Etelis coruscans (N = 787) and E. “marshi”  with 436–490 bp of mtDNA cytochrome b and 10–11 microsatellite loci. Phylogeographic analyses reveal no geographic structuring of mtDNA lineages and recent coalescence times that are typical of shallow reef fauna. Population genetic analyses reveal no overall structure across most of the archipelago, a pattern also typical of dispersive shallow fishes. However some sites in the mid-archipelago  had significant population differentiation. This pattern of no structure between ends of the Hawaiian range, and significant structure in the middle, was previously observed in a submesophotic snapper (Pristipomoides filamentosus) and a submesophotic grouper (Hyporthodus quernus). Three of these four species also have elevated genetic diversity in the mid-archipelago. Biophysical larval dispersal models from previous studies indicate that this elevated diversity may result from larval supplement from Johnston Atoll, ∼800 km southwest of Hawaii. In this case the boundaries of stocks for fishery management cannot be defined simply in terms of geography, and fishery management in Hawaii may need to incorporate external larval supply into management plans.The Hawaiian Archipelago has become a natural laboratory for understanding genetic connectivity in marine organisms as a result of the large number of population genetics studies that have been conducted across this island chain for a wide taxonomic range of organisms. However, population genetic studies have been conducted for only two species occurring in the mesophotic or submesophotic zones (30+m) in this archipelago. To gain a greater understanding of genetic connectivity in these deepwater habitats, we investigated the genetic structure of two submesophotic fish species (occurring ∼200–360 m) in this archipelago. We surveyed 16 locations across the archipelago for submesophotic snappers  Understanding the genetic connectivity of marine populations can provide valuable information about evolutionary mechanisms in the marine environment, as well as effective management and conservation strategies. The Hawaiian Archipelago has become a natural laboratory for understanding genetic connectivity of marine organisms, including more than 30 marine species across a wide taxonomic range  Pristipomoides filamentosus) The population genetic studies conducted thus far in the Hawaiian Archipelago have included only two species which occur at the lower margin of photosynthetic activity in the mesophotic and submesophotic zones, the Hawaiian Grouper, called “Hapu'upu'u” in Hawai'i  that shows strong genetic divergence among all islands sampled across the archipelago, and one surgeonfish (Zebrasoma flavescens) that shows low genetic structure The pattern of genetic structure and high genetic diversity in the mid-archipelago has not been observed in other fish species studied in Hawai'i thus far. These studies include 15 shallow-water reef fish species, most of which exhibit high connectivity across most or all of the archipelago, with the exception of one damselfish (Etelis coruscans) and the Ruby Snapper, called “Ehu” in Hawai'i . Adult E. coruscans and E. marshi typically occur at deeper depths  than adult P. filamentosus and H. quernus  To test the hypothesis that the pattern of genetic structure and high diversity in the mid-archipelago is consistent for other deepwater fishes, we conducted population genetic analyses of two submesophotic fishes which occur across the archipelago: the Flame Snapper, called “Onaga” in Hawai'i , or a suspected territorial, haremic social structure (H. quernus). In contrast, E. coruscans and P. filamentosus are more pelagic feeders that usually occur 2–50 meters above the bottom.The adult habitat of all four of these species is similar in that it consists of hard substrate with high structural complexity and/or high relief E. coruscans and E. marshi, we used mtDNA cytochrome b (cytb) sequences and 10–11 microsatellite loci from 16 sample sites across the archipelago (E. coruscans and E. marshi with the population genetic structure reported in previous studies for two other submesophotic fishes (P. filamentosus and H. quernus) and shallow-water fishes in the Hawaiian Archipelago. Additionally, submesophotic habitats may lie below the zone disrupted by sea level changes during glaciations, and thus communities in this deepwater habitat may be older and more stable than their shallow counterparts. We tested this hypothesis by estimating mtDNA coalescent times for E. coruscans and E. marshi, and comparing these values with previously published mtDNA coalescent times from shallow-water fishes.To investigate the population genetic structure of hipelago , Table 2E. coruscans (N = 787) (E. marshi (N = 770) (Specimens consisted of fin or muscle tissue collected throughout the Hawaiian Archipelago from 16 locations each for N = 787)  and E. mN = 770) . SpecimeN = 770) , Table 2b gene for both species. Primers used for E. coruscans were Cyb-05 L15020 (GCCAACGGCGCATCCTTCTTCTTAATAGGAAGTATCATTCGGGTTTGATGE. marshi were designed for this study: EhucybF (TCAGTCGCACACATCTGCCG) and EhucybR (AGTGCAACAAGGACGGCTGC) and amplified 524 bp. PCRs were performed in 15 µl volumes containing 1× MangoMix  and 0.2 µM each primer. For E. coruscans, cycle conditions were as follows: 94°C for 1 min; 35 cycles of 94°C for 30 sec, annealing temperature 55°C for 45 sec; and 72°C for 30 sec; and a final 72°C extension for 10 min. Cycle conditions for E. marshi were the same with the exception of the annealing temperature step, which was 68°C for 30 sec. To clean PCR products, 7.5 units of Exonuclease I and 0.75 units of FastAP alkaline phosphatase  were added to 7.5 µl of PCR product, and aliquots were incubated at 37°C for 60 minutes and 85°C for 15 minutes. PCR products were then sequenced in one direction with an ABI3730 automated sequencer . Questionable or low quality sequences were resequenced in the forward direction. Sequences were edited and aligned using GENEIOUS PRO 5.6.2 .PCRs amplified portions of mtDNA cytochrome E. coruscans and E. marshi through a procedure including restriction enzyme digestion, microsatellite enrichment, cloning of DNA fragments, and DNA sequencing of clones  diversities were obtained with ARLEQUIN. The nucleotide substitution models used to calculate genetic distance were Tamura Nei+gamma = 0.403 E. coruscans and Tamura Nei for E. marshi; these were the best-fit models chosen using the AIC method in jModeltest 0.1.1 For the mtDNA, nucleotide (π) and haplotype .To investigate the influence of temporal sampling on the estimation of allele frequencies and inference of genetic structure, we conducted pairwise 5 steps, followed by 106 steps. Analyses were run five times for each of K = 1 to K = 16 to test for consistency of estimates of P(X|K), where K is the number of clusters. Bayesian clustering analyses were conducted both with and without use of sampling location as a prior; using sampling location as a prior has been shown to recover population structure at lower levels of divergence, without bias towards assigning structure when it is not present Population genetic structure was further analyzed using Bayesian clustering analyses implemented in STRUCTURE 2.3.3 vs. genetic divergence (ΦST/(1−ΦST) Relationships between geographic distance and genetic divergence were investigated using GENEPOP 4.0.10 D and Fu's Fs tests b sequences. Large negative values of Tajima's D and Fu's Fs are expected to occur if populations have experienced selection or recent expansions D, a significant positive value is expected if populations are admixed or experiencing diversifying selection. Fu's Fs is expected to have greater power than Tajima's D for detecting population expansions b rate of 2% per million years between lineages (1% within lineages) calibrated with other marine fishes E. marshi in the Hawaiian Archipelago may mature at two years of age , and the maximum age for E. coruscans and E. marshi in the Hawaiian Archipelago is estimated at 13 years E. carbunculus in the Indian Ocean have lifespans of at least 35 years Etelis species, corresponding estimates of effective population size and population age should be interpreted with caution. However, they should suffice to provide first order approximations.To resolve evolutionary histories, Tajima's E. coruscans and in E. marshi.Microsatellite allele fragment lengths and cytb GenBank Accession No.'s (KF920464 to KF920552) are reported for each individual specimen in E. marshi specimens were present twice in the dataset: one specimen was present twice in the Maro Reef sample, and the other present twice in the Necker sample. One of each duplicate was removed from the dataset for subsequent mtDNA and microsatellite analyses. No E. coruscans specimens were present more than once in the dataset.Identification of matching microsatellite and mtDNA genotypes indicated that two E. coruscans, three loci showed evidence of deviation from HWE after Bonferroni correction, including locus EtelisO22O2 (seven geographic locations), EtelisG23O1 (four locations), and EtelisC19O2 (one location). These loci also exhibited an excess of homozygotes at two or more locations , indicating that deviations from HWE may result from null alleles. Three additional loci exhibited evidence of homozygote excess at one or more locations: EtelisI11O2 (five locations), EtelisM15E1 (one location), and EtelisG8E1 (one location). FREENA indicated a relatively low frequency of null alleles across loci and populations .For E. marshi, only one locus (EtelisG8E1) showed evidence of deviation from HWE after Bonferroni correction; this locus deviated from HWE at 12 geographic locations. This locus also exhibited an excess of homozygotes at 11 locations, indicating that null alleles may be responsible for deviations from HWE. Two additional loci exhibited evidence for homozygote excess: EtelisA14E2 (six locations) and EtelisE20O1 (one location). Loci which exhibited evidence for homozygote excess generally had higher null allele frequency estimates across populations  than other loci .For E. coruscans or E. marshi after Bonferroni correction. Global FST values calculated with and without correcting for null alleles had overlapping 95% confidence intervals for both species. Additionally, removing one locus at a time had little impact on pairwise FST results for either species. For E. coruscans, average deviations between all pairwise FST values calculated using all loci versus all except one locus ranged from 0.0021 (when removing EtelisE2E2) to 0.0059 (when removing EtelisE20O2). For E. marshi, average deviations between all pairwise FST values calculated using all loci versus all except one locus ranged from 0.0015 (when removing EtelisA14E2) to 0.0133 (when removing EtelisE6E1). For each species, the greatest deviations in average FST occurred with the removal of a locus that showed no evidence of null alleles. Below we report results from analyses using all loci.No loci showed evidence for linkage disequilibrium for either b DNA sequence data resulted in a 490 bp fragment for E. coruscans and a 436 bp fragment for E. marshi. Diversity values (h and π) were consistently higher for E. coruscans than E. marshi. For E. coruscans, h ranged from 0.67 to 0.84 and π ranged from 0.0019–0.0039  had zero diversity (h = π = 0); however, this low diversity may be related to the small sample sizes at these locations (n = 11 and n = 12) , E. coruscans still had consistently higher diversity values than E. marshi, except at Gardner, where haplotype diversity was slightly higher for E. marshi (data not shown).Editing of cyt9–0.0039 , with thhipelago . For E.  n = 12) . For E.  0.00185 , with thE. coruscans ranged from 0.643 to 0.739, and expected heterozygosity ranged from 0.726 to 0.813    Kaua'i and (2) all other locations (For s Banks) . For michoolawe) . For Bayocations .E. marshi, pairwise ΦST analyses for mtDNA revealed significant divergence for 9 out of 120 pairwise comparisons, and each of these comparisons involved Gardner (adjacent to St. Rogatien)  involving a location in the mid-archipelago (Gardner or Raita)  St. Rogatien in the middle of the archipelago, and (2) all other locations (For ogatien) . Pairwisr Raita) . For Bayocations .