in reaction mixtures containing 44 μl PlatinumInitial attempts to amplify an 841‐bp fragment of the mtDNA control region from all samples used primer L98 taken from Wenink, Baker, and Tilanus  and primWhen possible, a cytochrome b fragment of 712 bp was amplified using primers designed for Rock Sandpipers , the most closely related organism for which the entire mitochondrial genome is available. Sequences were trimmed to remove nucleotide positions with missing data, including a section of cytochrome b (n = 19 bp) that was not covered by the internal primers. All primers are listed in Table As mitochondrial DNA is inherited as a single locus, the control region and cytochrome b sequences were concatenated and analyzed as a single marker  and associated reagents . Individual reactions used 2 μmol/L primer concentrations in the following volumes: 0.4 μl forward primer, 1.6 μl reverse primer, 1.6 μl M13 primer, with 11.1 μl of water to bring the total volume to 20 μl. Duplex reactions used 2 μmol/L primer concentrations in the following volumes: 0.26 μl of the smaller fragment's forward primer, 0.2 μl of the larger fragment's forward primer, 1.0 μl of the smaller fragment's forward primer, 0.8 μl of the larger fragment's forward primer, 1.5 μl of the M13 primer, and 7.94 μl water to bring the volume up to 20 μl.All microsatellite reactions were performed in 20 μl volumes using Platinum® Taq polymerase and associated reagents . The product was then diluted 1:100 in water, and 5 μl was used as template for 10 singleplex reactions . Negative controls were included in all amplifications, and samples were amplified and genotyped three times to ensure accurate genotypes. All microsatellite PCR products were sent to McGill University and Génome Québec Innovation Centre, where they were genotyped using an ABI 3730xl DNA Analyzer.Because of limited quantity of DNA, the northern Nunavut samples were pre‐amplified  were treated as a separate wintering location for pairwise estimates. p‐Values to evaluate statistical significance were generated using 10,000 nonparametric permutations to obtain a null distribution of pairwise ΦST values. Estimated number of migrants between populations was calculated using Arlequin.Overall genetic differentiation among breeding populations and pairwise differentiation among all populations was calculated in Arlequin 3.5.1.3 . Five of these were found in wintering populations, which are known to violate Hardy–Weinberg assumptions due to the presence of individuals from several distinct breeding populations, all with null allele frequencies ranging from 5% to 17%. One was found in Iceland at locus CM2668, with a null allele frequency of 15%. Null allele frequencies below 20% have limited effect on general population structure analyses , and none of the significant Allelic diversity among the 10 microsatellite DNA loci ranged from 5 to 13 alleles, with a mean of 8.5 alleles/locus Table . Samples3.2ST calculated for mtDNA across all breeding populations was 0.089 (p = .004). Overall FST calculated for microsatellites across breeding populations was 0.080 (p < .001). Both of these values were significantly higher than expected under panmixia .Overall ΦST and FST values, again calculated for mtDNA and microsatellites, respectively, indicate differentiation between most breeding populations, and between Iceland/Svalbard populations and most wintering locations (see Table ST (p = .097) and FST (p = .999) values. Both Canadian breeding populations showed the opposite trend, with no measurable differentiation found between breeding population and wintering location. The exceptions to this were low but significant ΦST  and FST  values between northern Nunavut and Maine, and FST values between northern Nunavut and New Brunswick . Finally, the ΦST value for Iceland and western Newfoundland was much higher  than values found between Iceland and other North American wintering locations. In contrast, FST values between Iceland and western Newfoundland  were much lower, though still different, than those between Iceland and eastern Newfoundland .Pairwise