Pyrus ussriensis Maxim. is native to the northern part of China, but whose habitats are currently being destroyed by environmental changes and human deforestation. An investigation of population structure and genetic diversity of wild Ussurian pear is a priority in order to acquire fundamental knowledge for conservation. A total of 153 individuals of wild Ussurian pear from the main habitats, Heilongjiang, Jilin, and Inner Mongolia in China, possessed low genetic diversity as a result of habitat fragmentation. The genetic diversity of the populations in Inner Mongolia and north east of Heilongjiang was especially low and there was the possibility of inbreeding. Wild Ussurian pears were divided into 5 groups based on the Bayesian clustering method using 20 nuclear SSRs (nSSRs) and 5 groups by haplotype distributions using 16 chloroplast SSRs (cpSSRs), and the populations in Inner Mongolia and north east of Heilongjiang represented unique genotypes. AMOVA indicated there was a 20.05% variation in nSSRs and a 44.40% variation in cpSSRs among populations. These values are relatively high when compared to those of other tree species. Haplotype E, positioned in the center of the cpSSR analysis network and showed the largest number of connections with other haplotypes, represented the most important haplotype. Inner Mongolia and the north east of Heilongjiang are two areas that need urgent conservation because of their genetic vulnerability and peculiarity. We determined 4 conservation units based on the clustering by nSSRs and cpSSRs, and geographic factor. This information is helpful in deciding the conservation strategies for wild Ussurian pear in China. Investigating the genetic population structure, which includes genetic diversity, population differentiation and degree of gene flow , is veryPyrus in the tribe Pyreae of subfamily Spiraeoidae in the Rosaceae [Pyrus is considered to have originated during the Tertiary period (65–55 million years ago) in the mountain regions of southwestern China [Pyrus species in China. Main cultivars in China are (1) Sand pear (P. pyrifolia), (2) Xingjiang pear (P. sinkiangensis Yu), (3) White pear (P. bretschneideri Rehd.), and (4) Ussurian pear (P. ussuriensis Maxim.). Ussurian pear is the most important cultivated pears in northern part of China. The differences between Ussurian pear and the other Pyrus species are as follows; (1) Morphological feature: short peduncle and pedicle, small fruit, the fruit shape is round or oblate, that skin color is yellow or brown, and the diameter is ranging from 2 to 6 cm with a 1 to 2 cm short pedicle [P. ussuriensis cultivars is problematic. Floristic studies [P. pyrifolia and P. bretschneideri. However, many cultivars with longer fruit pedicels have been classified as P. ussuriensis in the horticultural literature [Pears, which are cultivated throughout the temperate regions and have spread all over the world, belong to the genus Rosaceae . The genrn China . China irn China , thus th pedicle . (2) Col studies  adopted et al. [et al.[Cultivated Ussurian pears are considered to be derived from wild Ussurian pear native to the northern part of China, the east of Russia, the north of Korea and the north east of the main island in Japan , 7, 9. Iet al.  and Wuyu. [et al., there w. [et al.. We needP. ussuriensis var. aromatica (syn. to P. ussuriensis Maxim.) which is called ‘Iwateyamanashi’ are distributed mainly in the northern east part of the main island. The trees of Iwateyamanashi are usually found in local farmyards, on grazing land, along old roads, and occasionally on mountains [P. ussuriensis var. aromatica and P. ussuriensis Maxim. in China is still unknown. The genetic investigation for wild P. ussuriensis Maxim. in China were especially deficient.In Japan, wild Ussurian pear trees and local varieties derived from ountains , 14. ThePyrus to help understand the genetic diversity and population genetic structure [et al. [et al. [Currently, many molecular biological approaches using nuclear and chloroplast DNA markers have been applied to tructure , 11, 20. [et al.  surveyed [et al.  researchPyrus [Malus [et al. [Pyrus. Chloroplast DNA marker is a useful tool for evolutionary studies and population structure analysis because of the non-recombinant and uniparentally inherited nature of organelle genomes [Pyrus was reported in the entire sequence data of P. pyrifolia cpDNA [Pyrus [Genetic analysis of population structure requires suitable markers. Microsatellite  has been used widely in construction of genetic maps, parentage and population genetic structure analysis . BecausePyrus , 27, 28 s [Malus , 30, 31, genomes , 33. Det genomes , 35. Theia cpDNA . The invA [Pyrus .P. ussuriensis var. aromatica in Japan and P. ussuriensis Maxim., and get an insight into the origin of P. ussuriensis.The aim of this study is (1) to reveal genetic structure of wild Ussurian pear in China, such as genetic diversity, gene flow, and genetic divergence. This study is an initial step in determining suitable conservation units, and considering possible conservation strategies for the wild Ussurian pear. The second aim of this study is (2) to reveal the phylogenetic relationships between Pyrus ussuriensis Maxim.) individuals were collected from their main habitat, Inner Mongolia, Heilongjiang, and Jilin in China. We collected 13 populations comprised as follows: 91 individuals from 6 populations in Inner Mongolia, 70 individuals from 6 populations in Heilongjiang, and 12 individuals from a single population in Jilin (Pyrus based on those of Yu (1979) and Ohwi (1965). To avoid multiple sampling of closely related individuals such as half sib or full sib., a minimum distance of 10 m between individuals was maintained.Almost no investigations of wild Ussurian pears in China have been conducted until now, so their habitats were unclear. After explorations based on the old documentary records and interviews, 13 populations of Ussurian pears growing wildly were recognized. A total of 153 wild Ussurian pear (in Jilin . All of in Jilin . ClassifP. pyrifolia: SL), 11 (9) Xingjiang pear cultivars (P. sinkiangensis Yu: XJL), 9 (7) White pear cultivars (P. bretschneideri Rehd.: BL), and 29 (16) Ussurian pear cultivars collected from 2 different places (P. ussuriensis Maxim.: QZL1 and QZL2). Furthermore, 12 (10) Japanese pear cultivars (P. pyrifolia: JAP), 15 (14) Japanese wild Ussurian pears (P. ussuriensis var. aromatica (Kikuchi et Nakai) Ohwi: Iwateyamanashi; IWT), and 11 (9) European pear cultivars (P. communis L.: EUR) were also used. The number shown in parentheses is the numbers of individuals used for cpSSRs analysis. No specific permissions were required for the described field studies: a) no specific permits were required for these locations/activities; b) locations were not protected; c) the field studies did not involve endangered or protected species. Young leaves were collected from the trees and DNA was extracted by A Plant Genomic DNA Kit .The Chinese cultivars used in this study as reference samples representative of cultivated pear gene pool were: 11 (10) Sand pear cultivars , was utilized to design primer pairs flanking SSRs. The major parameters for primer design were as follows: primer length, about 20 bp; PCR product size, between 100 to 250 bp; optimum annealing temperature, 60°C; GC content, about 50%. Synthesized primer pairs (Life Technologies Japan Ltd) were tested to detect polymorphisms within 95 individuals of wild Ussurian pear endemic in Japan, and 16 cpSSR primers were selected for the present study  which were developed from a Japanese pear cultivar ‘Hosui’, an European pear cultivar ‘Bartlett’, and Apple  using POPGENE 32 version 1.3.2 [FIS and their significance (FIS ≠ 0) was examined using 1,000 permutation tests. For cpSSRs, the number of haplotypes per locus (Na), effective number of haplotypes (Ne) and Nei’s gene diversity were also calculated using POPGENE 32 version 1.3.2.The genetic diversity as defined by nSSRs in each population was quantified in terms of the number of alleles per locus (Na), effective number of alleles (Ne), allelic richness (Ar), the mean observed heterozygosity (Ho), expected heterozygosity (He), and fixation index  was performed to examine the hierarchical genetic structure using the program Arlequin version 3.11  for wildp / ms) as described by Mitsui et al (2010) using only wild Ussurian pears in China. The equation of mp / ms is based on the prediction that the levels of population differentiation can differ between nuclear and cytoplasmic markers because of their different modes of inheritance and that the extent of population differentiation is related to the relative levels of pollen and seed migration among populations [To estimate the relative amounts of pollen and seed flow, we calculated the pollen to seed migration ratio  values against the geographic distances between the respective pairs of populations [http://lab.uribou.net/ll2dist/).Mantel tests  were carulations . The geoA dendrogram was constructed using nSSRs data to interpret relationships among populations by a neighbor-joining (NJ) method  based onA median-joining network including potential median vectors was constructed with wild Ussrian pears in China using Network 4.5.1.6  using cpFive flower morphologies: (1) Flower diameter, (2) Petal length, (3) Petal width, (4) Petal length/ Petal width, and (5) Peduncle length were measured in 7 populations  and 26 Ussurian pear cultivars. We measured up to 10 randomly selected flowers from each individual . Six fruTo determine the morphological features for each population, we performed a discriminant analysis, using the program JMP 8 . Discriminant analysis was performed using all of the morphological traits except for calyx existence.Pyrus species. Twenty five haplotypes were represented in wild Ussurian pears in China , therefore haplotype E was the most common haplotype for wild Ussurian pears in the north east of Heilongjiang , Table 3The relationships between 25 cpSSR haplotypes detected in wild Ussurian pears in China were analyzed by a median-joining network model . The hapThe results of the AMOVA for nSSR showed that 20.05% of the variation was due to differences among populations, and 79.95% within each population. For cpSSR, 44.40% of the total genetic variation was due to differences among populations, and 55.60% within each population. Differentiation among populations for cpSSRs was more than twice as large as that of nSSRs. The calculated ratio of pollen flow to seed flow were 5.8: 1 in 13 wild populations from China.In the STRUCTURE analysis, the plot of the average log-likelihood values reached plateau at K = 10 Figs  and 3. AMost of the cultivars formed admixtures at K = 10, but QZL1, QZL2, and JAP were made up of relatively uniform gene pool .FST / (1-FST) values were significantly correlated with geographic distance among populations for nSSR  and cpSSR .As a result of the Mantel test, IBD pattern was detected for both nSSR and cpSSR. Pairwise Pyrus species . Two populations from Heilongjiang (HLYCS3 and HLFYX) showed significant deviations from the Hardy-Weinberg equilibrium. In cpSSRs, wild Ussurian populations from China also showed less genetic diversity than Chinese Ussurian cultivars (QZL) and Iwateyamanashi (IWT). JLGZL and HLYCS2 showed high genetic diversity, and all of the populations in Inner Mongolia had low diversity  and HLYCS2 had the second highest values for most indicators . All the populations from Inner Mongolia indicated low genetic diversity   as did