ΦST or FST values for any location or any genetic marker , indicating that temporal sampling had little impact on the estimation of allele frequencies for those locations. For all other locations, all specimens were collected exclusively  from one time period (i.e. within three years of each other). The finding of little genetic structure across the archipelago for all markers across samples collected during different time periods suggests that temporal sampling had little impact on allele frequency estimates.For both species, analyses comparing samples collected during different time periods within a given geographic location resulted in no significant pairwise E. coruscans, genetic divergence was significantly correlated with geographic distance for both mtDNA (P = 0.044) and microsatellites (P = 0.042), although the correlation coefficient (r) was low for both marker types   or microsatellites (P = 0.833).For = 0.171) . For E. Fs and most values of Tajima's D were negative and significant (P<0.05) for all sites for both species; locations which did not have a significant Tajima's D included Pearl & Hermes, Twin Banks, and Kaho'olawe for E. coruscans . E. coruscans was the only species with evidence of a genetically divergent population outside of the mid-archipelago (Kaua'i).Population genetic analyses across the Hawaiian Archipelago for the deepwater fishes E. coruscans is much younger than sympatric E. marshi , although P. filamentosus has a slightly longer spawning period (approximately ten months) H. quernus, which peaks in the spring.Life history traits that might influence the population structure of these four species  are similar but not identical across species. The most dispersive life stage for these fishes is probably the larval stage. Spawning timing may influence dispersal of pelagic eggs and larvae due to seasonality of oceanographic currents in the Hawaiian Archipelago H. quernus, P. filamentosus, E. coruscans, and E. marshi remain in the water column no more than 48 hours before hatching H. quernus, the PLD is estimated to be 35–45 days (R. Nichols and E. DeMartini pers. comm.). PLD is not known for P. filamentosus, E. coruscans, or E. marshi, but eteline lutjanids typically have longer PLDs than H. quernusP. filamentosus, a captive rearing study suggests a potential PLD estimate of 52–120 days, based on observations of larvae associating with the bottom as early as 52 days and the completion of larval metamorphosis by 120 days after hatching The length of time that larvae are in the water column  also likely influences dispersal and genetic connectivity for these four deepwater fish E. marshi and H. quernus) might be expected to have more structure than fishes that are epibenthic as adults (E. coruscans and P. filamentosus). Contrary to this expectation, neither of the bottom-dwelling species showed higher structure in pairwise FST comparisons. The significant structure at mid-archipelago (based on microsatellites) was FST = 0.008–0.010 and FST 0.008–0.013 for the bottom dwellers, and FST = 0.011–0.013 and FST = 0.004–0.066 for the pelagic roamers. Further, significant isolation by distance was detected in one bottom dweller (H. quernus) and one pelagic feeder (E. coruscans), providing no clear pattern based on habitat preference. Despite modest differences in spawning time, PLD, and habitat use, Hawaiian submesophotic fishes have concordant population structure that is not shared with fishes that inhabit adjacent shallow habitat.Fishes that are benthic as adults (H. quernus and P. filamentosus) in the Hawaiian Archipelago, the authors hypothesized that the pattern of population structure and high diversity in the mid-archipelago is driven by connectivity with Johnston Atoll E. coruscans. In addition, biophysical modeling of larval dispersal for the submesophotic fish H. quernus indicated connectivity between Johnston and Gardner, but not other locations in Hawai'i In previous genetic studies of submesophotic reef fishes . These subtle differences in genetic structure for different markers and analyses and the overall pattern of weak genetic structure across the archipelago for all four species are consistent with a scenario in which migration across the archipelago is around the level at which genetic methods lose power to detect restrictions in gene flow. Genetic methods lose power at levels of gene flow that are still low enough to maintain demographically independent populations; this is especially true when effective population sizes are large, as in many marine fish and invertebrate populations H. quernus, E. coruscans). Therefore the structure detected for deepwater fish using genetic methods is likely biologically relevant on an ecological timescale.Although the pattern of genetic structure and high diversity in the mid-archipelago was consistent across the four submesophotic reef fishes in the Hawaiian Archipelago, not all genetic markers or analyses consistently revealed these patterns within species. For example, mtDNA analyses of Pristipimoides multidens) in Indonesia and northern Australia supported this idea, with surprisingly strong genetic structure found over short geographic distances  Etelis specimens have been documented below that depth; this would seem to be ample buffer against disturbance in the upper 130 m. Furthermore, the primary habitat of these species consists of rocky substrate, not living coral, and therefore the abundance of suitable habitat may not be substantially influenced by sea level changes. Nevertheless, these fish show the short coalescence times that are typical of shallow reef populations , and cytb GenBank Accession numbers for Etelis coruscans specimens. Missing data coded by “0”.(TXT)Click here for additional data file.Table S2Specimen ID, sampling location, microsatellite allele lengths (bp), and cytb GenBank Accession numbers for Etelis marshi specimens. Missing data coded by “0”.(TXT)Click here for additional data file.


Pieris rapae L.  sampled from the center to the periphery of Marseille. We utilize established and novel environmental correlation approaches to scan genetic variation for evidence of selection. We find significant morphological differences in urban populations, as well as weak genetic structure and decreased genetic diversity in urban versus non-urban sites. However, environmental correlation tests provide little support for selection in our dataset. Our comparison of different methods and allele frequency clines suggests that loci identified as significant are false positives. Although there is some indication that selection may be acting on wing size in urban butterflies, genetic analyses suggest P. rapae are undergoing neutral drift.Urban areas are increasing globally, providing opportunities for biodiversity researchers to study the process in which species become established in novel, highly disturbed habitats. This ecological process can be understood through analyses of morphological and genetic variation, which can shed light on patterns of neutral and adaptive evolution. Previous studies have shown that urban populations often diverge genetically from non-urban source populations. This could occur due to neutral genetic drift, but an alternative is that selection could lead to allele frequency changes in urban populations. The development of genome scan methods provides an opportunity to investigate these outcomes from samples of genetic variation taken along an urbanization gradient. Here we examine morphological variation in wing size and diversity at neutral amplified fragment length polymorphisms in the butterfly  Human populations have grown rapidly since 1950, with urban populations increasing nearly five-fold in size at an average rate of 2.6% per year Perhaps one of the main challenges facing urban species is to maintain viable, interconnected populations when suitable habitats are highly fragmented et al. While neutral divergence is one possible response to urbanization, a compelling alternative is that rapid evolutionary adaptation occurs, inducing directional changes in genetic and morphological variation environmental correlation approach, explicitly tests for correlations between environmental factors and allele frequency distributions environmental correlation approach have attempted to correct for correlations among individuals due to recent shared ancestry Genome scans are a promising approach to determine whether population genetic patterns result from adaptive genetic changes or genetic drift Pieris rapae L.  sampled near Marseille, to test whether phenotypic changes in wing shape and differences in genetic diversity emerge in urban populations. Urban gradients are known to influence butterfly species diversity P. rapae.Our study focuses on the butterfly No permits were required for the collection of butterflies in this study and the individuals sampled were killed instantly to minimize suffering.P. rapae along an urbanization gradient in Marseille, focusing on two neutral models and one selection model. Under a neutral divergence with gene flow model, morphological differences should not be evident among urban and non-urban sites, genetic diversity should be reduced in urban sites, and changes in genetic structure among urban and non-urban sites should reflect weak genetic drift. Under a neutral geographical isolation model, morphological differences should not be evident among urban and non-urban sites and changes in genetic structure should reflect the isolation of urban populations , while a decline in genetic diversity is not necessarily expected. Finally, under a model of local adaptation, morphological divergence should be evident in urban sites and a subset of loci should exhibit allele frequency changes that are significantly correlated with changes in environmental variation.We propose to test hypotheses of drift versus selection in P. rapae individuals from 41 sites along a 100 km transect, extending from the center of Marseille northeast through fields and shrubland/forest . Digital images were scanned alongside a metric ruler and measurements were made in Adobe Photoshop CS3 software.We measured wing size variation among the sampled male butterflies, as females represented a small proportion of our total sample size (26%). Six wing size characters were recorded, including forewing length (FL), forewing width (FW), forewing area (FA), hindwing length (HL), hindwing width (HW), and hindwing area  were obtained from MapInfo (MapInfo Corporation); in addition, the density and number of buildings in a radius of 150 m were combined as a composite measurement of urbanization (var2). Land cover category (var3) and percent tree cover (var4) were obtained from GlobalMap (NASA Earth Observatory). The free database WorldClim was used to extract 19 climatic variables representing temperature and precipitation averages measured during 1950–2000. To reduce cross-correlations among climatic variables, we identified 16 highly correlated (Pearson's r>0.8) WorldClim variables (Table S1), combined them in a principal component analysis after standardization, and retained the first two eigenvectors  as variables (var5 & 6). The three remaining uncorrelated climatic variables, isothermality (var7), temperature seasonality (var8) and mean diurnal temperature range (var9) were analyzed individually.Data on road width .t-test to assess statistical significance. We also grouped individuals into distance classes, 0–25 km, 25–50 km, 50–75 km, and 75–100 km from the center of Marseille to the periphery, and compared group means for each wing character in an analysis of variance (ANOVA) allowing for unequal variance among groups. We used an F-test to assess the significance of differences among group means. The ANOVA was repeated excluding the 25–50 km distance class, due to its smaller sample size. Finally, we calculated the Pearson correlation