Φ3.3The Bayesian Figure , MLB, anp‐distance between individuals within this clade and individuals outside the clade was .05 (or 0.5%).Clade A contained five haplotypes and was present in both the Bayesian and MLB trees. This clade has a PP of 96% and a bootstrap value of 52%. Clade B contained nine haplotypes and was found in all three trees. PP for this clade was 79%, and bootstrap values were 59% and 44% for MLB and MPBs, respectively. Notably, this clade was monophyletic for the two Canadian breeding populations of Purple Sandpipers , which may indicate that these haplotypes are unique to Canadian breeders. The remaining clades observed in these trees each contained only two haplotype groupings and had posterior probabilities or bootstrap values between 31% and 74%. The Clade B was also present in the haplotype network. Haplotypes from all breeding locations were dispersed throughout most of the network outside Clade B, indicating limited geographic structure of Purple Sandpiper breeding populations. Branches were also quite short, with many singleton or low‐frequency haplotypes differing from a common haplotype by a single substitution.Three circular connections were found in the haplotype network Figure a. In an 3.4When all samples (breeding and wintering) were considered without a priori population information, the most likely number of clusters was four. However, only 43 individuals 15%) were assigned to a cluster with greater than or equal to 80% probability, and 127 (46%) birds were assigned to a cluster with 60% or greater probability. Iceland samples consistently showed highest assignment to one cluster, with Svalbard samples showing lower assignment to the same cluster , and thus, this result should be interpreted with caution.None of the Wilcoxon tests for heterozygosity excess was significant, and the mode shift test was significant only for southern Hudson Bay (data not shown). However, this population was below the minimum recommended number of samples  all showed a genetic signature that could reflect recent expansion of the Purple Sandpiper global breeding range, recovery from a strong bottleneck, or a recent selective sweep  than values between Iceland/Svalbard and other North American wintering populations. These large differences suggest either that Purple Sandpipers from Iceland/Svalbard do not migrate to these areas of North America, or that they do so in relatively small numbers compared to migrants from northern Canada, Hudson Bay, the United Kingdom, and possibly unsampled areas such as Greenland. Purple Sandpipers from Iceland have been hypothesized to migrate to western Newfoundland in the winter, due to the large size of individuals in that particular region of Atlantic Canada , the Iceland population of Purple Sandpipers qualifies as an “evolutionarily significant unit”  for both FST and ΦST (with the exception of northern Nunavut for ΦST). It is not possible to discount the comparison between Svalbard and northern Nunavut given that the sample size was larger for northern Nunavut than southern Hudson Bay (as referred to above). We suggest that conservatively, Svalbard should be recognized as a separate MU while requiring further analysis to determine whether it should also be recognized as a distinct ESU. Similarly, the presence of a distinct mitochondrial lineage  and mussels (Mytilus spp.), as well as other arthropods, a diet similar to those studied in Europe. Wintering Purple Sandpipers rely heavily on habitat containing intertidal red seaweed (Mastocarpus stellatus), and to a lesser extent on Rockweed (Ascophyllum nodosum), and commercial harvesting of marine alga  in Atlantic Canada could also negatively affect wintering populations, and consequently breeding populations from Canadian and European locations that winter here, by depleting mussels and periwinkle populations, both of which have been shown to be favored prey of this crab .Control Region and Cytochrome B sequences for Purple Sandpipers and Rock Sandpipers have been submitted to GenBank under accession numbers  Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.