Hiversity . When thiversity .P. pyrifolia, P. bretchneideri, P. sinkiangensis, and P. communis. Japanese cultivated pears (P. pyrifolia) and wild Ussurian pear from Japan (Iwateyamanashi) were positioned far away from the other groups (6)  The 6 populations  originating from Inner Mongolia which were all closely related. (2) The 5 populations  from Heilongjiang and the population JLGZL from Jilin also showed close relationships. Group (3) including HLMTZ from Mudanjiang, Heilongjiang was distantly related the other populations from Heilongjiang, and was positioned near the cultivated Ussurian pear group (4). Group (5) contained the other cultivated pears such as oups (6) .In order to demonstrate the relationships between populations based on flower and fruit morphologies, discriminant analysis was carried out using the values of 5 floral and 4 fruit morphological characters . The 5 pMalus sieversii and M. sylvestris), African fruit tree of Rosaceae (Prunus africana), wild Callery pear , and the average of many perennial and outcross species [Using nSSR markers, we have shown that wild Ussurian pears from China have much lower genetic diversity Iwateyamanashi. The genetic diversity of wild Ussurian pear in China inferred from the values of na, ne, Ho, and He was low compared to the values of wild grapevine from Spain, wild apple  on Yakushima Island, Japan, Quercus semiserrata Roxb. in northern Thailand, Pinus albicaulis Engelm. (whitebark pine), Mexican pinyon pine (Pinus nelsonii Shaw), Magnolia stella, and Apricot [For cpSSRs, the genetic diversity within each wild Ussurian pear population was lower than that of the other trees such as  Apricot , 59, 60.FST were also significantly different among populations with the exception of several populations within Inner Mongolia , referring to previous studies such as pulation , P. ussupulation . For cpSpulation  and 4B. pulation . The cirpulation . HLSWX, pulation .P. ussuriensis from China  [P. calleryana from China [A significant correlation between genetic distance and geographic distance was detected by a Mantel test using both nSSRs and cpSSRs. Isolation by distance was expected to be the main factor causing the high level of population divergence in wild Ussurian pears, due to limited gene flow and the effects of genetic drift as a result of habitat fragmentation. Geographic distance influences migration of pollen and seed and restricts the gene flow among populations. The ratio of pollen to seed flow was calculated using the equation of Ennos , and the; 24: 1) . This isom China . The rat‘Group 1’ included all of the populations from Inner Mongolia . Wild InP. ussuriensis growing wild in Northern part of Japan was introgressed from Japanese pear cultivars [‘Group 5’ included HLMTZ, from Muling, Mudanjiang City, Heilongjiang Province. Based on STRUCTURE analysis, this population was separated from the other wild populations in Heilongjiang, and positioned near the Ussurian pear cultivar group in the phylogenic tree . Moreoveultivars , 62.et al. [Haplotype E which is the representative haplotype of Heilongjiang, and according to coalescent theory , there iet al.  using hyet al [Although the flower shape in the population ‘HLFYX’ represented intermediate flower morphology between wild and its derived cultivars, wild Ussurian pear and cultivated Ussurian pear in China were genetically divergent from each other Figs  and 6I. et al  using M1et al . From moThe phylogenetic relationship of Ussurian pear in China and IwateyamanashiP. ussuriensis Maxim. (wild Ussurian pear in China) and P. ussuriensis var. aromatica (Iwateyamanashi) were genetically divergent from the result of STRUCTURE analysis and phylogenetic tree  and P. ussuriensis var. aromatica (Iwateyamanashi), then each of these Pyrus followed the evolutionally different way.In this study, ree Figs  and 5. Tf Eocene . Neogenef Eocene . At thatThe detection of population differentiation using cpSSRs haplotypes and STRUCTURE analysis using nSSRs can help to define appropriate conservation units, and provide a good focus for conservation management . Wild Usin situ level. This study also revealed that the genetic diversity of group 1 was especially low , 10 individuals of Iwateyamanashi , and 16 individuals of cultivated Ussurian pears in China (AA-PP). The haplotypes are indicated by yellow circles, and small red circles show median vectors. The size of each pie chart is proportional to the frequency of corresponding haplotype.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S7 Table(DOCX)Click here for additional data file.S8 Table(DOCX)Click here for additional data file.S9 Table(DOCX)Click here for additional data file.


Myodes (=Clethrionomys) glareolus populations were studied. Isolation and pollution are considered to have important effects on biodiversity. Animals were collected from ten populations in isolated (island), mainland, and metal-polluted areas. Three populations were in areas near zinc and lead smelters; four were on islands in the relatively unpolluted Mazurian Lake District and in the Bieszczady Mountains; and three were in clean-mainland areas in the Mazurian Lake District, the Niepołomice Forest, and the Bieszczady Mountains. Cadmium and lead concentrations in liver and kidney were measured to assess the animals’ exposure to metals. The metal concentrations were greater in animals from areas classed as polluted than in animals from the clean-mainland areas and islands. The genetic diversity of each population was analyzed using eight microsatellite markers. The results confirmed that isolation adversely affects genetic diversity in M. glareolus populations , but the effect of metal exposure on genetic diversity was not strong. Of the samples from polluted areas, only the Katowice population, which is exposed to high levels of metal pollution and is also isolated because of human activity, showed genetic variation parameters that were similar to those for the island populations. Nei’s genetic distances indicated that the island populations were genetically distant from each other and from the other populations, and there were noticeable inbreeding effects that would have been caused by the isolation of these populations.The effects of isolation and heavy-metal pollution on genetic diversity in  Ecological and ecotoxicological studies are being increasingly focused on diversity loss, which has been studied during the last few decades at various organization levels: genetic diversity in a single species, species richness in communities, and biodiversity in ecosystems  by Berckmoes et al.  showing a significant negative relationship between genetic diversity and metal concentration  and environmental factors  affect these variations. Isolation by natural barriers , artificial barriers , and geographic distance have been shown to be important factors in the loss of diversity. Geographic distance sometimes has a less significant effect than do other barriers, such as isolation by a stretch of water. Kozakiewicz et al.  showed tM. glareolus, chosen for the study, is one of many common species that have been used as model species in research into universal physiological processes in organisms in threatened environments. The main reason for choosing this species is that it is very common and that it inhabits a variety of environments, including areas close to industrial regions  animals from areas near zinc/lead smelters have greater metal concentrations in their tissues than animals from other populations; (2) populations on islands have lower levels of genetic diversity than do populations in clean and open site, because of the isolation effect; and (3) populations in polluted areas have low levels of genetic diversity because of the direct and indirect effects of metal pollution. We selected three types of bank vole populations in southern and northeastern Poland. The populations inhabit chronically polluted sites, clean-mainland areas, and islands in lakes.M. glareolus individuals (both sexes) from ten study sites between August and November 2009 using a standard live-trapping technique. The trapped animals were transported to the laboratory in plastic cages and then killed by decapitation. Kidney and liver tissues were collected and stored at −75 °C until they were subjected to chemical analysis. Part of the auricle from each individual was preserved in 96 % ethyl alcohol solution  and stored at −75 °C, for molecular analysis. All procedures were performed according to EC Directive 86/609/EEC for animal experiments.We collected 197 The collection sites were in northeastern and southern Poland, and they are shown in Fig. Dobskie (IDo)—54°5′N, 21°36′E, area 1.5 ha, approximately 500 m from the shore, North;Dejguny (IDe)—54°2′N, 21°37′E, area 9.3 ha, approximately 250 m from the shore, North;Święcajty (ISw)—54°11′N, 21°45′E, area 7.4 ha, approximately 400 m from the shore, North;Skalista (ISo)—49°21′N, 22°30′E, area 3.0 ha, approximately 250 m from the shore, South.Three of the island populations that were sampled were in the Mazurian Lake District in northern Poland (labelled North), and one was in the Bieszczady Mountains in southern Poland (labelled South). The three North islands do not have geographical names, so we labeled them with the names of the lakes they are in:Olkusz (POl)—50°19′N, 19°30′E;Miasteczko Śląskie (PMi)—50°31′N, 18°55′E;Katowice (PKa)—50°15′N, 19°4′E.The three polluted sites were close to zinc and lead smelters in Silesia Province (southern Poland), and the environment in each area was contaminated with a similar combination of pollutants. The polluted sites were as follows:We were unable to find a set of similarly polluted areas in northern Poland. The POl site is near the Bolesław zinc/lead mine and smelter, and high concentrations of contaminants have been reported in this area for several decades —53°46′N, 21°30′E, Mazurian Lake District, North, approximately 30 km from the closest of the island populations that were studied);Niepołomice (CNi)—50°0′N, 20°20′E, South, approximately 20 km from Kraków);Teleśnica Oszwarowa (CTe)—49°22′N, 22°32′E, Bieszczady Mountains, South, 2 km from ISo Island).The clean-mainland populations were near these areas:Samples from a total of 115 individuals were collected for metal analyses (Table N = 197) using the DNeasy blood and tissue kit  using a protocol designed for animal tissue. Lysis was performed at 55 °C in a Thermomixer . The DNA concentration was measured using a Nanodrop ND-1000 spectrophotometer .DNA was isolated from an auricle fragment from each of the individuals that had been caught .The genetic diversity was assessed using eight polymorphic loci: MSCg-4, MSCg-9 . The fragment analysis of each sample was performed using a reaction mixture of 14.3 µL of formamide, 0.2 µL of Applied Biosystems GeneScan 500LIZ Size Standard (Applied Biosystems), and 0.5 µL of previously amplified DNA.The PCR products were identified based on their sizes using an Applied Biosystems 3130The distributions of the metal concentrations in bank vole liver and kidney samples from each population were checked for normality using the Kolmogorov–Smirnov test . Most of the data did not form a normal distribution, so they were log-transformed. We used a two-way analysis of variance (ANOVA) to test the significance of the effects of the sex of the individuals and the site on metal accumulation in the tissues of the animals from different populations. Sex was not a significant factor, so the rest of the analyses were performed on the combined data for