coefficient between each morphological character and genotypes at each AFLP locus. These correlation tests were corrected for multiple testing using the Dunn-Šidák correction (1– (1– α)1/n), where n is the number of variables multiplied by the number of loci (6×366), and the adjusted p-value is 2.34×10–5. This correction assumes independence among tests and provides a similar, though slightly more conservative correction, than a Bonferroni adjustment.We calculated the Pearson correlation coefficient between each morphological variable and the distance of the sampled butterfly from the center of Marseille, and used a Hj), using the software Genalex v.6.41 STF and Nei's genetic distance. These analyses were conducted in the software AFLP-Surv STF is typically robust to small deviations from demographic equilibrium and provides a measure of genetic differentiation among populations STF and Nei's gene diversity.Genetic diversity was measured at each collecting site as the percentage of polymorphic loci and the expected heterozygosity or Nei's gene diversity (K) while allowing for admixture and correlated allele frequencies across populations. We examined 20 runs at each K to determine the maximum log-likelihood, setting the burnin to 100,000 and the MCMC sampling to 500,000 steps. Finally, we tested for isolation by distance (IBD) using a Mantel test of the correlation between genetic distance among individuals and Euclidean geographic distance in Genalex. Significance of IBD was tested using 999 permutations of the data.To examine population structure graphically, we used principal coordinate analysis (PCoA) on all individuals, calculating independent coordinates using a standardized covariance matrix of genetic distance, and plotted the first two coordinates to identify spatial clusters. We then used the Bayesian clustering program Structure v.2.3.3 Methods S1). The logistic regression model was implemented using a binomial error distribution and the logit link function, and significance was assessed using an analysis of variance F-test: iy  =  logit-1(ißX). The GEE approach was implemented using autocorrelation correlation structure 1 (‘ar1’), which corrects for relatedness among individuals sampled from neighboring localities. The GEE model also uses a binomial error distribution and a Wald test was used to assess significance: iy  =  logit-1(ißX),Var(iy)  =  iφα, where the scaling parameter (φ) and variance function (ia) are used to model the covariance structure. For the two linear mixed models, the environmental variable was tested as a fixed effect, while the intercept was allowed to vary across groups. In one version of a LMM , individuals were grouped into four distance classes defined previously: iy  =  ß0j[i] + ß1j[i] iX + iε, where fixed effects and intercepts vary for j geographical groups. In the second version, the value of each environmental variable  at the sample site was used to group individuals into classes : iy  =  ß0j[i] + ß1j[i] iX + iε, where fixed effects and intercepts vary for j environmental classifications. Each LMM was implemented with autocorrelation correlation structure 1 (‘ar1’) and the binomial error distribution. The fixed effect of each variable was compared to a null model without the variable using a likelihood-ratio test to assess significance. All environmental correlation tests were corrected for multiple testing using a Dunn-Šidák correction (n = 9 variables ×366 loci) with the adjusted p-value of 1.56×10−5. For loci with significant correlations, we examined allele frequency clines along the Marseille transect using the hzar package in R. We plotted and estimated parameters for one-dimensional (1-D) clines, comparing this 1-D model to a null model of frequency changes independent of location using a likelihood ratio test.We used four approaches to test for environmental correlations, including logistic regression . Although two loci (14 and 339) were weakly correlated (r>0.25) with morphological characters, neither was significant after correction for multiple testing.Measurements of the wing size of male butterflies were significantly negatively correlated with distance from the center of Marseille Table 1.r = 0.573, p-value  = 0.0003, Table S2). Expected heterozygosity decreased from urban to non-urban samples, but this was largely due to small sample sizes in urban sampling locations. When butterflies were grouped into distance classes, the expected heterozygosity increased from urban sites at 0–25 km (mean 0.10526±0.00638) to non-urban sites at 25–50 km (mean 0.12518±0.00747), 50–75 km (mean 0.12604±0.00687), and 75–100 km (mean 0.11605±0.00668), though this relationship is not significant. Non-urban populations  were not differentiated based on STF or Nei's genetic distance (STF (0.0038) of urban and non-urban samples was statistically significant . Additionally, Nei's gene diversity was significantly higher  in non-urban (0.1617±0.0068) compared to urban populations (0.1457±0.0066).The percentage of polymorphic loci was high , with polymorphism increasing from urban to non-urban sites  = −25839.7). However, individuals were not assigned to discrete clusters when K>1 .A plot of the first two principal coordinates (27.6% and 25.5% of the variation) provided no evidence of population structure Figure 2Figure S4), nor were they correlated with morphological characters.We found significant correlations using three of the four statistical approaches Table 3.Urban environments provide an important experimental system in which to investigate the role of ecological and evolutionary processes as species become established in highly disturbed habitats Pieris rapae exhibits changes in wing morphology across an urban-to-rural gradient in Marseille . Given the observed shifts in morphological traits, it is possible that evidence of adaptation might be found with more comprehensive coverage. The recent developments in next generation sequencing in non-model organisms P. rapae.Recent simulation studies suggest that environmental correlation tests might suffer from a high rate of false positives P. rapae are heritable and under selection.Numerous studies have focused on how biodiversity and ecological traits change along urban gradients Figure S1Measurements of wing size taken on male butterflies. FL  =  forewing length, FW  =  forewing width, HL  =  hindwing length, HW  =  hindwing width.(PDF)Click here for additional data file.Figure S2Principal component analysis (PCA) of temperature and precipitation variables at the sample sites.(PDF)Click here for additional data file.Figure S3Boxplots of morphological variation in male butterflies taken at distances measured from the center of Marseille along a gradient of urbanization.(PDF)Click here for additional data file.Figure S4Plots of the allele frequency clines of significant loci as a function of distance (meters) from Marseille.(PDF)Click here for additional data file.Table S1Pearson correlations among variables, showing in bold climatic variables with significant correlations (r) greater than 0.80.(PDF)Click here for additional data file.Table S2Summary of AFLP genetic variation at each sampling site.(PDF)Click here for additional data file.Methods S1Statistical model formulas and details of implementation in the computing environment R.(PDF)Click here for additional data file.


The expansion of intensive livestock production systems in developing countries has increased the introduction of highly productive exotic breeds facilitating indiscriminate crossbreeding with local breeds. In this study, we set out to investigate the genetic status of the Vietnamese Black H’mong pig breed by evaluating (1) genetic diversity and (2) introgression from exotic breeds. Two exotic breeds, namely Landrace and Yorkshire used for crossbreeding, and the H’mong pig population from Ha Giang (HG) province were investigated using microsatellite markers. Within the province, three phenotypes were observed: a White, a Spotted and a Black phenotype. Genetic differentiation between phenotypes was low (0.5–6.1%). The White phenotypes showed intermediate admixture values between exotic breeds and the Black HG population (0.53), indicating a crossbreed status. Management practices were used to predict the rate of private diversity loss due to exotic gene introgressions. After 60 generations, 100% of Black private alleles will be lost. This loss is accelerated if the admixture rate is increased but can be slowed down if the mortality rate  is decreased. Our study showed that a large number of markers are needed for accurately identifying hybrid classes for closely related populations. While our estimate of admixture still seems underestimated, genetic erosion can occur very fast even through indiscriminate crossbreeding. Since the domestication of animal species, livestock movements and trade have been important driving factors for the genetic make-up of existing breeds, with selection and drift. One of the best examples is the high diversity of African cattle breeds, which results from different migration waves of taurine and zebu cattle . HoweverToward the end of the 20th century, gene flow reached developing countries, resulting in an expansion of intensive livestock production systems; an expansion referred to as the “livestock revolution” . IncreasIn Vietnam, pig production is divided into smallholder production and small-medium, medium, and large production. Medium and large production involves more than 20 sows and use of exotic breeds. These farming systems are centralized in areas of high population density and amount to 10% of the Vietnamese pig population . SmallhoSurrounded by exotic pigs, the H’mong black pig may not resist and little is yet known about it. What is its genetic pattern? Is it already crossbred? What is the effect of indiscriminate crossbreeding on its genetic pool? How long will it take to lose its specific genetic pool if no conservation programes are launched? These are the main questions that this study focused on. Genetic data, phenotypic data, and management practices were combined to estimate the degree of introgression to date and to predict the effects of a prolonged introgression.F-statistics were computed. According to the National Institute of Animal Husbandry (NIAH), improvement programes have been implemented in northern provinces using two exotic breeds: Landrace and Yorkshire. Samples from these breeds were collected at the NIAH in Hanoi: 22 and 25 individuals for Landrace and Yorkshire, respectively.The remote Vietnamese province of HG (Genomic DNA was extracted from tissue samples using the QIAamp Kit from QIAGEN. The PCR products were labeled with fluorescent dyes and genotyped using a capillary sequencer (Beckman Coulter CEQ8000) for 16 microsatellite markers recommended by FAO . These markers belonged to different chromosomes, except SW1067 and SW122, both mapping to chromosome 6. Genotypes from this study are available on request.r≥ 0.2 were considered to be potentially problematic for calculations. Expected and Observed heterozygosities were calculated using GENETIX 4.4  were assessed according to D·ΔK) criterion after testing for K= 1 using the Pritchard approach (q) of each individual's admixture coefficient. STRUCTURE software was run first for the exotic and HG dataset , then for the HG dataset , and finally for the Black population only.We investigated the genetic structure and individual assignments using a Bayesian clustering procedure implemented in STRUCTURE , with thapproach . The proijF) were regressed on ijd, where ijd was the spatial distance between individuals i and j. Standard errors were assessed by jackknifing data for each locus. Spatial correlation between ijF and ijd was tested by permutation (9999) on individuals. Significant correlations were associated with the IBD process.We tested the genetic pattern of the Black HG phenotype for isolation by distance (IBD) based on pairwise kinship coefficients between individuals using SPAGeDi 1.3 software . As suggIn order to assess the discrimination power of our