Cichorium intybus (chicory) which was introduced to North America in the 18th century and which now can be found in all 48 continental U.S. states and much of Canada. We genotyped a Eurasian collection of 11 chicory cultivars, nine native populations and a North American collection of 20 introduced wild populations which span the species range . To detect the geographic sources of North American chicory populations and to assess the genetic diversity among cultivars, native, and introduced populations, we used both a sequenced cpDNA region and 12 nuclear simple sequence repeat (SSR), microsatellite loci. Four cpDNA haplotypes were identified and revealed clear geographic subdivisions in the chicory native range and an interspecific hybrid origin of Radicchio group. Nuclear data suggested that domesticated lines deliberately introduced to North America were major contributors to extant weedy populations, although unintended sources such as seed contaminants likely also played important roles. The high private allelic richness and novel genetic groups were detected in some introduced populations, suggesting the potential for local adaptation in natural sites such as deserts and nature reserves. Our findings suggest that the current populations of weedy U.S. chicory have evolved primarily from several sources of domesticated and weedy ancestors and subsequent admixture among escaped lineages.Plant invasions are recognized as major drivers of ecosystem change, yet the precise cause of these invasions remains unknown for many species. Frequency and modes of introductions during the first, transport and colonization, stages of the invasion process as well as phenotypic changes due to plasticity or changing genetic diversity and adaptation during later establishment and expansion stages can all influence the “success” of invasion. Here, we examine some of these factors in, and the origin of, a very successful weed,  Much haFactors such as the number and source of introductions into new environments have also proven to be important and often are a consequence of human socioeconomic activities. Most noxious invaders have shown a lag time between first introductions, establishment, and the range expansion. With historical data, Aikio, Duncan, and Hulme  quantifiCichorium intybus (chicory) is an established, cosmopolitan weed. It is diploid (2n = 18), perennial, self‐incompatible, and possesses extensive phenotypic diversity. Chicory is grown for its roots which are used as a coffee surrogate, a source of polysaccharide inulin, and as a leafy vegetable . Chicory also became a weedy/invasive species in North America and Australia and is labeled a noxious weed in the state of Colorado. Weedy chicory can be found across North America in 48 continental states of the United States and most provinces of Canada (USDA Plants Database). Chicory was also collected in 1956 on O'ahu Island (www.hear.org/vouchers/pier/bish0000011844.htm), but is not currently reported in Hawaii. Chicory exhibits a great range of phenotypes for leaf shape, color, leaf surface, hairiness, as well as plant size and, based on greenhouse experiments with variable soil types, temperatures and climatic conditions much of the phenotypic diversity can be attributed to environmental plasticity : var. sativum (1) Root chicory, and the remaining groups used for leaves: var. foliosum (2) Witloof (or Belgian endive), (3) Pain de Sucre, (4) Radicchio, and (5) Catalogna. All the red types of radicchio are believed to come from red‐leafed var. foliosum, while plants with spotted or variegated leaves likely originated from spontaneous or controlled crosses between red‐leafed chicory var. foliosum and broadleaved endive Cichorium endivia  with 10 or more repeats, and 4‐bp SSRs with 5 or more repeats. Primers flanking the SSRs were designed in Primer3 program , but no evidence for LD was detected. The final set of twelve most polymorphic markers for C. intybus was employed for genotyping all the individuals.In this study, we used twelve microsatellite nuclear markers Table  and the 2, and 0.2 μl of Taq polymerase. The final reaction volume was brought to 25 μl with sterile water. We used a touchdown protocol with following cycles: 5‐min denaturation at 95°C, ten cycles of 30 s at 94°C, 30 s at 60°C, and 45 s at 72 °C, annealing temperature decreasing to 50°C by 1°C per cycle, followed by 30 cycles of 30 s at 94°C, 30 s at 50°C for 30 s, 45 s at 72°C for 30 s, followed by a final extension at 72°C for 5 min.Seeds for chicory cultivars and Eurasian populations were grown in the greenhouse, and leaf tissue was harvested for DNA extractions. North American chicory DNA was extracted from dried leaves. Collectors dried partial or full leaves from flowering chicory populations either by placing them into 15 ml tubes containing DriRite  or by pressing the leaves in newspaper. Samples were mailed to authors together with population location information. FastDNA extraction kit  was used for DNA extractions according to manufacturer's protocol. Chloroplast primers were