both sexes. We used one-way ANOVA and Tukey post hoc test to determine the significance of the differences between the populations and the effects of the population type. We used main-effects ANOVA to study the effects of the geographical region (South versus North populations) and the site . These statistical analyses were also performed using Statistica ver. 9.He), the unbiased expected heterozygosity (UHe), the observed heterozygosity (Ho), the average number of alleles at each locus (Na), the number of private alleles, and the fixation index /He). We performed analysis of molecular variance (AMOVA) based on the Fst values. FSTAT software (http://www2.unil.ch/popgen/softwares/fstat.htm) was used to assess the allelic richness, the gene diversity (Hs)  Nei , and difK = 1 to K = 10, using a burn-in period of 100,000 iterations, and we collected data for 500,000 iterations. STRUCTURE HARVESTER software was used for collating results generated with STRUCTURE   Table . It was Lead concentrations were generally greater in kidneys than in livers: from 0.74 to 1.32 mg/kg dw in the animals from the polluted sites and from 0.12 to 0.47 mg/kg dw in the animals from the clean sites .Mean UHes Table  indicatees Table  ; other factors act indirectly by decreasing the population size. Contamination and isolation can both affect the genome in two ways, either decreasing or increasing genetic diversity. Biomarkers, such as microsatellites, have high mutation rates and high degrees of variability, and they can be used to determine changes in genetic diversity  was several times greater than those of individuals from the clean sites in this study and in other studies: 0.3 mg/kg dw  and allelic richness (Table Na) were slightly lower only for the PKa population than for the other mainland populations, including those from polluted areas. This may be explained in two ways. Metals may affect genetic diversity (1) by causing a decrease in effective population size and leading to a bottleneck effect or (2) by selection (Van Straalen and Timmermans We analyzed the microsatellite DNA of our bank vole populations and found that exposure to a contaminated environment had no effect on genetic variation, as expressed as the average number of alleles per locus or as heterozygosity, in ss Table . The popss Table  and 4. Civersity  by causiDipodomys merriami exposed to radionuclides (Theodorakis et al. Sigmodon hispidus showed no differences in genetic diversity between populations of clean sites and those of oil refinery sites (Pfau et al. Genetic diversity parameters for the populations of the other two polluted sites, POl and PMi, did not appear to be different from the those for the clean-mainland populations Table . One expHs between the island and the polluted site populations [the effect of population type (Table M. glareolus populations living close to a copper smelter in the Ural Mountains was found to be the result of decreased populations (Gileva et al. The lack of difference in pe Table B] could Genetic diversity parameters showed that island populations had low levels of genetic variation, confirming that water is an effective barrier for bank vole populations. Nei’s genetic distance results Table  and Baye


Amphiprion omanensis, throughout its entire species range. Using genetic assignment tests we demonstrate bidirectional exchange of first generation migrants, with subsequent social and reproductive integration, between two populations separated by over 400 km. Immigration was 5.4% and 0.7% in each region, suggesting a biased southward exchange, and matched predictions from a physically-coupled dispersal model. This rare opportunity to measure long-distance dispersal demonstrates connectivity of isolated marine populations over distances of 100 s of kilometres and provides a unique insight into the processes of biogeography, speciation and adaptation.Dispersal is a crucial ecological process, driving population dynamics and defining the structure and persistence of populations. Measuring demographic connectivity between discreet populations remains a long-standing challenge for most marine organisms because it involves tracking the movement of pelagic larvae. Recent studies demonstrate local connectivity of reef fish populations via the dispersal of planktonic larvae, while biogeography indicates some larvae must disperse 100–1000 s kilometres. To date, empirical measures of long-distance dispersal are lacking and the full scale of dispersal is unknown. Here we provide the first measure of long-distance dispersal in a coral reef fish, the Omani clownfish  Dispersal drives population dynamics, allows the replenishment of harvested marine species and defines the structure and persistence of marine populations across fragmented and often ephemeral habitat landscapes Many recent studies have focussed on coral reef fish, which are generally highly site-specific to naturally fragmented habitats, thus providing a valuable model system for studying dispersal and connectivity in marine ecosystems. Findings show that recruitment on coral reefs is largely driven by the retention of larvae within their populations of origin Amphiprion omanensis  or 66°C (multiplex B), and 45 s at 72°C, followed by 20 cycles of 45 s at 94°C, 45 s at 55°C (multiplex A) or 61°C (multiplex B), and 45 s at 72°C, with a final elongation period of 45 min at 72°C. Amplified PCR products were analysed on a CEQ 8000 Genetic Analysis System  and the resulting electrophoregrams were scored using CEQ 8000 software. Alleles were scored manually  to eliminate scoring errors.Genomic DNA was extracted from fin tissue samples using standard proteinase K digestion . Samples were genotyped with a panel of six microsatellite loci previously described for two congeneric species ISFv.2.2.3 STF) were computed in ARLEQUIN. Missing data accounted for 0.9% and 1.5% of the data in northern and southern populations, respectively.Samples were grouped within northern and southern regions. Nei’s unbiased expected and observed heterozygosity structure 2.3.3 We applied a model-based Bayesian clustering method implemented in hybridlab v1.0 To determine population-specific thresholds of assignment we first used a conservative threshold of 0.9 a priori dispersal model to simulate dispersal of the larval stage of A. omanensis. The model was forced using daily surface current data obtained from the Navy Coastal Ocean Model  for the period 2005–2008, and simulations of larvae released from both northern and southern locations were made for the A. omanensis spawning period during three successive seasons .A total of 136 Using assignment tests, we detected an asymmetrical dispersal pattern between the two regions with a higher occurrence of southward dispersal than vice versa . Our anaIn addition to first-generation migrants, we identified 17 north-south hybrids, 10  in the south and 7  in the north. The majority of individuals sampled in each population (221 or 85.0% in the south and 127 or 93.4% in the north) were identified as local-type . An addia priori predictions of a physically-coupled individual-based dispersal model parameterised for the larval stage of A. omanensis and forced using seasonal current data. Movement of larvae released into the model between December to February, when A. omanensis usually reproduces, was predominantly southward as a result of the winter monsoon , which simulated passive larval transport in realistic current fields during the reproductive season. Local migrants were found at all life stages suggesting that these long-distance dispersal events occurred over multiple events and are a regular phenomenon rather than a process limited to chance Geography, oceanography, ecology and behaviour combined favourably in this study system to provide a unique opportunity to empirically measure connectivity between distant populations of a coral reef fish. We found demographically relevant bidirectional exchange of A. omnanensis individuals, providing subsequent analyses with sufficient discriminatory power. With more markers and a higher proportion of individuals sampled we would expect intra-regional patterns in dispersal and retention to also become evident as in previous local-scale studies of congeneric species The combination of rarity and conspicuousness meant that our team of 22 divers over 92 dives sampled an estimated 5–20% of all Harvested and threatened populations under spatially variable levels of exploitation, or exposed to environmental stressors including fluctuations in habitat quality and climate change, are offered substantial resilience if replenishment from far beyond the range of local impact is possible. While there are obvious benefits to staying close to home The persistence of spatially structured populations is a factor of both local growth rates and connectivity between populations Our study also demonstrates that simple physically-forced models can give valuable predictions for realised patterns of connectivity. It is likely that if ocean currents are important for driving the long-distance dispersal events we observed, for fishes with prolonged pelagic larval durations (weeks to months), including many commercially important coastal fishes, long-distance dispersal may be even more prevalent Figure S1Frequency distributions of simulated genotypes for the derivation of population-specific assignment thresholds. A total of 5,000 northern-type, 5,000 southern-type and 10,000 north-south hybrid genotypes were simulated using location specific allelic frequencies. Bayesian clustering analysis determined the posterior probability of assignment of each simulated individual to either northern or southern populations.(TIFF)Click here for additional data file.Figure S2Scatterplot of the two main components of the discriminant analysis of principal components in four populations of the Omani clownfish Amphiprion omanensis. Sampled populations are shown using different colours and 95% inertia ellipses and dots represent each individual in the sample. The x-axis represents 82.9% and the y-axis represents 9.6% of genetic information retained in each discriminant function (inset).(TIFF)Click here for additional data file.Table S1Sampling sites and number of samples collected.(PDF)Click here for additional data file.Table S2Details of six polymorphic dinucleotide microsatellite loci developed for Amphiprion omanensis.(PDF)Click here for additional data file.Table S3Details of genetic analysis of each marker. Number of alleles (Na), observed heterozygosity (Ho), expected heterozygosity (He), the inbreeding coefficient (F), and departure from Hardy-Weinberg’s equilibrium (HWE) were calculated for each locus.(PDF)Click here for additional data file.Table S4Connectivity matrices (% of larvae successfully reaching a reef) for simulated dispersal events. The upper three panes are yearly averages ; the lower panes are overall means, maximum values, standard deviation, coefficient of variation of connectivity and number of non-zero larval transport events over all 30 realisations. Matrices read from-row to-column; grey cells indicate long-distance dispersal. Mirbat and Halaaniyat combine to form the southern region; Masirah and Bar Al Hickmann (MasBAH) combine to form the northern region (see gion see .(PDF)Click here for additional data file.Information S1Physically-coupled individual-based Lagrangian stochastic dispersal model; Graphical representation of genetic variation in the Omani clownfish; Supplementary References.(DOCX)Click here for additional data file.