micro-satellite set, we generated 100 individuals for each of four genotypic classes that originated after two generations of hybridization, that is, first-generation hybrids (F1), second-generation hybrids (F2), backcrosses of F1 with the first parental population (BE), backcrosses of F1 with the second parental population (BL). Parental source populations were the Exotic (Landrace and Yorkshire mixed) breed and the local Black HG population. Genotypes were generated using HYBRIDLAB software .q′) of each individual belonging to each of six genotypic classes that originated after two generations of hybridization as described above. We ran the program for two datasets: (1) the simulated dataset with Exotic and Black parental populations and (2) the real datasets with Exotic (P0), Black (P1), Spotted, and White pigs. For both runs, we provide a priori population information for all the Exotic samples and for 10 individuals from the Black HG population. The Black HG individuals were selected as the ones showing the highest q-values from STRUCTURE analyses. We used a burn-in period of 150,000 followed by 500,000 iterations. The simulated dataset was also then evaluated using STRUCTURE in a similar manner to the empirical data from K= 1 to K= 7.We then used NEWHYBRIDS software  to compun+ 1, because an overlapping generation existed, the new generation participated in the reproductive stock at the mortality rate (MR): MR × (1 – AR), (1 – MR) × (1 – AR) gametes from parental population Bn– 1 were randomly selected, and AR gametes from W0 stock, which remained constant as these pigs were bought from outside and only kept for one year. Our model considered a constant population size and took into account genetic drift by randomly selected gametes from the gamete pool. The parameters were one batch of 1000 individuals for 100 generations in a constant population size. The model was replicated 100 times. We tested three values for the AR, AR = 0 as no admixture, AR = 0.25, as estimated by us, and AR = 0.5, and two for the MR, MR = 0.4, following our interviews, and MR = 0.2. In the following analysis, an allele was considered as lost when its frequency was equal to 0.Householders were questioned about their management practices. They were asked to estimate the age at first parturition, the farrow interval, and the mortality rate (MR). According to the interviews, the householders did not practice any selection for reproduction. Reproductive sows were raised freely, while most of the fattening pigs were shut in sheds. However, personal observations and interviews revealed that they often escaped. This information, plus the admixture rate (AR) estimated from genetic data, was used to create a model designed to estimate the evolution of allelic frequencies and admixture in the Black population. The model was run using R software  and madeFIS values were at least four times higher for the Black and Spotted populations  than for the White population. The highest genetic differentiation was found between Landrace and Black HG populations (18%). Genetic differentiation between the White and Black HG populations was 6.1% and 4.1% between the White and Spotted HG populations. The FST value was low (0.5%) between the Black and Spotted HG populations.The expected heterozygosity was similar for all three phenotypes (0.791–0.804) . RegardiD·ΔK was the highest for K= 2; q1 probability >0.99 , we obtained a subdivision into two clusters. (ty >0.99 . The Bla 0.18 SD . HistogrK= 2 with one cluster represented by the White population (WC) and the other by the Black population (BC) (CBq > 0.9) or the black cluster (49% with CWq > 0.9). Only 10% of Spotted pigs showed intermediate values (0.3 < q < 0.7). The CBq values averaged across individuals with Black and Spotted phenotypes reached a mean AR of 0.25 with the White phenotype cluster. Following our results, we put forward the hypothesis that the White phenotype is the result of an admixture between exotic and local breeds, and the Spotted phenotype is the result of multigeneration crossing.When analyzing the HG population alone, we found ion (CB) . Within K= 1 following Pritchard's procedure. The values of the kinship coefficient averaged over a set of distance classes were plotted against geographical distance (b± SE = 0.0097 ± 0.0018 was highly significant (P < 0.00001). Individual kinship coefficients negatively correlated with geographical distance showing a logarithmic relationship, indicating an IBD process.The Bayesian procedure on Black HG pigs revealed no genetic subpopulations. The value of the criterion used was the highest for distance . ConsideK= 2, thereby representing the two parental clusters, Exotic (E) and Black (B). As such, we expected that parental  individuals would show q-values > 0.9 and all hybrid classes should have q-values < 0.9 and near to 0.5, at least for F1 and F2. The results showed that 92% of HG Black could be identified as parental individuals  were assigned as parents. In addition, no hybrid of any class was assigned as a parent. Of the individuals from class F1, 55% were assigned correctly and 11% missassigned to other hybrid classes. F2 individuals showed a substantially higher rate of assignment at q′ > 0.9 compared to other hybrid classes. This difference was attenuated when considering an assignment threshold of q′ > 0.7. The lowest correct rate (15%), at q′ > 0.9, was found for Black HG backcrosses (BL). In addition, they also showed the lowest similar missassignment rate (13%). Of the individuals assigned to F1 and F2 classes, 83% and 80%, respectively, were true F1 and F2 for q′ > 0.7.While STRUCTURE is used to estimate ARs, NEWHYBRIDS is assumed to distinguish first- or second-generation crosses more accurately. Using NEWHYBRIDS on the simulated dataset, the efficiency of our marker set in discriminating between crossbreeding classes was very variable . More thq′ values below 0.7 and were thus not assigned to any category.NEWHYBRIDS run on the empirical data identified more than 98% of Black HG as phenotypic Black HG and 100% of Exotic pigs as Exotic ones . AccordiAccording to the householder interviews, the average age of first parturition was at 18 months and the farrow interval was about 8.5 months. On average, each sow was used for two parturitions, and the natural MR in the population averaged 0.4. Mortality was used as a recruitment rate for the reproductive stock in the model, leading to a constant population size , the remaining pigs being sold as meat carcasses.We first tested our model with no admixture and only genetic drift. After 100 generations, 17% of alleles were lost. Among alleles lost, 38% were from exotic origin from previous admixture. Only four of the 15 privates Black alleles were lost.CBq= 0.25 averaged across Black and Spotted phenotypes; see above). This frequency was considered constant across generations; in other words, reproduction with White pigs always amounted to 25% in the HG pig population.According to the householders, White pigs were raised for less than one year and only for fattening. In other words, each year the householder bought new White pigs. Consequently, the allele frequencies of the White pigs were assumed to remain constant over the years. The householders did not state that they used White pigs for reproduction but escapes and uncontrolled reproduction can happen. We therefore considered that the probability of crossbreeding was equal to the mean AR . Consequently, according to FST values (0.18). It can be expected that for populations with a very similar genetic pool, the discrimination of hybrids might only be possible when the contribution of one parental population becomes significantly greater, as in the case of backcrosses. Nonetheless, it can also quickly become impossible to distinguish backcrosses from parental classes , The justification for conserving the genetic diversity of local breeds is straightforward. Genetic traits that are or may be useful to husbandry are worth conserving, including those that affect disease resistance, environmental tolerance, and taste. However, farmers are more inclined to raise breeds that are popular on the local market and for which economic gain will be the highest. Many studies have shown that the introduction of exotic breeds is mainly driven by economic forces. In Vietnam, almost 80% of pig production is still in the hands of smallholders, but commercial pig production, raising exotic prolific breeds, is growing fast . A populMany studies on wild species have demonstrated the consequences of genetic dilution by introgressive hybridization, ultimately leading to extinction . The conFST between contemporary brown trouts decreased around twofold compared to ancestral samples. While genetic differentiation was still very low between White and Black HG pigs (0.061), it disappeared after 100 generations (FST= 0.0002). Conversely, differences in expected heterozygosity (from 0.80 to 0.78) and effective alleles (5 to 4.5) were not so pronounced. The apparent status quo in genetic diversity with exotic introgression has to be taken with caution. Indeed, in our study this was due to the contribution of new alleles from exotic breeds. Similar stability of genetic diversity has been observed in Atlantic salmon with crossbreeding occurring with individuals from multiple farms collectively containing a large genetic variation that mitigates genetic erosion  phenotypic observation, (2) householder interviews, and (3) genetic data. Such a global procedure should be recommended to gain precision for characterizing and establishing AnGR conservation policies. Our results suggest major gene flow between exotic breeds and the H’mong pig breed. We expect that future AnGR management and improvement projects will carefully consider the use of exotic genetic pools. Our estimation of the number of generations for homogenization of the introduced and local populations could provide some clues for conservation biologists in wildlife management. Translocated populations may genetically homogenize faster that we thought with local populations and, as a consequence, greatly modify the prior management units and induce the disappearance of local adaptations. In our study, we believe that admixture was underestimated due to tool limitations in the case of closely related populations. Further studies using high-throughput SNP chips available in many species such as the pig  might im