used as described by Taberlet et al. . PCRs wi2.3Avant sequencer . cpDNA sequence editing and alignment was performed using the program Sequencher 4.9 (http://genecodes.com/). We used Peak Scanner software for microsatellite fragment length scoring (Applied Biosystems). Peaks were assigned numbers by Peak Scanner based on the 400HD Rox size ladder which approximated the length of amplicons. Each individual peak size was confirmed visually. The observed (Ho) and the expected (He) heterozygosity, inbreeding coefficient (F), and the analysis of molecular variance (AMOVA) were calculated using Arlequin v. 3.5.1.3 .Chloroplast DNA (cpDNA) fragments were sequenced and PCR products targeting microsatellite regions were assayed on the 3100‐K set from 1 to 7), and with a complete data set  with K set from 1 to 10, with 10 iterations for each K in each independent run. Structure results were run through STRUCTURE HARVESTER v. 0.6.93  haplotype. None of the North American chicory populations (NA) possessed more than one haplotype in our analysis. Hap 1 was the most common and found in 13 of 20 populations. Hap 2 was detected in five populations  and Hap 3 in two populations  in North America  of all populations was generally high ranging from 0.44 to 0.76 (Tables He = 0.617), EU (He = 0.658), and NA (He = 0.610) were similar, although slightly higher in the EU group (Table F = 0.23) among all CC and EU accessions  and the wild invasive NA (FST = 0.0238) groups, than these two wild groups had with each other (FST = 0.0442), suggesting that the domesticated lines may have been a key vector in the North American invasion history. The AMOVA  was varied from one‐seven. The highest likelihood in the partial analysis was obtained when K was set to four, and also the maximal ΔK occurred at K = 4 using the method of Evanno et al. (K) was varied from one to twelve. Using the method of Evanno et al. . Wild Eurasian populations showed mosaic genotypes, and no clear nuclear genotype division was observed between northern and southern populations as was detected for the chloroplast data. Radicchio It1) and Pain de Sucre (Zuc) genetic cluster was present in Ge, Po1, Ru, Cz, Yu, and Ir populations . The most widespread genetic group, Catalogna, is present throughout the east but highly represented in only NM in the west. One rather dominant genetic group in three of the NA populations  does not seem to be well represented in the CC or EU collections. Several North American populations showed multiple distinct genetic clusters within a population . Mosaic genotypes suggesting high levels of gene flow were detected in MV, Amf, UNH, MEP, STL, and OH populations. The presence of apparent mixed‐ancestry is clearly visible in urban areas with disturbed habitats. Populations with single ancestry seem more common in less disturbed more natural sites such as deserts and nature reserves , similar to another North American non‐native taxon in the Asteraceae  and only three others exceeded 0.10. Thus, we have no indication that we missed a major contributor to the North American invasion.While a high genetic diversity is not necessarily a prerequisite for successful habitat colonization  as they were to wild NA accessions (FST = 0.0238). Conversely, the wild accessions of Eurasia and wild NA accessions showed nearly twice the FST value (0.0442). This suggests that North American chicory evolved primarily from introduced domesticated lines over the past two hundred years. However, some NA populations (NV and CO) seemed to have a closer affinity to wild accessions from EU and other populations in NA seemed relatively distinct from the major domesticated lines surveyed in this study. This together with the high private allelic richness found in the invaded region indicate additional introductions and sources of diversity as well as the possibility of local adaptation to the new environments.Our results are consistent with previous studies in European chicory which showed high levels of gene flow between cultivated and wild chicory accessions  to that in the Iran region of the EU collection as well as some of the highest private allelic richness scores  could indicate the potential for adaptation for drier habitats or other local conditions. Mosaic genotypes detected in urban populations may imply multiple introductions and an extensive gene flow in these areas.Cichorium intybus populations. Chicory possesses many traits that contribute to its ability to spread and adapt to a variety of habitats. These favorable traits include a self‐incompatibility system that promotes outcrossing, plasticity, tolerance of different environmental conditions, and also recently discovered allelopathy (Wang et al., There are no previous studies examining genetic diversity and population structure in North American chicory. The objective was to determine the genetic relationships and structuring between domesticated, wild, and introduced None declared. Click here for additional data file. Click here for additional data file. Click here for additional data file.