Capra hircus), one of the most ancient breeds in China, has historically been a critical source of meat and cashmere production for local farmers. To adapt to the high-altitude area, extremely harsh climate, and hypoxic environment that the Tibetan cashmere goat lives in, this goat has developed distinct phenotypic traits compared to lowland breeds. However, the genetic components underlying this phenotypic adaptation remain largely unknown.The Tibetan cashmere goat , which has been previously reported to be involved in complex oxygen sensing and significantly associated with high-altitude adaptation of human, dog, and grey wolf. The missense mutation Q579L that we identified in EPAS1, which occurs next to the Hypoxia-Inducible Factor-1 (HIF-1) domain, was exclusively enriched in the high-altitude populations.We obtained 118,700 autosomal SNPs through exome sequencing of 330 cashmere goats located at a wide geographic range, including the Tibetan Plateau and low-altitude regions in China. The great majority of SNPs showed low genetic differentiation among populations; however, approximately 2-3 % of the loci showed more genetic differentiation than expected under a selectively neutral model. Together with a combined analysis of high- and low-altitude breeds, we revealed 339 genes potentially under high-altitude selection. Genes associated with cardiovascular system development were significantly enriched in our study. Among these genes, the most evident one was Our study provides insights concerning the population variation in six different cashmere goat populations in China. The variants in cardiovascular system-related genes may explain the observed phenotypic adaptation of the Tibetan cashmere goat.The online version of this article (doi:10.1186/s12864-016-2449-0) contains supplementary material, which is available to authorized users. EPAS1  gene as the common selected locus [Among the most severe environmental challenges to confront animals is the low oxygen availability of high-altitude regions such as the Tibetan Plateau, which is at an elevation above 5,000 m. The partial pressure of oxygen at 5,000 m is only approximately 50 % of the value at sea-level, and the resultant hypoxia imposes severe constraints on aerobic metabolism and leads to high-altitude illness –3. Thus ed locus –9, in otCashmere goat has the broadest altitudinal range of all herbivores in mainland China , as it iOur previous studies regarding the Chinese cashmere goat, based on markers from both mitochondrial DNA and microsatellites –27, showP: P value; R: Pearson’s correlation coefficient; RBG = 0.865, PBG < 2.20E-16; RRT = 0.933, PRT < 2.20E-16; RCDM = 0.903, PCDM < 2.20E-16; RNJ = 0.928, PNJ = 2.20E-16; RIM = 0.885, P IM < 2.20E-16; RLN = 0.872, PLN < 2.20E-16; Additional file We sequenced six pools of captured exomes from goats living at different altitudes Table  using a The proportion of polymorphic loci and the average heterozygosity ranged from 84.3 % to 94.0 % and from 22.4 % to 24.0 %, respectively, with the lowest heterozygosity value in LN and the highest heterozygosity in CDM Table , indicatFst for each identified SNP, and this estimation was termed the global Fst analysis. To evaluate the population diversity we calculated the average genetic differentiation coefficient (Fst), the average overall genetic diversity (Ht) and the average diversity within a population (Hs), which were 0.0531, 0.2116 and 0.2007, respectively. This calculation indicated that only 5.31 % of the total genetic diversity was partitioned among breeds and that most of the variability (94.69 %) was within populations (Hs = 0.2007).Because both natural selection and artificial selection have shaped the goat genomes and because genetic adaptations can occur due to many different variables, we first focused on the genetic differentiation in a global scenario to obtain a more comprehensive understanding of the selection patterns among all six populations. We estimated the global Fst, which was identical to that observed in our global Fst dataset (mean Fst = 0.0531). Generally, the simulated data were consistent with the observed data  had extreme Fst values in the observed data compared with the simulation  had Fst values greater than 0.1723, whereas the corresponding value in the simulated data were 0.5 % (n = 290). Thus, approximately 2-3 % of the loci in our exome re-sequencing data showed greater genetic differentiation than expected under a selectively neutral model. Therefore, a threshold of the top 2.5 % was used to search for the loci under selection, and 1,449 SNPs, which overlapped with 880 genes, were found in the global Fst analysis.To elucidate whether the observed genetic differentiation is primarily driven by drift or selection, a simulation test was conducted. The simulation was based on six subpopulations, each with an effective population size of 23,231 individuals who had been separated for 3,000 generations, and on the average simulated ata Fig. . More thion Fig. . Only 66P_FDR = 6.90E-06), regulation of molecular function  and multicellular organismal development  , response to cell communication involved in cardiac conduction , blood vessel development , and circulatory system process (P_FDR = 4.49E-02), which may be related to adaptation the local environment adaptation.Gene Ontology (GO) analysis of the 880 genes revealed 33 significantly enriched GO categories. Among these categories, the most significantly enriched GO categories were cell adhesion  and χ2 test (FDR-adjusted p < 0.01) as the high-lowland analysis, we obtained 1,134 outlier SNPs that were genetically differentiated between highland and lowland goat populations , cell adhesion , regulation of molecular function , regulation of multicellular organismal process  and cardiac muscle cell action potential involved in contraction  , intercellular cell adhesion molecule-1 (ICAM1), endothelin receptor type A (EDNRA), Yamaguchi sarcoma viral-related oncogene homolog 1 (YES1), and plakoglobin (JUP)  from the global SNP dataset only. Interestingly, categories that were associated with the cellular component organization and biological adhesion were still significantly enriched, but the categories related to cardiovascular system were not , are hypoxia-related genes according to a study by Zhang [EPAS1, EDNRA, SIRT1, RYR1, desmoglein 2 (DSG2), cardiomyopathy associated 5 (CMYA5), receptor-type tyrosine-protein phosphatase eta (PTPRJ), FUT1, heart development protein with EGF-like domains 1 (HEG1), tyrosine phosphatase receptor-type Z polypeptide 1 (PTPRZ1), PAS domain-containing serine/threonine kinase (PASK), sialic acid-binding Ig-like lectin 1 sialoadhesin (SIGLEC1), Niemann-Pick disease type C1 gene-like 1 (NPC1L1) and nestin (NES), were previously reported to be associated with the cardiovascular system through literature mining ; and 54, at the intergenic regions. Among these 235 coding SNPs, 86 non-synonymous SNPs were found in 73 genes, with one to four SNPs per gene. Among these 73 candidate genes, four genes, by Zhang  , which is a member of the protein tyrosine phosphatase (PTP) family and which is present in all hematopoietic lineages [PTPRJ gene; one in the 3’-UTR and five in the coding region. Of these five SNPs, four were non-synonymous, and three SNPs changed the physicochemical properties of the amino acid residues. However, multi-species alignment of the non-synonymous substitutions encoded in the PTPRJ genes showed that none of these four non-synonymous SNPs were present at evolutionary conserved sites. Surprisingly, the same type of SNPs occurred in the NPC1L1, PTPRZ1, NES, and PASK gene. For the EDNRA and FUT1, the non-synonymous substitutions were present at extremely evolutionary conserved sites; however, the variants in EDNRA and FUT1 did not present increased allele frequencies in different goat populations as the altitude increased. Therefore, no further validation was conducted on these genes  from other groups. This result is consistent with previous reports from our own studies and other studies based on microsatellite and mitochondrial data , 34. TheFst value for each identified SNP; the average Fst value was 0.0531, indicating that a small portion of the genetic diversity in the total population was attributed to differences among the populations. In addition, all six breeds had a high proportion of polymorphic loci and a similar average heterozygosity value, suggesting that a high level of variation has been maintained within these Chinese cashmere goat breeds. Our overall global Fst value was slightly lower than those of previous studies of Chinese cashmere goats  and of s . The reFst analysis  gene had the highest Fst value (0.41) among the goat breeds; the gene plays an important role in maintaining the normal structure and function of hair [P = 2.72E-2) may be relative to the differential cashmere production in these breeds  in our analysis. The GO categories previously reported to be associated with high-altitude adaptation in grey wolf [P < 2.26E-02). Further analysis was performed to determine whether the above loci presenting significant genetic differentiation, such as cardiovascular system-related genes, are associated with high-altitude adaptation.In addition, many genes with high n in pig , such asrey wolf , such asAdaptation to a hypoxic environment manifests as a large phenotypic shift observed in the Tibetan cashmere goat compared with the goats living at low altitudes. Previous studies have showen that the Tibetan cashmere goat has a higher adult Hb concentration, a heavier heart, lung and trachea, lower heart rate, and a high rate of arrhythmia –20. ThesFst analysis  on different continents show distinct patterns of adaptation to high-altitude hypoxia , and difEPAS1 gene is the most prominent candidate gene identified in this study. EPAS1, a member of the transcription factor family characterized by a basic helix-loop-helix (bHLH) and by a (Per-AhR-Arnt-Sim) PAS domain, plays a prominent role in process of adaptation to hypoxia and in the promotion of survival during hypoxic stress. EPAS1 represents a “hot spot” of high-altitude adaptation research since several previously published studies provided evidence of an association between a few SNPs near or in EPAS1 and the high-altitude adaption [EPAS1 polymorphisms in the native Tibetan people were found to be associated with their lower Hb concentrations [EPAS1 have been identified in the Tibetan dog, including a key amino acid mutation thought to be associated with the lower blood flow resistance [The adaption , 30, 56.sistance .EPAS1 is pleiotropic; therefore, other responses to hypoxia may be similarly affected. A few individuals living at low altitudes have been reported to have gain of function mutations in the HIF domains of EPAS1; these individuals exhibit excessive erythrocytosis and elevated