Mazzaella laminarioides, a low-dispersal benthic intertidal red seaweed that inhabits along ~3,700 km of the Chilean coastal rocky shore.The Chilean shoreline, a nearly strait line of coast expanding across 35 latitudinal degrees, represents an interesting region to assess historical processes using phylogeographic analyses. Stretching along the temperate section of the East Pacific margin, the region is characterized by intense geologic activity and has experienced drastic geomorphological transformations linked to eustatic and isostatic changes during the Quaternary. In this study, we used two molecular markers to evaluate the existence of phylogeographic discontinuities and detect the genetic footprints of Pleistocene glaciations among Patagonian populations of M. laminarioides. They are distributed along the Chilean coast in strict parapatry. The deep divergence among lineages suggests that they could be considered putative genetic sibling species. Unexpectedly, genetic breaks were not strictly concordant with the biogeographic breaks described in the region. A Northern lineage was restricted to a broad transition zone located between 30°S and 33°S and showed signals of a recent bottleneck. The reduction of population size could be related to warm events linked to El Niño Southern Oscillation, which is known to cause massive seaweed mortality in this region. To the south, we propose that transient habitat discontinuities driven by episodic tectonic uplifting of the shoreline around the Arauco region (37°S-38°S); one of the most active forearc-basins in the South East Pacific; could be at the origin of the Central/South genetic break. The large beaches, located around 38°S, are likely to contribute to the lineages’ integrity by limiting present gene flow. Finally, the Southern lineage, occupies an area affected by ice-cover during the last glaciations. Phylogeny suggested it is a derived clade and demographic analyses showed the lineage has a typical signature of postglacial recolonization from a northern glacial refugium area.Three main genetic lineages were found within M. laminarioides, low dispersal capacity and small population size are sufficient to generate phylogeographic discontinuities determined by genetic drift alone. Interestingly, our results confirm that seaweed population connectivity over large geographic scales does not rely only on dispersal capacity but also seem to depend highly on substratum availability and population density of the receiving locality.Even if environmental adaptation could have strengthened divergence among lineages in  Relying on the assumption that genealogical breaks arose only through vicariant processes, strong phylogenetic breaks are generally associated to longstanding population separations and are thus used to pinpoint the location of (past or current) geographic barriers to dispersion. Indeed, phylogeographic discontinuities congruent across multiple taxonomic groups are often in concordance with biogeographic boundaries. Numerous studies have revealed deep genetic separation for coastal marine taxa that concur with well-known Transition Zones such as: the Isthmus of Panama , the souMarine intertidal species have characteristics that make them particularly susceptible to neutral processes of genetic differentiation. They have narrow and linear geographic distributions that may restrict dispersal to a stepping-stone process in species without long-lived planktonic larvae. Dispersal occurs in an advective environment (i.e. dominated by a more or less continuously oriented flow), which limits the effective population size . SeaweedThe South East Pacific coast (SEP) is subdivided in two major regions. North of 42°S, the coastline is linear, continuous and largely dominated by rocky shores. The coastline is also intersected by sandy beaches of up to 50 km long and a few small rivers. The oceanography and coastal climate of this region are largely defined by coastal upwelling dynamics associated with temperate east-boundary coasts ,24. The Macrocystis pyrifera, , we found 19 polymorphic sites and 10 haplotypes [GenBank: JQ408398-JQ408407],  separated by 15 to 45 bp and 6 to 10 bp for COI and rbcL, respectively. Within each haplogroup, pairs of haplotypes were separated by 1 to 13 bp for COI and 1 to 3 bp for rbcL ; a Central haplogroup between 34°05'S and 37°38'S ; and lastly, a Southern haplogroup ranging from 39°40'S to 54°03'S  . For the Southern haplogroup, the R10 chlorotype was present in all local populations analyzed, while the C15 mitotype was detected in all but one  were higher for COI than for rbcL, regardless of the haplogroup considered . Within the Southern haplogroup, a higher genetic diversity was observed in populations located in the non-glaciated area  than in the five southernmost populations located within the region covered by ice during the LGM  than the southernmost populations (H = 0.284 +/− 0.054 and π = 0.0005 +/− 0.0006) . Second, for rbcL, even if the comparison between the two regions was not significant , samples from local populations south of 42°S lacked genetic diversity (both H and π = 0) while three haplotypes were found in the populations north of 42°S .Even though there was a slight tendency for a reduction of genetic diversity from the Northern haplogroup, to the Central and then to the Southern haplogroup  indicated, in congruence with the results from the haplotype networks Figure A, B, thaD and Fu's Fs statistics  further support a scenario of population expansion. The Bayesian skyline plot showed evidence of a subtle but persistent population growth for the Southern haplogroup . The Bayesian skyline plots detected a recent population decrease in the Northern haplogroup and stable population size for the Central haplogroup , and the mismatch distribution was unimodal  and the support values for the population expansion models were low (between 0.11 and 0.23) . Within this fraction, only one substitution was detected between these sequences and the Northern haplotypes from our study, which is also congruent with the sampling location of these specimens (~ 32°S).The GenBank sequence proposed as the COI barcode for the ecies by  falls wirbcL : 13.63 +/− 1.41% for the COI and 4.16 +/− 0.66% for the rbcL  between COI in plants and the intergenic spacer Cox2-Cox3 in red algae [rbcL, the divergence between the Central and Southern haplogroups was dated between 8.2 and 9.6 Myr.The K2P distance within each lineage ranges from 0.33 to 1.23% for the COI and from 0.15 to 0.22% for the ed algae ,52. DiveMazzaella laminarioides, with three genetic clades distributed in strict parapatry along the Chilean coast. There was no strict concordance between the geographic distribution of lineages and two recognized biogeographic transition zones along the coast of Chile. A Southern lineage, that occupies areas that were glaciated during the LGM, showed a clear signature of postglacial recolonization from a northern refugium area.Our study revealed deep genetic structure within the red alga M. laminarioides were all grouped within a single monophyletic lineage, clearly separated from congener species. Haplotype networks of COI and rbcL were in complete congruence and revealed a within-species structure including three deeply divergent haplogroups with a strict parapatric distribution. The among-haplogroup distances remained largely inferior to the distance among the congener species included in our study . However, the mean genetic distance among the three haplogroups of M. laminarioides is close to 7-fold of that within-haplogroups , confirming a deep divergence between them. The non-overlapping values of divergence of intra-lineages vs. inter-lineages in M. laminarioides are congruent with general barcode rules [Mazzaella using rbcL indicated an among-species divergence ranging from 1 to 6.5%, with most values around 1 to 3% of divergence [Despite the marker, phylogenetic trees confirmed that haplotypes of de rules  suggestide rules . In the vergence .M. laminarioides lineages should be taken with caution and as a heuristic tool. In the kelp L. nigrescens, a deep divergence among lineages occurring along the SEP coast has been dated to be between 0.2 and 1.7 Myr [M. laminarioides lineages, ranging from 0.5 to 12.1 Myr (depending on the marker and the divergence event considered), seems to predate Pleistocene glaciations. This is further supported by the complete lineage sorting of mitotypes and the high number of mutations accumulated between haplogroups (more than 15 for the COI). Indeed, models suggest it is unlikely that so many steps could have evolved in only 20,000 years. This is assuming a typical per gene mutation rate for the mitochondria, where a period of ≈ 50,000 years is considered a minimum for the fixation of different mitotypes in diverging populations [Given the absence of known fossils, estimates of mutation rates are very limited and uncertain in macroalgae and our estimation of the time of divergence between  1.7 Myr . The autulations . Howeverulations .rbcL revealed some slight discordance in their topologies. For the COI, three well-supported and reciprocally monophyletic lineages were observed, while in the rbcL tree, the Northern haplotypes formed a set of basal parapatric branches. In fast-evolving markers (e.g. COI), the reciprocal monophyly is reached more rapidly than in slow-evolving markers (e.g. rbcL) and differences in mutation rates possibly explain this difference between markers [rbcL suggests a scenario of budding speciation or parapatric speciation process [Gene tree reconstructions of both COI and  markers ,59. The  process  related Lessonia nigrescens[Durvillaea antarctica[D. potatorum on both sides of the Bassian Isthmus  [Fucus spiralis[F. ceranoides[L. nigrescens, authors have proposed that transient allopatry, as the result of geographic isolation of populations in several refugia during the Pleistocene cold cycles and subsequent recolonization, could have participated in the parapatric pattern of sister-species pair’s distribution actually observed. In the Southern hemisphere, morphological and ecological differences were noted between some of the five distinct paraphyletic clades of D. antarctica[D. potatorum in Tasmania [Fucus[L. nigrescens, taxa that were recently recognized as different biological species [M. laminarioides, new studies will be necessary, such as controlled crosses and/or estimation of potential inter-lineage gene flow considering a smaller spatial scale and hypervariable nuclear markers.The presence of highly divergent lineages found in parapatry has been observed in other brown seaweeds such as: igrescens and Durvntarctica in the Sittoral) , and Fuc spiralis and F. ceranoides along thntarctica and betwTasmania . Howeveria [Fucus and this species . To testvs. Central haplogroups) and (2) between 37-39°S  for M. laminarioides, however, the biogeographic breaks along the Chilean coast are considered to be located at 30°S and 42°S.Two phylogeographic breaks were located: (1) between 32-34°S .Camus  . The Northern haplotypes were geographically very restricted. All mitotypes and all but one cytotypes were private. Likely, unstable populations and habitats and biotic conditions that are at the tolerance limit of M. laminarioides[M. laminarioides revealed a strong spatial patchiness at meso and local scales. At mesoscale, M. laminarioides’ populations are clearly fragmented; no individuals have been found along the 200 km of coastline separating LBR and CDA (~ 29°S) from FRJ (31°S) (pers. obs. of the authors). At local scales, within the almost continuous rocky shores of the LBR/CDA region, populations are patchy. Contrastingly, populations located south of 31°S form nearly continuous beds where rocky shores are present. The SEP coast north of 35°S is characterized by interannual cold and warm ocean temperature events linked to El Niño Southern Oscillation (ENSO), a global ocean-climate disturbance present since the Pliocene [narioides, explainPliocene . The NorPliocene , affectiM. pyrifera: [L. nigrescens: [C. concholepas[J. cirratus[A. monodon[M. laminarioides is likely to contribute to haplogroup divergence. Correspondingly, large beaches have been proposed as potential barriers to gene flow for two other marine organisms, with habitat restricted to rocky intertidal shores: the southern bull-kelp Durvillaea antarctica[Lontra felina[M. laminarioides.Our study revealed an ancient genetic discontinuity between the Central and Southern sister lineages. These lineages formed reciprocally monophyletic groups in both marker trees. However, between 37°S and 39°S, no biogeographic break has been described in marine organisms ,27 and nyrifera: ; L. nigrrescens: ; invertencholepas; J. cirr cirratus, A. mono. monodon). The re. monodon. Only smntarctica and the ra felina. By limiM. laminarioides. Along the Arauco region, the small population sizes of M. laminarioides (due to habitat fragmentation) together with its low dispersal capacity could have lead to phylogeographic discontinuities determined by genetic drift alone [Uplift and subsidence are common phenomena along the temperate Eastern Pacific margin, a coastline shaped by the subduction of the oceanic Nazca plate under the South American continent. Coastal geomorphology bears evidence of the complex interplay between local tectonic uplift and glacio-eustatic sea-level changes . The Araft alone ,13. It ift alone .Acanthina monodon, brooding gastropod, [Durvillaea antarctica, buoyant kelp, [Lontra provocax[Macrocystis pyrifera[Concholepas concholepas a gastropod with an extended pelagic larval phase [M. laminarioides (this study). The existence of a genetic break at 42°S seems to be less