Hb levels [However, b levels .EPAS1 in high-altitude goat populations; multi-species alignment indicated that this mutation is near to a well-defined domain (HIF) and is present at an evolutionary conserved site  were collected and tested individually at Tibet Autonomous Region People’s Hospital within 24 hours using a Sysmex XE-2100 Autoanalyzer . Hb and MCHC values were measured by blood tests. The 60 BG cashmere goats whose Hb levels were measured were the same goats used in the exome re-sequencing.The validation in the larger population included the following samples: Tibetan goat , CDM cashmere goat (17), NJ cashmere goat (10), IM cashmere goat (17), GF goat (9), LN cashmere goat (20), and CWG goat (10). All animal experiments in this study were fully approved by the Animal Care and Use Committee of the Ministry of Agriculture of the People’s Republic of China.For each DNA pool, a targeted exome library with an insert size of 150–200 bp was constructed by an exome capture strategy, using a GenCap custom exome enrichment kit . An Illumina HiSeq 2000 platform was used to generate paired-end 100-bp raw reads for each enriched library according to the manufacturer’s protocol. Quality filtering of the raw reads was performed using NGSQC Tookit v2.3.3 . For eacAfter removing low quality reads, we used BWA v0.7.10)  software0  softwaFst values across six populations were calculated using the method from Nei  [Fst values under a perfect neutral model, a simulation was conducted using the POWSIM software [s subpopulations of equal effective size (Ne) through random sampling of 2Ne genes. After drift for t generations for each subpopulation, the expected degree of divergence is Fst = 1 - (1- 1/2Ne)t [We estimated the allele frequency of each SNP site for each population by analyzing read count data for the sequencing pool. For each SNP site that had a minimum of 30 reads from each of the six sampled breeds, global , p.191) . To descsoftware . This pr 1/2Ne)t .Fst, 57,948 SNPs with the minimum coverage ≥ 30 for each of the six sampled populations were used to perform the simulation. Over all these SNPs, the average frequency of the most common allele was ~0.8619, and the average Fst was 0.0531.To reduce the sampling bias of Ne have little effect on Fst distribution [t to 3000 generations for the Chinese cashmere goat. According to the above-mentioned information and formula, we tested various combinations of Ne and t to reach Fst ≈ 0.0531 and simulated a relative large base population where a single biallelic locus segregated at a frequency of 0.8619. The base population was split into six subpopulations of effective size Ne = 23,231, and then these subpopulations were allowed to drift apart for t = 3,000 generations to arrive at an expected Fst = 0.0531 for neutral loci, with 1 allele sampled from each subpopulation. If we had used Ne = 23,231 and t = 3,000, the simulation would take a long time ; therefore, we changed the Ne and t values to 2,749 and 300, respectively. These values can produce almost an identical Fst distribution. After Fst was calculated and this process was repeated 57,948 times, we compared the distribution of simulated Fst values with the observed values. The largest simulated Fst value was Fst =0.2953, and the SNPs exhibiting Fst > 0.2953 were considered under directional selection.The objective evidence from archaeological excavations and historical records demonstrated that Tibetan cashmere goat was first recorded at Chamdo County in Tibet and has resided in the plateau area since 4,000-5,000 years ago , 67, 68.ribution . Moreoveribution , while fribution . TherefoFst analysis. Six goat populations were divided into two groups based on the altitude; the highland group (BG + RT) consisted of goat populations originating from Tibet, and the lowland group (CDM + NJ + IM + LN) consisted of goat populations originating from other locations. To retrieve candidate SNPs under selection in highland breeds, the di values, which combined the Fst value for each breed were calculated for each SNP as described in Akey et al. (2010). A general statistic di [χ2 test [2 test, as a second empirical outlier analysis, combining the highland population group (BG + RT) as the case population and treating the other lowland population group (CDM + NJ + IM + LN) as a control group. In total, 1,449 SNPs that were within the top 2.5 % of the empirical distribution were identified in each analysis. Due to a smaller effective population size than the autosomes, X chromosomal and unplaced regions were not included in the analysis. The complete workflow of the data analysis is presented in Additional file To define the candidate positions that have undergone directional selection during high-altitude adaption, 57,948 SNPs with minimum coverage ≥ 30 for each of the six populations were used in the high-lowland istic di  was base[χ2 test  was usedet al. 20. A generBased on the genetic distance matrix that was generated from the population allele frequency data of SNPs in the six populations, the neighbor-joining phylogenetic trees was constructed using POPTREE software . The phyThe primers for polymerase chain reaction (PCR) were designed for genotyping of the individual goat to measure the accuracy of the site frequency survey using a pooled strategy. After PCR amplification, Sanger sequencing technology was employed to verify the target SNPs in ten different populations.EPAS1 (chr11: 28306765) was genotyped on a larger population using traditional Sanger sequencing technology. All PCR primers used in this study are listed in Additional file The protein domains of EPAS1 were predicted by the Pfam web service . The fulP values, which indicate the significance of the overlap between various gene sets, were calculated. Only the terms with a FDR-adjusted P value < 0.05 were considered statistically significant and were listed. Genes with LOC symbols were not used in the enrichment analysis.GO categories and function association networks were defined with the STRING v9.1 program using standard parameters , and remAll raw sequencing data from this study have been deposited in the Sequence Read Archive (SRA) database at the NCBI under accession number: SRX1009748, SRX1013181, SRX1013324, SRX1013922, SRX1013947, and SRX1013952.


More than two decades of archaeological research at the site of Sagalassos, in southwest Turkey, resulted in the study of the former urban settlement in all its features. Originally settled in late Classical/early Hellenistic times, possibly from the later fifth century BCE onwards, the city of Sagalassos and its surrounding territory saw empires come and go. The Plague of Justinian in the sixth century CE, which is considered to have caused the death of up to a third of the population in Anatolia, and an earthquake in the seventh century CE, which is attested to have devastated many monuments in the city, may have severely affected the contemporary Sagalassos community. Human occupation continued, however, and Byzantine Sagalassos was eventually abandoned around 1200 CE. In order to investigate whether these historical events resulted in demographic changes across time, we compared the mitochondrial DNA variation of two population samples from Sagalassos (Roman and Middle Byzantine) and a modern sample from the nearby town of Ağlasun. Our analyses revealed no genetic discontinuity across two millennia in the region and Bayesian coalescence-based simulations indicated that a major population decline in the area coincided with the final abandonment of Sagalassos, rather than with the Plague of Justinian or the mentioned earthquake. The time-depth provided by the analysis of genetic variation in past populations is key to circumventing some of the limitations of interpreting present-day genetic patterns alone. In particular, the study of the maternally inherited mitochondrial DNA (mtDNA) has contributed to reveal past demographic scenarios previously undetected in ancient human communities.Within the Eurasian continent, most aDNA studies on human populations have addressed various levels of temporal and geographical resolution, from wide-scale prehistoric population dynamics ,2 to smaAcross multiple temporal layers of the history of our species, the importance of the Anatolian peninsula as a bridge between continents and cultures is unquestionable. The remains of the ancient city of sagalassos are situated in southwest Turkey, at an altitude of 1450–1600 m above sea level in the Taurus mountain range. Located 7 km north of the town of Ağlasun, in the Turkish province of Burdur , Sagalassos played a key role in the past of the wider region then known as Pisidia, as almost 30 years of archaeological research have revealed [The first indications of human presence in the Ağlasun Valley can be placed at approximately 10 000 BCE in Epi-Palaeolithic times. The study region, which forms part of the Turkish Lake District, contains a range of well-studied Late Neolithic and Bronze Age sites . The so-Pax Romana, a period of peace and reduced military engagement spanning more than two centuries from 25 BCE up to 180 CE, Sagalassos experienced one of its most flourishing periods. This is testified by an intense pottery production, large-scale exchange [During the Two main factors are considered to have contributed to the traditional scenario of late antique (demographic) decline and the overall restructuring of Sagalassos’ community. One factor is the first recorded outbreak of plague—the Plague of Justinian—which between 541 and 543 CE spread into Europe through the Mediterranean Basin , causingThe second factor is seismic activity in the area. A severe earthquake destroyed several monuments in the urban centre in the course of the first half of the seventh century CE . The comkastron was destroyed, and the local population is considered to have shifted towards Ağlasun. By that time, Ağlasun was occupied by the Seljuk Turks [Whereas the demographic trajectories of the following centuries remain quite elusive, at least based on the current archaeological and historical evidence, the final abandonment of Sagalassos can be dated to the early thirteenth century CE, when a fortified settlement or uk Turks  and is ahttp://www.tursaga.com/). Finally, we applied Bayesian coalescence-based approaches to identify the most plausible demographic scenarios supported by available DNA, historical and archaeological evidence.To better elucidate the demographic changes across time that affected the people living in Sagalassos and its territory, we have analysed the mtDNA variation in 24 individuals dated to Roman and Early Byzantine times, ranging from the early second up to the sixth century CE. The mtDNA sequences obtained were compared with a Middle Byzantine sample from Sagalassos previously studied  and with2.2.1Bone and teeth samples of 44 individuals dated to late Hellenistic (late first century BCE), Roman Imperial (first–third centuries CE), Late Roman (fourth–fifth centuries CE) and Early Byzantine times (sixth–seventh centuries CE), as determined by direct accelerator mass spectrometry radiocarbon dating, stratigraphic associations and contextual archaeological evidence, were available for aDNA analyses .Analyses were carried out in dedicated aDNA facilities of the Forensic Genetics and Molecular Archaeology Department of the University of Leuven (Belgium). The two hypervariable segments (HVS-I and HVS-II) of the mtDNA and eight diagnostic SNPs of the mtDNA coding region were amplified and sequenced in multiple independent experiments as described elsewhere . Further2.2rlequin v. 3.5 [FST genetic distances. The Slatkin’s linearized FST values were represented in a two-dimensional multidimensional scaling plot with Statistica v. 8 (Statsoft) and for Sagalassos and Ağlasun they were plotted on a geographical map with Surfer v. 6 (Golden Software) using the Kriging method. The relative frequencies of haplotypes from Sagalassos and Ağlasun (excluded CRS haplotypes) shared with the modern populations were estimated with Arlequin and displayed on a geographical map with Surfer. A median-joining network [etwork v. 4.6 (www.fluxus-engineering.com).To unravel the genetic affinities of two ancient population samples from Sagalassos (Roman and Middle Byzantine) and the modern sample from Ağlasun to present-day populations, we generated an in-house database with more than 11 000 concatenated HVS-1 and HVS-2 mtDNA sequences of contemporary populations from the literature . We used An v. 3.5  to compu network  of concaFST genetic distances analyses .To include a larger number of populations and ancient samples from the literature, a second database of more than 17 000 HVS-1 sequences was used for tatistica . Detailed information about the statistical methods and the databases is given in the electronic supplementary material.Haplogroup frequencies from more than 180 ancient and modern populations were used for a principal component analysis (PCA) and represented in a two-dimensional plot with S2.3imcoal software [In order to identify the major events that took place during the history of Sagalassos, we first built demographic models  involvinsoftware ,21, a prFurther details regarding the ABC validation, continuity tests and additional simulations accounting for potential maternal relationships in the Roman sample are fully described in the electronic supplementary material.3.3.1Authentic aDNA sequences were obtained for 22 Roman and two Early Byzantine individuals , which were pooled in one population sample addressed here as Roman. Haplogroup frequencies in Roman and Middle Byzantine Sagalassos and Ağlasun  show oveThe high diversity of mtDNA lineages observed in Sagalassos and Ağlasun is exhibited by the network . In several instances, haplotypes of the three samples  differ by one or two mutational steps.When compared with haplotypes of the database, Sagalassos shows a lower fraction of unique haplotypes  than Ağlasun (57%) and overall the haplotypes shared with present-day populations have a West Eurasian distribution .a) and the FST values indicate in particular higher affinity with populations from the Caucasus, Southwest Asia, the Eastern Mediterranean, Southern Italy and some countries of continental Europe . Northern Asian populations exhibited the largest values. A similar pattern of genetic affinities is observed in Middle Byzantine Sagalassos and in Ağlasun .figure 2A Southwest Asian affinity of Sagalassos and Ağlasun is also detected when contrasting genetic distances with a larger number of populations at the level of HVS-1 variation . Ancient Mesolithic, Neolithic and Bronze Age samples from Europe and Asia were included in the analysis and some from Early–Middle Neolithic central Europe [FST analysis, the haplogroup frequencies PCA  reveals affinity between ancient Sagalassos and Southwest Asian populations, and an even closer relationship with samples from Caucasus, Southern Italy  and the ancient samples of Early and Middle Neolithic central European farmers and Bronze Age Crete.Concordant with the 3.2a, the model with the highest support is the AM (91%), where the population contraction corresponding to the abandonment of the city of Sagalassos is very strong  reveal a clear Southwest Asian affinity, in particular with the Caucasus, Iraq, Iran, the Levant and the general Anatolian region. Genetic distances increase eastward across the Asian continent, and westward in Europe, though a genetic affinity was observed with continental Western Europe (France and Germany) and Southern Italy. Furthermore, the analyses reveal a common genetic thread along Southwest Asia, the Mediterranean basin  up to ancient central European farming communities, a pattern that may reflect the complex connections that took place in these areas across time since the Neolithic [The maternal genetic composition of Sagalassos in Roman times shows a typical West Eurasian pool, as previously observed in the Middle Byzantine sample . The muleolithic ,24.Roman times were certainly dynamic in the whole Mediterranean region. In that period, Sagalassos grew into the major regional centre of Pisidia. The city expanded and grounded its economic success in local agricultural and artisanal production . As a dyThe mtDNA pool of Sagalassos and Ağlasun remained homogeneous across time, in fact no significant differences in haplogroup frequencies were observed, except for macro-haplogroup M. This is corroborated by the network analysis, which showed close genealogical relationships between most of the mtDNA lineages in the three samples.In agreement with published data from modern Turkish populations , lineageMiddle Byzantine Sagalassos overall showed lower genetic distances with the modern populations as opposed to the Roman sample and Ağlasun . This co4.2Our results support population continuity until the present in the study region of Sagalassos since Roman times, suggesting that, even though migratory events may have taken place across the centuries (e.g. linked to the introduction of Asian lineages), no significant major changes that could not be explained by a relatively simple demographic model occurred in the mtDNA pool of the people inhabiting the area.There is very scarce direct evidence of human presence in the immediate surroundings of Sagalassos and the Ağlasun Valley before the origins of the human communities in the later fifth century BCE. However, in all demographic scenarios tested we set the BO-phase (approx. 1000 BCE) as the initial date of human occupation of the region in light of the strong increase in cereal cultivation, arboriculture and grazing grounds for livestock, which are indicative of a significant increase of human activities in the area .a).Our ABC analyses strongly support a scenario in which a population decline in the study region coincided with the abandonment of Sagalassos in the early thirteenth century CE, rather than with the Plague of Justinian or the earthquake in the seventh century CE a.b). Our simulations did not make it possible to discriminate with high support any of the three alternative scenarios investigated. However, cumulative posterior probabilities of the models with two contractions (71%) suggest that a decline coincident with the plague and/or the earthquake may have taken place. If it occurred, then it was moderate, with a reduction of 10–20% in effective population size in the general study region. Whether this contraction was due to an increased death rate or to migration out of the region remains to be investigated.We further explored this scenario in more depth by investigating whether the plague and/or the earthquake contributed to a reduction in population size in the region prior to the abandonment of the city. We also included a scenario of slight recovery in population size between the two contractions b. Our sihamam in Ağlasun in the 1230s CE [Most likely, either the plague and/or the earthquake in the seventh century CE determined a general loss of social complexity of Sagalassos’ community , which w1230s CE . We cannAs a final consideration, after accounting for the effective population size of mtDNA  and the effective size as approximately one-third of the census size, the results of our simulations indicate a wide discrepancy with the census size estimated through archaeological survey—up to 50 000 including the urban and rural area —prior to any possible demographic contraction. These results should be treated with caution, owing to possible underestimations in the methodology of the archaeological survey, and to the limited power of demographic reconstructions based on a single genetic locus. In addition, the effective population sizes estimated from genetic data can be difficult to relate to actual population sizes when populations are structured. In such cases, the estimated effective size will be influenced by the area over which gene flow occurs.The discrepancy observed might suggest that the mtDNA pool of people in the region was enriched across time by the introduction of novel lineages, thus indicating more complex scenarios and supporting the hypothesis of genetic signatures left by mobility into the area. The models tested are clearly not exhaustive of all possible demographic factors that may have taken place across time in the study region. Modelling the source and extent of gene flow in the study region of Sagalassos was beyond the scope of this study, but may become an interesting focus of future studies when additional aDNA data can be obtained across wider regions of Anatolia and the surrounding regions.5.In this study, we addressed questions regarding the demographic trajectories across the last two millennia of a human population at the very fine scale of one city—Sagalassos, in southwest Turkey—and its surrounding region. By comparing mtDNA variation in three period groups, Roman, Middle Byzantine and modern, and by simulating possible scenarios inferred by historical and archaeological evidence, our data suggest that concurrently with the abandonment of the city in the early thirteenth century CE, the population of the region may have been drastically reduced by almost 90%, most likely owing to migrations towards farther regions in southwest Anatolia. Furthermore, an earlier but milder contraction in population size may have taken place in the sixth–seventh centuries CE, either owing to the Plague of Justinian and/or an earthquake in the region. Whether this hypothetical contraction was due to increased death rate or migration out of the region remains unclear, and—together with the overall demographic scenario described here—needs to be better addressed by incorporating nuclear markers on a large genomic scale.