related to species dispersal capacity than to its Quaternary history in Patagonia. Indeed, for A. monodonD. antarctica and L. provocax, the 42°S biogeographic break seems to represent a secondary contact zone between haplotypes that had diverged in distinct refugia. In these species, the southernmost populations represent a recent expansion from coastal refugia located within the SEP glaciated region [A strong biogeographic discontinuity along the Chilean coast has been described within the 41-42°S zone . This distropod, ; Durvillnt kelp,  and the  pyrifera, in Concal phase  and in Md region ,42 or frd region ).M. laminarioides, the Southern lineage appears strongly affected by the Quaternary oscillations and shows, as expected, a star-like topology consistent with a recent demographic expansion. Populations located south of 43°S (i.e. region previously covered by ice) exhibit a very low genetic diversity. The common haplotypes present in the southernmost populations were also encountered in the Southern haplogroup populations north of 43°S. This pattern is recurrent in leading edge expansions. This type of expansion occurs when rare long-distance colonizers spread ahead of a core population, and characterizes the post LGM poleward expansion of the Northern hemisphere temperate terrestrial and marine biota [M. laminarioides. Ice scouring and winter freezing has a drastic impact on sessile organisms living in the high intertidal [M. laminarioides shows a complete population extirpation in Patagonia during the LGM and a severe colonization sweep in this region. The absence of a genetic break at 42°S may then be explained by the short time frame available to accumulate diversity (via new mutation) since the post LGM recolonization for Patagonian populations.In ne biota ,35,37. Wtertidal , and indM. laminarioides[M. laminarioides[M. laminarioides is in direct contrast to the evidence of long-distance dispersal ability during the recolonization of the region previously covered by ice. Mature fronds of M. laminarioides, although not buoyant, could disperse via rafting with individuals fixed on drifting wood or fronds entangled between mats of M. pyrifera or D. antarctica (Authors’ pers. obs.).In our study, we detected sharp genetic differences between nearby populations, especially in the northern part of the species distribution, concordant with the poor dispersal capacity of narioides. These rnarioides. Additionarioides,84. The D. antarctica, [F. ceranoides[M. laminarioides, the existence of a blocking effect could explain the “rapid” habitat tracking of new suitable habitats during the last 17,000 years in Patagonia through rare events of large-distance dispersal by a handful of individuals on high-intertidal shores almost completely depopulated. This process may be contributing to the complete parapatric distribution of the three lineages and the lack of connectivity among established populations along the Northern part of the Chilean coast not affected by ice.Paradoxical dispersal capabilities have also been reported in other marine algae , but seeranoides. Indeed,M. laminarioides, a red alga inhabiting 28° of latitude  along the SEP coast, consisted in a complex cluster of three sibling lineages. The existence of strict parapatry suggests mechanisms limiting gene flow among close populations. A high-density blocking-effect or reproductive isolation between lineages may have helped to preserve historical phylogeographic disjunction patterns. Because of its life cycle and restricted vertical distribution in the mid-high intertidal zone, M. laminarioides appears highly sensitive to ENSO events and ice scour during glaciations. Both mechanisms are capable of causing massive mortality. Macroalgae endemic to the SEP coast seem to provide excellent species complexes to test hypotheses involving diversification and speciation forces. Comparative studies contrasting both sides of the Arauco region should be the focus of tests investigating geological processes as drivers of gene flow and drift.Our study reveals that M. laminarioides, collected from 18 localities covering the whole distribution range of the species  enzyme, using the primers F-rbcL and R-rbcS [Additionally, we amplified a 922 bp region of the chloroplastic gene d R-rbcS  and the d R-rbcS . SequencTM DNA Purification kits  and sequenced using the forward and the reverse amplification primers by Macrogen Inc. . Sequences were edited using Chromas v. 2.33 [For both markers, PCR amplifications were performed in a Perkin Elmer Gene Amp PCR System 9700 . All PCR products were purified using UltraClean v. 2.33  and alig v. 2.33 .h); the number of private haplotypes ; the number of polymorphic sites (S); gene diversity , we realized a non-parametric Mann–Whitney U test : the nusity  and nuc . To tessoftware ).φST among locations were calculated and their significance was tested using 1000 permutations. Moreover, a nested Analysis of Molecular Variance  and geographic distance , and its significance was tested using 1000 permutations. All these analyses were performed in Arlequin.To characterize the population genetic structure, pairwise values of   was imp  and were thus not included in our phylogenetic reconstructions.Phylogenetic reconstructions for each marker dataset were performed with the maximum likelihood (ML) method using TreeFinder  and a Banarioides,88,106: mbranacea,106 and . affinis represenus clade ,106. We rbcL data were TN for the first and second codon positions and J3 + I for the third codon position. For COI the selected models were J1, TN and HKY for first to third codon positions, respectively. Using TreeFinder, we performed a heuristic search in order to reconstruct two separate trees (i.e. COI and rbcL) and node supports were assessed by non-parametric bootstrap .Bayesian inference was performed using the general type of the best fit model parameters defined for each dataset. Four independent analyses were run with four chains each, for ten million generations. Trees and parameters were sampled every 1000 generations and the default parameters were used to fit temperature and swapping. The first 25% of sampled trees were discarded as “burn-in” to ensure stabilization. The remaining trees were used to compute a consensus topology and posterior probability values. The split frequency (variance between the four independent runs) was below 0.0005, confirming that sampling was from the posterior probability distribution.M. laminarioides. For the rbcL marker, a divergence rate of 0.109-0.127% per million years (Myr) has been proposed for the red alga Caloglossa leprieurii[Cox2-Cox3 (a non-coding sequence neighboring the COI marker) for the red algae Bostrychia calliptera[We estimated the Kimura-two-parameters (K2P) distance , using Meprieurii. No divealliptera and a dialliptera) as loweAMOVA: Analysis of molecular variance; BI: Bayesian inference; bp: Base pairs; COI: Cytochrome c oxidase I; df: Degree of freedom; ENSO: El Niño Southern Oscillations; IA: Intermediate Area; K2P, Kamura-2-parameters; LGM, Last Glacial Maximum; ML, Maximum Likelihood; MP: Magellanic Province; Myr: Million years; PP: Peruvian Province; rbcL: Large subunit of the RUBISCO; RUBISCO: Ribulose-1,5-bisphosphate carboxylase/oxygenase; SEP: South East Pacific; SS: Sum of squares.The authors declare that they have no competing interests.MLG, AM and SF designed the research. All authors contributed to field collection. AM generated sequence data and performed molecular and statistical analysis. MLG led the writing; and AM, FT and SF assisted with the writing. PH and BB revised critically the manuscript. All authors read and approved the final manuscript.Geographic distribution of COI haplotypes. Number of individuals bearing each reported haplotype for COI in each sampling site . Click here for file


Hydromantes) and whether intraspecific genetic structure parallels geographic distance within and among caves. We generated mitochondrial cytochrome b gene sequences from 184 individuals representing 48 populations, and used a Bayesian phylogeographic approach to infer possible areas of cladogenesis for these species and reconstruct historical and current dispersal routes among distinct populations. Our results show deep genetic divergence within and among all Sardinian cave salamander species, which can mostly be attributed to the effects of mountains and discontinuities in major calcareous areas and cave systems acting as barriers to gene flow. While these salamander species can also occur outside caves, our results indicate that there is a very poor dispersal of these species between separate cave systems.Detecting the factors that determine the interruption of gene flow between populations is key to understanding how speciation occurs. In this context, caves are an excellent system for studying processes of colonization, differentiation and speciation, since they represent discrete geographical units often with known geological histories. Here, we asked whether discontinuous calcareous areas and cave systems represent major barriers to gene flow within and among the five species of Sardinian cave salamanders (genus  Allopatric speciation, the interruption of gene flow between populations, is one of the most widely accepted modes of speciation e.g., . TherefoCompared to other vertebrate groups, amphibians are commonly considered poor dispersers and are characterized by strong site fidelity Hydromantes) endemic to Sardinia offer an ideal system to investigate the intra- and interspecific genetic divergence of troglophiles. Adult plethodontid salamanders are lungless, and therefore strongly sensitive to environmental conditions. They require cool and moist environments for optimal breathing through the skin Hydromantes are troglophilic organisms so that in addition to caves, they can also be found in other humid environments HydromantesPlethodontid cave salamanders  discontinuities between calcareous areas represent the major barrier to gene flow within and among species and/or 2) intraspecific genetic structure parallels geographic distances. A Bayesian phylogeography approach was applied to infer possible area(s) of cladogenesis for the Sardinian species and to infer the historical and current dispersal routes among distinct populations.We here investigate inter- and intraspecific genetic variation in Sardinian cave salamanders using samples from the entire distribution range of all five Sardinian species. Our work consists of a much larger sample size than previous genetic studies Animals used in this study were not sacrificed and were all released at the sampling sites after sampling. Sampling permits were obtained prior to field work and tissue sampling followed sampling permit requirements.Hydromantes  was a result of a vicariant event associated with the ancient microplate that made up the island. At a later date, but before the cladogenesis of the Eastern clade, Hydromantes invaded mainland Italy.All phylogenetic analyses using different genetic markers have always, however, recovered flavus, nine supramontis, three sarrabusensis, 14 imperialis, and seven genei, corresponding to a total of 21% of the samples used in the current study and to 15 populations) were already included in a previous study b fragment PCR amplification, sequencing, and chromatogram checking are described in b gene as a marker for this study due to the availability of this marker for other species (the outgroups used in this study as well as mainland European Hydromantes) for which we did not have tissue samples and because in plethodontids, the cytochrome b marker has been observed to have faster rates of molecular evolution than other mitochondrial genes Sampling was carried out in Sardinia in 2007 and 2008. Sampling localities are indicated in b gene common to all species and with less than 5% of missing data for 83 haplotypes  and five outgroups were used for phylogenetic analyses. Cytochrome b sequences of the mainland Italian species were from b sequences of each Sardinian and mainland species separately, and then by