Rhodiola kirilowii, a perennial herb with its distribution ranging from the southeastern QTP and the Hengduan Mountains (HM) to adjacent northern China and central Asia, provides an excellent model to examine and disentangle the effect of both geological orogeny and climatic oscillation on the evolutionary history of species with such distribution patterns. We here conducted a phylogeographic study using sequences of two chloroplast fragments (trnL-F and trnS-G) and internal transcribed spacers in 29 populations of R. kirilowii. A total of 25 plastid haplotypes and 12 ITS ribotypes were found. Molecular clock estimation revealed deep divergence between the central Asian populations and other populations from the HM and northern China; this split occurred ca. 2.84 million year ago. The majority of populations from the mountains of northern China were dominated by a single haplotype or ribotype, while populations of the HM harbored both high genetic diversity and high haplotype diversity. This distribution pattern indicates that HM was either a diversification center or a refugium for R. kirilowii during the Quaternary climatic oscillations. The present distribution of this species on mountains in northern China may have resulted from a rapid glacial population expansion from the HM. This expansion was confirmed by the mismatch distribution analysis and negative Tajima's D and Fu's FS values, and was dated to ca. 168 thousand years ago. High genetic diversity and population differentiation in both plastid and ITS sequences were revealed; these imply restricted gene flow between populations. A distinct isolation-by-distance pattern was suggested by the Mantel test. Our results show that in old lineages, populations may harbour divergent genetic forms that are sufficient to maintain or even increase overall genetic diversity despite fragmentation and low within-population variation.The evolution and current distribution of the Sino-Tibetan flora have been greatly affected by historical geological events, such as the uplift of the Qinghai-Tibetan Plateau (QTP), and Quaternary climatic oscillations.  The evolution and genetic structure of extant species have been greatly affected by both geological history and climate oscillations Rhodiola kirilowii (Regel) Maxim., Incarvillea sinensis Lam., Caragana jubata  Poir., Hypecoum leptocarpum Hook. f. et Thoms.), some of which could have originated and expanded northeast from the Hengduan Mountains The Hengduan Mountains (HM), which are located at the southern and eastern edge of the QTP Habitat fragmentation could dramatically influence the demographic history of plant species Rhodiola L. (Crassulaceae) consists of about 70 species mainly found at high- elevation in cold regions of the Northern Hemisphere, with the QTP and adjacent area representing its distribution center Rhodiola and examined the biogeographic relationships between the QTP and other regions of the Northern Hemisphere. The results showed that the origin and diversification times of this group are largely correlated with the extensive uplifts of the Qinghai-Tibetan Plateau, and the ancestral area of Rhodiola is on the QTP, from where it dispersed to other regions of the Northern Hemisphere. Rhodiola kirilowii, one of the most widespread species of this genus, is distributed through the HM, northwest to the QTP platform and the Tianshan Mountains, and northeast to the plain of northern China R. kirilowii would be very helpful for exploring the floristic relationship between the QTP plus its neighboring area and the vast adjacent northeastern plain in northern China, and to reveal the consequences of natural habitat fragmentation. Our objectives included: (1) inferring the historical demography that could explain the current distribution pattern of R. kirilowii by examining the spatial pattern of plastid DNA (pDNA) and nrDNA ITS variation; (2) assessing the floristic relationship between the Hengduan Mountains and northern China; (3) revealing the consequences of natural habitat fragmentation on genetic diversity of R. kirilowii.R. kirilowii populations sampled were shown in No specific permits were required for the described locations in China because all researchers collecting the samples had introduction letters from College of Life Sciences, Peking University, Beijing. The field studies did not involve endangered or protected species. All locations of R. kirilowii during the summers of 2009 to 2012, with the exact GPS coordinates and altitude shown in R. kirilowii were sampled and leaves were dried with silica gel. In addition, four individuals of R. rosea from different populations were sampled as outgroups.Population sampling was conducted throughout the whole distribution range of trnL-F were c and f trnS-G were trnS and trnG DNA was extracted from silica-gel dried leaves with a Plant Genomic DNA Kit . The amplification primers used for ITS were ITS-1 and ITS-4 R. rosea were chosen as the outgroup for the phylogenetic analysis based on previous phylogeny studies trnL-F and trnS-G) were concatenated into a matrix. Chloroplast haplotypes and ITS ribotypes were determined from both indels and nucleotide substitutions using DnaSP v5 Four accessions from In order to detect genealogical relationships among sequences with shallow genetic divergences, we constructed pDNA and ITS haplotype networks with NETWORK ver. 4.2.0.1 χ2 distribution with n-2 degrees of freedom, where n is the number of taxa (haplotypes plus outgroups). A molecular clock could not be rejected for the ITS data . We then used BEAST v.1.7.5 R. kirilowii and its relatives were available, we used substitution rates to estimate divergence times. The substitution rate of nrITS in shrubs and herbs varies from 3.46×10−9 to 8.69×10−9 s s−1 yr−1−9, and SD of 1.590×10−9.To infer the divergence time between lineages, we used the ITS data set to conduct a dating analysis with the BEAST software h) and nucleotide diversity (π) for each population were calculated using DnaSP v5 HS), total gene diversity (TH) and between population divergence  were estimated by the program PERMUT (available at www.pierroton.inra.fr/genetics/labo/Software/) with 1000 permutations tests. A higher NST than GST indicates the presence of phylogeographic structure, which means that closely related haplotypes will be found more often in the same area than more divergent haplotypes FST) were calculated with Arlequin version 3.5 FST values and the geographical distances was performed using Arlequin ver. 3.5 Haplotype diversity (K) of groups of geographically adjacent populations that maximize the FCT value (differences among groups of populations). We determined the value of K by repeatedly running the program with K = 2–10, and choosing the one which gave the maximum FCT value. The amount of variation among populations within a region and within a population was calculated by the hierarchical analysis of molecular variance (AMOVA) framework carried out using Arlequin version 3.5 The spatial genetic pattern was examined by spatial analysis of molecular variance (SAMOVA) using SAMOVA v. 1.0 D and Fu's Fs to identify signatures of demographic expansion of each clade recovered based on the plastid data set D and Fs statistic should have large negative values as a result of an excess of rare new mutations. We also conducted a mismatch distribution analysis R. kirilowii. We pooled all haplotypes of each clade together without considering their frequencies because population structure has little effect on mismatch distribution SSD) between observed and expected mismatch distributions, and the raggedness index (HRag) of Harpending τ) to estimate time (in generations) since expansion with the equation t = τ/2uu was calculated as u = µ×k×g, where µ is the number of substitutions per site per year (s s−1 yr−1), k is the average sequence length in the present study and g is the generation time in years (age when first reproduction occurs). In the present study, our value for k was 1549 bp. The pDNA substitution rates for most angiosperm species have been estimated to be in the range 1−3×10−9 substitutions per site per year (s s−1 yr−1) −9 s s−1 yr−1 for the plastid data set g .We calculated Tajima's trnL-F, and 609 bp for trnS-G. After combining the two data sets, we detected 25 haplotypes (H1–H25) from the 306 sampled individuals of R. kirilowii, determined by 36 single nucleotide polymorphic sites and three indels. The length of these haplotypes varied between 1424 and 1491 bp. Eight haplotypes were unique to single populations, while the rest were shared by different populations  was much lower than total gene diversity (HT) , but it varied across populations, ranging from 0.000 to 0.0016, with the LHS population having the highest π value (HS) was much lower than total gene diversity (HT)  were recovered (R1–R12), three of which occurred in not more than one population. These ribotypes were determined by a total of 30 substitutions, and their length was in the range 610–618 bp. Among the ribotypes determined, only three occurred in a single population, while the other nine were shared by at least two populations . The mos h value . At the  π value . Like thR. kirilowii was well supported. The 25 haplotypes clustered into three multiple-haplotype clades    and four, and C)  depictedFor the ITS data, phylogenetic relationships reconstructed by Bayesian and MP methods were consistent . All 12 R. kirilowii was particularly high, with GST values of 0.599 and 0.822 for pDNA and ITS data, respectively (NST than GST was detected by the permutation tests examining the ITS data (P<0.01), indicating a strong phylogeographic structure in R. kirilowii. In contrast, the permutation tests of pDNA showed that NST was not significantly higher than GST  and lineage divergence. Thus, we divided the 29 populations collected into three geographic groups as mentioned above. Pairwise FST between the three geographic regions varied from 0.5387 to 0.9163, and from 0.1937 to 0.5768 for pDNA and ITS data, respectively .Population differentiation of ectively . A signihan GST . In the o to ten , making ectively , suggestSSD) and the raggedness index for Clade A were not significantly different from the expectation under the population expansion model  to the QTP area. However, after the recent intensive uplifts, the flora of the northern and western part of the QTP was replaced by alpine scrub grasslands and desert R. kirilowii was fragmented by the intensive uplift of the QTP in the late Pliocene. Pliocene uplift of the QTP has been suggested as a driving force for intraspecific divergence of several alpine species in this region Taxus wallichiana Zucc. Picea likiangensis (Franch.) E. Pritz. Both our phylogenetic and NETWORK analysis showed that the central Asia populations (Population WLMQ and NS) are clearly divergent from the other populations of Pliocene , during The HM area has long been recognized not only as an important species center for Tertiary elements, but also a principal glacial refugium for many plants in East Asia R. kirilowii's distribution extending from the HM to the mountains of northern China, we have the chance to discuss this issue. Both our pDNA and ITS results show that most populations from northern China harbor only one haplotype or ribotype . A high total genetic diversity has also been documented in two previous phylogeographic studies involving two other congeneric species  S. H. Fu and R. dumulosa (Franch.) S. H. Fu; HT = 0.950 and 0.981, respectively) Pedicularis longiflora Rudolph (HT = 0.770) Aconitum gymnandrum Maxim. (HT = 0.739) Incarvillea sinensis Lam. (HT = 0.677) Taxus wallichiana Zucc. (HT = 0.809) R. kirilowii, the hypothesis that genetic variability decreased as a result of population fragmentation was not supported. Although numerous studies have revealed that habitat fragmentation can reduce genetic diversity due to restricted gene flow, inbreeding and genetic drift in isolated small populations Rhodiola kirilowii grows exclusively at forest margins, on grassy slopes near mountain peaks at an elevation of 2,000 to 5,600 m, effectively occupying ‘ecological islands’ which are naturally fragmented. These conditions are found especially in the HM area, where severe uplift created high mountains and deeps valleys, providing great opportunities for species like R. krilowii to undergo allopatric divergence. A high genetic differentiation among populations could be the result of spatially isolated populations with restricted gene flow FST and geographic distance , indicating a significant and strong isolation-by-distance pattern.For both pDNA and ITS data sets, we detected a significant high total genetic diversity  from the SAMOVA results. (a) results based on pDNA haplotypes; (b) results based on ITS ribotypes.(TIF)Click here for additional data file.Figure S2Mismatch distribution analyses of the three multiple-haplotype pDNA clades. The histogram of observed mismatch frequencies and the best-fit curve of the sudden expansion model was shown.(TIF)Click here for additional data file.Table S1Locations of populations of R. kirilowii sampled, sample sizes (n), frequencies of cpDNA haplotypes and ITS sequences per population, and estimates of haplotype diversity and nucleotide diversity for chlorotypes and ribotypes within populations.(DOCX)Click here for additional data file.Table S2Haplotype composition of 29 sampled populations of Rhodiola kirilowii.(DOCX)Click here for additional data file.Table S3Ribotypes composition of 29 sampled populations of Rhodiola kirilowii.(DOCX)Click here for additional data file.