combining all haplotypes to account for species introgression , H. shastae (accession # U89611), H. platycephalus (accession # U89612), Ensatina eschscholtzii (accession # FJ151870), and Salamandra salamandra (accession # AY035819) were used as outgroups. The software DAMBE was used to test for saturation at the third codon position http://beast.bio.ed.ac.uk/Tracer) and checked for convergence and appropriateness of the burn in period. Uncorrected genetic p-distances were computed in MEGA A total of 511 base pairs (bp) of the cytochrome Hydromantes are currently available Hydromantes as a calibration point in the Three distinct time estimates for the divergence between American and European The Bayesian phylogeographic analysis was run in BEAST v1.6.1 H. flavus, H. supramontis, H. imperialis, H. sarrabusensis) to avoid confounding effects due to the mixture of two separate cladogenetic events (see “Study system” and Discussion for further explanations). A ML unrooted tree was obtained as described above using 511 bp of the cytochrome b and all the haplotypes belonging to distinct populations of the four analyzed species (69 haplotypes). This tree was used as a starting tree for the Bayesian phylogeographic analysis Hydromantes belonging to the Eastern clade and the dispersal routes within and among these species. Due to the uncertainty associated with the available calibration points for the most accurate dating of our data  for a non-recombining genetic region, e.g. cytochrome b, is defined as one plus the number of segregating sites (S+1) h is greater than S+1 then convergent mutations are implicated at a subset of sites. Species datasets were used to make a statistical parsimony-based network using TCS Population genetic analyses were performed on a species by species level using the maximum fragment length without missing data available for each species see . SequencK) that are geographically adjacent, genetically homogeneous within each group, and maximally differentiated from each other in terms of total genetic variance (high CTF index). A simulated annealing process for each K value was repeated 1023 times for each of 100 sets of initial conditions in order to assure that the final population groups were not affected by the initial configuration. The configuration with the higher CTF after the independent annealing processes was then retained as the best grouping of populations. K values were tested for each species, starting from two until the value for which CTF reached a plateau. Alternative population groupings  were tested by applying an analysis of genetic variance (AMOVA) as implemented in Arlequin Spatial analysis of molecular variance (SAMOVA) was performed using SAMOVA v1.0 http://ibdws.sdsu.edu/~ibdws/distances.html). Linear geographic distances (measured in Km as a linear distance between two locations on the ground) among populations of the same species were calculated by inserting the geographic coordinates of each sampling site in Google Earth. For each species  20.000 permutations and population average uncorrected p-distance values were used for this analysis. Population average uncorrected distance was calculated as the average of all the pairwise distances among individuals of two distinct populations  was run by using the Isolation by Distance Web Service IBDWS, , availabrd codon position was rejected (data not shown). All species were recovered as monophyletic groups with high bootstrap and posterior probability support by the ML and BI analyses . Uncorrected p-distances among full Hydromantes species ranged from 0.065 (between H. ambrosii and H. strinatii) to 0.178   was almost always higher (>0.123) than the maximum distance among any of the Eastern species . Intraspecific p-distances among clades (as indicated in H. genei 4A from the other clades of H. genei (H. genei B (genei 2B+genei 3B) had a maximum genetic distance from samples belonging to the H. genei 1A that was below the minimum distance among full Hydromantes species  . H. italH. genei . The cla species .supramontis-flavus and imperialis  and genetic differentiation of H. sarrabusensis in its current distribution range in the South-East of Sardinia  indicating that multiple mutations occurred at some sites. In fact, while in H. flavus and H. sarrabusensis all the polymorphic sites were two-fold variants, in the other three species between one and three sites were three-fold variants. We obtained a total of 65 haplotypes for five species. Haplotype diversity was highest in H. imperialis and H. genei and lowest in H. sarrabusensis  were recovered as a network separate from the rest . The correlation coefficient, r, indicated either a strong , a medium , or a weak  correlation. In H. flavus, two distinct groups with correlated genetic and geographic distances were found . A similar, even if less marked pattern was observed in H. supramontis, while in H. imperialis and H. genei high genetic distances were almost uniformly distributed across geographical distances (data not shown).Genetic population structure was found to be correlated, except in one case, with geographic distances , the ring-shaped area around the Gennargentu  for H. supramontis and H. imperialis , and the area of the Iglesiente for H. genei (corresponding to the Western part of the current range of distribution of the species). After initial divergence in allopatry, Cimmaruta et al. H. imperialis and H. supramontis into restricted genetic islands corresponding to separated mountain chains , followed by few episodes of secondary contacts.Our study revealed strong genetic differentiation among populations of Mountain chains and elevational gradients have been reported to act as barriers to gene flow in amphibians H. supramontis-H. flavus from an ancestor of the other species and further cladogenesis following a stepping-stone mode. Intraspecific divergence can be explained by dispersal events to multiple adjacent areas, with few secondary contacts or by genetic differentiation and consequent extinction of an ancestral genotype among populations with different population size. Only one area of dispersal or of persistence of an ancestral genotype, corresponding to the center of the Monte Albo, was found for H. flavus. Dispersal to adjacent areas followed by genetic divergence within this species or genetic differentiation from the ancestral genotype due to habitat isolation and different effective population sizes are also supported by our parsimony-based analysis. A major area of dispersal  corresponding to the calcareous massif and to the mountain complex of Oliena-Orgosolo-Urzulei (populations 30–35) was recovered for H. supramontis. From there, an initial range expansion to adjacent areas and a secondary stepping-stone migration and genetic differentiation in new localities, including separate calcareous areas and mountains (populations 33 and 36–37), or local genetic differentiation took place. Back migration or persistence of an ancestral genotype are suggested between some of the localities within the same calcareous complex. In H. imperialis, the initial diversification probably occurred in the calcareous area and mountain complex of the Ogliastra (populations 17 and 19), from which individuals dispersed to adjacent areas within and to separate calcareous massifs or where genetic differentiation occurred locally. Finally, our results support an early diversification of an ancestor of H. sarrabusensis and secondary dispersal of this species to its current distribution area. We cannot, however, exclude genetic differentiation of H. sarrabusensis occurring in its current habitat paralleled by the extinction of his ancestral genotype.Our results suggest divergence of an ancestor for Eastern Sardinian species to have occurred near the Gennargentu mountains. Allopatric speciation in separate mountain areas as proposed by Cimmaruta et al. Hydromantes also occur outside caves . However, in both species we also found genetically strongly diverged lineages at a small geographic distance. The reasons for these patterns may be found in local environmental heterogeneity, which might have restricted gene flow, or in a more complex evolutionary history of the populations. In the case of H. genei, additional sampling from other sites and further geological investigation may help to identify possible barriers to gene flow. In fact, the strongly divergent samples of H. genei represented the westernmost part of its distribution.Calcareous areas and cave systems have been shown to offer refugia during times of changing environmental conditions and to be associated with genetic divergence and speciation in several taxa e.g., . In SardHydromantes populations  was extremely low and occurred mostly among geographically proximate sites. This suggests that phylogeographic breaks also occur in continuous areas without any obvious long-term geographic barriers. This phenomenon may result from the combination of low dispersal capacity and low population size Gene flow among Sardinian Hydromantes, including the extra-European species, is beyond the scope of this article, and was reported by Wake et al. H. genei called H. genei A and H. genei B that suggested the existence of cryptic species. This result was also confirmed by later studies H. genei, these do not correspond to the clades defined by Nascetti et al. H. genei containing two individuals from the westernmost part of its range that were recovered as genetically most divergent. When excluding these samples, genetic distances among clades A and B became similar to the intraspecific genetic divergence in other Sardinian species. Hence, the interpretation of these clades as cryptic species might have been influenced by insufficient knowledge of the intraspecific genetic divergence in other Hydromantes species and insufficient sampling of H. genei. We agree with Speybroeck et al. Hydromantes may be premature at this time. Further studies with even finer sampling, including populations from potential contact zones among genetically divergent lineages are required.A detailed review of the genus Hydromantes species among the amphibians that are likely to suffer most from future climate changes due to the strong environmental and temperature sensitivity. However, cave habitats may be strongly buffered against climate fluctuations in comparison to other habitats. All Sardinian Hydromantes species are currently listed in the annexes II and IV of the habitats directive 43/92/EEC, as well as listed as Vulnerable/Lower Risk in the IUCN Red Data book of Italy Plethodontid salamanders are considered excellent biological indicators of environmental change due to their ecological characteristics and life-history traits. Among these are site fidelity and poor dispersal, sensitivity to habitat disturbance and destruction, generally high population density, and longevity Click here for additional data file.Information S2Genetic p-distances among species (a) and intraspecific clades (b) of Italian Hydromantes. Average p-distances (minimum and maximum p-distances in parenthesis) based on 511 bp of the cytochrome b gene. Numbers with each species refer to clades as in (DOC)Click here for additional data file.Information S3Intraspecific haplotype networks distances. Minimum number of steps (absolute genetic distances) to connect intraspecific single haplotype networks.(DOC)Click here for additional data file.Information S4Results of the SAMOVA/AMOVA analyses. Intraspecific genetic structure for each Hydromantes species on the base of the within and among population genetic variance, the geographic location and sampling site. This was not tested for H. sarrabusensis, as only one haplotype network was recovered for this species. Populations belonging to distinct clusters (gene pools) are indicated in parenthesis with numbers referring to populations as in STF, CTF, and SCF indicate the proportion of total variance among populations, among groups, and among populations within groups, respectively. * indicates significant p-values<0.05.(DOC)Click here for additional data file.


M. integrifolia based on the chloroplast DNA (cpDNA) trnL-trnF and trnfM-trnS regions, and defined 26 haplotypes that were phylogenetically divided into six clades dated to the late Tertiary. The six clades correspond, respectively, to highly differentiated population groups that do not overlap in geographic distribution, implying that the mountain ranges acting as corridors or barriers greatly affected the evolutionary history of the QTP plants. The older clade of M. integrifolia only occurs in the southwest of the species' range, whereas the distributions of younger clades extend northeastward in the eastern QTP, suggesting that climatic divergence resulting from the uplift of the QTP triggered the initial divergence of M. integrifolia native to the plateau. Also, the nrDNA ITS region was used to clarify the unexpected phylogenetic relationships of cpDNA haplotypes between M. integrifolia and M. betonicifolia. The topological incongruence between the two phylogenies suggests an ancestral hybridization between the two species. Our study indicates that geographic isolation and hybridization are two important mechanisms responsible for the population differentiation and speciation of Meconopsis, a species-rich genus with complex polyploids.The complex tectonic events and climatic oscillations in the Qinghai-Tibetan Plateau (QTP), the largest and highest plateau in the world, are thought to have had great effects on the evolutionary history of the native plants. Of great interest is to investigate plant population genetic divergence in the QTP and its correlation with the geologic and climatic changes. We conducted a range-wide phylogeographical analysis of  The evolutionary history of plant species could be dramatically influenced by habitat fragmentation The phased uplift of the Qinghai-Tibetan Plateau (QTP) continuing from the middle Tertiary has greatly changed the geology and topography of East Asia, leading to the unique geomorphological configuration and complex land conditions To date, the geographic patterns of population genetic variation were investigated on more than ten plant species from different lineages in the QTP Meconopsis comprises about 50 species that are mainly distributed in the QTP and the neighboring mountains, with the only European species M. cambrica inhabiting in the humid and shady deciduous forest from Ireland, south-west England to Northern Spain Meconopsis integrifolia, one of the most widely distributed species of the genus, is a flagship species of the alpine scree in the QTP. It is scattered in the scree along mountain ridges, with an island-like distribution at altitudes of 4000–5200 m above sea level. In the eastern edge of its range, the species often grows in patchy habitat under azaleas at lower altitudes of 2700–4000 m above sea level. The bright yellow flower of this species makes it easy to be distinguished from the blue (or sky blue) flower relatives M. betonicifolia and M. simplicifolia, two quite narrowly distributed species in the southeast QTP  in Beijing.Meconopsis integrifolia during the summers of 2005–2009. Fresh leaves were collected from 35 populations and, with few exceptions, 15–29 individuals were sampled from each population .Population sampling was conducted throughout the range of pulation . To avoiectively . Ten indtrnfM-trnS trnL-trnF M. integrifolia clade that was nested within M. betonicifolia in the cpDNA phylogeny, 26 individuals of M. simplicifolia, M. betonicifolia and M. cheilanthifolia and 151 samples from 20 populations that can represent the seven geographic groups  GST and NST using program HAPLONST GST taking into account haplotype frequency and NST taking into account both frequency and similarity of the haplotypes. Besides, the U-statistic was performed to compare the GST and NST, with a higher NST than GST indicating the presence of phylogeographic structure The DNA sequences were aligned with Clustal X 2 distribution with n – 2 degrees of freedom, where n is the number of taxa (haplotypes of M. integrifolia plus outgroups). When M. chelidonifolia served as outgroup, the results of the likelihood ratio tests rejected a clock-like evolution for both cpDNA (P<0.01) and ITS haplotypes (P<0.01). If M. betonicifolia and M. simplicifolia were chosen as outgroups, the results of the likelihood ratio tests rejected a clock-like evolution for cpDNA (P<0.01) but not for ITS haplotypes (P = 0.096). As no fossil of Meconopsis and its close relatives was available, the crown group age : 15.2–3.8 Ma) M. integrifolia and its close relatives was used to date lineage ages of M. integrifolia in the cpDNA and nrDNA ITS phylogenies. Given the wide confidence interval of this constraint age, the average nrITS substitution rate for herbaceous plants  M. integrifolia and its close relatives. Molecular clock analysis of the age and confidence interval was performed using BEAST v1.6.2 −9 subs/site/yr) with a 95% confidence interval for the mean rate, the substitution rate was set to a normally distributed prior with a mean of 4.13×10−9 subs/site/yr and a standard deviation of 1.47×10−9 subs/site/yr. The Bayesian Markov chain Monte Carlo simulation was run for 20,000,000 generations with a sample frequency of 1000, and the first 2,000,000 generations were discarded as ‘burnin’. Trace file and statistics of estimated parameters were created using Tracer version 1.5 M. integrifolia and its close relatives was dated to 6.20 Ma (95% HPD: 15.23–2.78 Ma) (not shown). Then, a relaxed clock model M. chelidonifolia as outgroup. An uncorrelated lognormal distribution model with a Yule speciation process and a Hasegawa-Kishino-Yano plus Gamma site heterogeneity model was applied in the analyses. To cover the time range (9.2–6.2 Ma) with a 95% confidence interval for the mean age, the crown group age of M. integrifolia and its relatives was set to a normally distributed prior with an average of 7.7 Ma and a standard deviation of 0.9 Ma. The remaining settings and procedures were the same as in the nrITS data set described earlier.To examine the constancy of molecular evolution rate among lineages of the phylogenies, a likelihood ratio test M. integrifolia, which defined 26 cpDNA haplotypes (A–Z)  . For phyes (A–Z) , BEAST (es (A–Z) , and maxes (A–Z) , and fivThe 26 cpDNA haplotypes are distributed in seven non-overlapped geographic regions . The fouM. integrifolia, twenty-two (62.86%) harbored pure haplotype, ten (28.57%) fixed two haplotypes, and only three (8.57%) had more than two haplotypes. Significant population differentiation was observed, with a GST = 0.843 and a NST = 0.887. The U-statistic showed that GST and NST were significantly different from each other . The analysis of molecular variance (AMOVA) revealed that 80.18% of the total variation occurred among groups, 16.82% among populations, and 3.00% within populations  with high bootstrap and posterior probability values were recognized. In the ITS tree (nu) is a natural group of M. integrifolia rather than being nested into M. betonicifolia as in the cpDNA tree (nu and IInu of the ITS haplotypes are exclusively restricted to population groups WNY and LZ (corresponding to clades I and II in the cpDNA phylogeny), respectively (nu includes seven haplotypes (H8–H14) that only occur in population group ET (corresponding to clade IV in the cpDNA phylogeny), but two haplotypes  of clade VIInu occur in population 2 of this group. The monophyletic relationships of groups SLL, DX and EQ were not supported by the nrDNA ITS analysis.Twenty-five nrDNA ITS haplotypes  were detected from 151 individuals representing the seven population groups . PhylogeITS tree , the groDNA tree . A signiDNA tree , the claectively . Clade IM. integrifolia and its close relatives was set to 7.7(±1.5) Ma as a constraint, the most recent common ancestor of M. integrifolia was dated to about 5.77 Ma (95% highest posterior density (HPD): 7.86–3.38 Ma) based on the cpDNA haplotypes (nu and clade IVnu was dated to 2.92 Ma (95% HPD: 4.45–1.73 Ma), and that between clade VIInu and its sister clade was estimated to be 3.71 Ma (95% HPD: 5.37–2.26 Ma).When the crown group age of plotypes  and arouplotypes . The croTsuga dumosaIt is well known that complex topography could have great effects on the patterns of population genetic variation in space and time M. integrifolia indicates a subtle geographic genetic structure and remarkable genetic differentiation among lineage-specific population groups of the species   indicates that populations of the species have experienced genetic drift. An increasing number of studies indicated that habitat fragmentation could play important roles in the divergence and speciation for plants M. integrifolia occurred during late Miocene (5.77 Ma) to early Quaternary (2.32 Ma) . Given the uncertainty with the constraint age used in the present study, this scenario should be accepted with caution. However, our inference is consistent with previous reports that lineage divergence and speciation of plants in the QTP could be attributed to the topography changes resulting from the rapid uplift of the plateau M. integrifolia may imply that habitat fragmentation arising from the uplift of the QTP has played a key role in speciation and extinction of the QTP plants.Moreover, individuals in relatively low altitudes (3000–4000 m) often inhabit in forest gaps or under shrub, a habitat quite different from the alpine scree at higher altitudes of the QTP. The great population genetic differentiation (FST)  and exte2.32 Ma) , a time 2.32 Ma) . The estM. integrifolia only occurs in southeast Tibet, with the distributions of younger groups  extending northeastward in the eastern QTP (M. integrifolia dated to around 5.77 (95% HPD: 7.86–3.38) Ma based on cpDNA or 5.33 (95% HPD: 7.35–3.45) Ma based on ITS seems to have occurred synchronously with the climatic divergence in the QTP M. integrifolia.Another interesting finding is the rough correlation between the haplogroups' distribution and the regional climate in the QTP . A typictern QTP , 3. Thertern QTP  [47]. ItM. integrifolia is very different between the cpDNA (clade I) and ITS (clade Inu) phylogenies constructed based on population sampling  and ×M. harleyana (M. integrifolia×M. simplicifolia) were reported about eighty years ago M. integrifoliaM. harleyana was identified as a yellow-flowered variety of M. simplicifolia rather than a hybrid, since it was often difficult to distinguish hybrids of Meconopsis only based on morphological characters M. pseudointegrifolia, a variety of M. integrifolia or M. pseudointegrifoliaM. integrifolia and M. betonicifolia Click here for additional data file.