Lednia tumana (Ricker) of Montana, USA, a cold-water obligate species. We sequenced 1530 bp of mtDNA from 116 L. tumana individuals representing “historic” (>10 yr old) and 2010 populations. The dominant haplotype was common in both time periods, while the second-most-common haplotype was found only in historic samples, having been lost in the interim. The 2010 populations also showed reduced gene and nucleotide diversity and increased genetic isolation. We found lower genetic diversity in L. tumana compared to two other North American stonefly species, Amphinemura linda (Ricker) and Pteronarcys californica Newport. Our results imply small effective sizes, increased fragmentation, limited gene flow, and loss of genetic variation among contemporary L. tumana populations, which can lead to reduced adaptive capacity and increased extinction risk. This study reinforces concerns that ongoing glacier loss threatens the persistence of L. tumana, and provides baseline data and analysis of how future environmental change could impact populations of similar organisms.Much remains unknown about the genetic status and population connectivity of high-elevation and high-latitude freshwater invertebrates, which often persist near snow and ice masses that are disappearing due to climate change. Here we report on the conservation genetics of the meltwater stonefly  Climate change is rapidly altering physical and biological systems worldwide , 2. WarmLednia contains four species, all of which are known from high-elevation regions of the western United States [Lednia tumana (Plecoptera: Nemouridae) is endemic to the Waterton-Glacier International Peace Park region of Montana, Alberta, and British Columbia and is an obligate denizen of cold meltwater streams immediately below glaciers, permanent snowfields, and alpine springs [L. tumana is likely tied to that of the region’s rapidly disappearing glaciers [L. tumana by over 80% (from ~23.2 to ~4.5 km2) of its predicted current range [The stonefly genus d States , 15. Led springs . Recent nt range .L. tumana in the foreseeable future due to significant range contraction. As such, the species is currently a candidate for listing under the United States Endangered Species Act [L. tumana resembles Zapada glacier (Baumann and Gaufin), which is also in family Nemouridae, and occupies a similar range and habitats. Our recent work has found this species to be experiencing range contraction in the face of melting glaciers, with further population reductions predicted [These major habitat reductions imply a greatly increased probability of extinction of cies Act . In manyredicted .L. tumana, and test the hypothesis that subdivision is increasing due to fragmentation over time. We chose to analyze mtDNA because it is easy to extract from historical samples , and because only mtDNA data exist for comparisons to related species. To help qualitatively test patterns of diversity and subdivision, we compare results to published mtDNA data from two other stonefly species, one from the same family and found in a high-latitude environment, and another from the same geographic region. Our data help clarify species and population status, inform endangered species listing considerations, and offer valuable insight into the potential impacts of climate warming on the genetic diversity of cold-climate freshwater invertebrates.Here, we use analyses of mitochondrial DNA (mtDNA) sequences to explore population genetic structure and connectivity among 10 populations of L. tumana from Glacier National Park (GNP) and its environs in 2010 . We PCR-amplified two protein-coding mitochondrial genes, COI and CytB. COI primers were modified for this study from LCO1490 and HCO2198 of Folmer et al. : F–TGTCCst and 3rd codon positions, suggesting strong stabilizing selection on a functional gene. Sequences have been deposited in GenBank (accession numbers KX212679-KX212864).We explored for the presence of nuclear mitochondrial pseudogenes (numts) in our data using the recommendations from Fst values were calculated using Arlequin 3.5 [Molecular diversity measures including gene and nucleotide diversity and pairwise population quin 3.5 . Gene diquin 3.5 .st estimates and straight-line geographic distances. We ran the Mantel test for the seven 2010 populations only, using 100,000 permutations. We implemented AMOVA in Arlequin with each of the ten aggregated populations considered to be a group.Arlequin analyses were run twice to test both fine- and coarse-scale genetic differentiation. First, we performed a within-stream analysis that kept all sampling sites separate to see if substantial fine-scale local (<2 km) genetic differentiation could be identified. For example, in Lunch Creek, we sampled four within-stream locations separated by 0.5 km or less. We calculated genetic diversity within and differentiation between each of these locations separately. Second, we performed within-basin analyses by combining sampling sites that were near each other (<2 km) as long as they were not separated by potential geographic barriers such as mountain ridges. This reduced the number of populations from 16 to 10 . We alsoPteronarcys californica Newport (Pteronarcyidae) collected across the western United States, including mountainous regions [Amphinemura linda (Nemouridae) collected near Churchill, Manitoba, Canada [P. californica dataset included 219 individuals from 27 populations, with sample sizes ranging from five to 10 individuals per population. Populations were separated from each other by between 16 and 978 km. The A. linda dataset included 179 individuals from three populations, with sample sizes ranging from three to 119 individuals. Populations were separated from each other by between 0.7 and 10.5 km.For qualitative comparisons of mtDNA diversity and subdivision with other species, we analyzed two previously published Plecoptera datasets using the same computational methods, including CytB data from  regions  and COI , Canada . The P. L. tumana, two from A. linda, and 23 from P. californica. We used SMOGD [ST, G’ST, and Dest for both modern and historic populations, and to calculate 95% confidence ranges for these estimates.In order to better compare allelic richness between populations and species, we used HP-Rare  to standed SMOGD  to estimL. tumana individuals, respectively. The concatenated dataset had a total length of 1530 bp and 16 haplotypes. One of these haplotypes (LtA) was shared by 88 individuals, including many 2010 and historic samples. Each of the other 15 haplotypes differed from this dominant haplotype by only one substitution , which were separated by only 130 m, was 0.026, and was not statistically significant (p>0.05), meaning there was no population structure within streams. These results justified our approach of combining sampling locales within a few km of each other along the same creek or basin .Pairwise L. tumana revealed an overall Fst of 0.32  was intermediate compared to that for the more widespread P. californica (0.35) and localized A. linda (-0.07), while the overall Fst of L. tumana was higher than the other two species . The mean Fst between historic L. tumana population samples and 2010 samples was 0.23.AMOVA analysis of  of 0.32 , with 19 species . The meaST, G’ST, and Dest for modern and historic populations revealed nearly complete overlap between these intervals, failing to reject the hypothesis that by these metrics, diversity has remained constant over time (The Mantel test was highly significant (p = 0.01), with a correlation coefficient between the matrices of 0.91. The SMOGD calculations of 95% confidence intervals of Gver time .Lednia tumana has a restricted geographic and environmental range, being found in isolated sky-island populations in one of the most mountainous areas of North America, the Crown of the Continent Ecosystem [The results of our population genetic analyses are consistent with those found in a small but growing body of literature addressing genetic diversity and divergence in alpine-adapted aquatic invertebrates threatened by climate change. Such studies suggest that the loss of alpine glaciers is causing significant declines in both taxonomic  and genecosystem , 30. ThiL. tumana populations bear the signature of recent declines in genetic diversity. Mean nucleotide and gene diversity for historic samples were higher than those for the 2010 samples . Furthermore, in spite of extensive sampling, we found no LtB haplotypes in 2010 populations, including many that were within 3 km of historic sampling sites where LtB was found. Additionally, historic populations had higher mean allelic richness than 2010 populations (2.5 vs. 2.0). This apparent loss of genetic diversity in L. tumana is consistent with small effective population sizes, which can lead to reduced fitness and evolutionary potential and increased genetic drift and extinction risk [Extant ion risk –33.L. tumana than in either of the comparison species. Mean gene and nucleotide diversity estimates were two to five times lower (L. tumana (2.2) was much lower than in P. californica (3.6) and A. linda  is generally considered to be a more variable locus than COI [Both metrics of genetic diversity were lower in es lower . Likewisnda 6.1; . Becausethan COI .L. tumana over our 13-year study period. The mean Fst value between historic L. tumana populations and modern populations was 0.23, a value influenced by the presence of the LtB allele in the historic samples and suggesting substantial genetic change over time. Similarly, mean Fst values between historic L. tumana populations was 0.14, while the mean Fst among 2010 populations was 0.18, suggesting a possible decrease in gene flow and/or local effective population size. Rapidly changing genetic composition, reduced gene flow, and loss of haplotypes suggest that L. tumana is vulnerable to extinction.As species’ ranges are reduced and fragmented, we expect to see increased population isolation as indicated by decreased gene flow . Our temBaetis alpinus across a range of stream “glacialities” on the Iberian Penninsula. They found significantly lower regional genetic diversity in recently deglaciated streams compared to those with “high glaciality” characteristics. Similar findings have emerged in other systems as well. For instance, reductions in genetic diversity have also been identified in an obligate alpine chipmunk  that has experienced documented, climate-change-induced range contraction in Yosemite National Park [Fragmentation, range contraction and rapid genetic change are increasingly problematic in many species . Our datnal Park .L. tumana. For example, the two well-sampled populations of A. linda, Ramsay Creek (n = 119) and Eastern Creek (n = 57), are separated by only 10.1 km, a value comparable to many of the L. tumana sites in this study. The terrain around Churchill has few dispersal barriers, being quite flat, and having a great deal of suitable intervening habitat. It is therefore not surprising that the Fst value between these A. linda populations was effectively zero, indicating panmixia. The mean pairwise Fst value from the combined analysis among 2010 L. tumana populations with similar separation (<17 km) was higher at 0.04, and may be the result of partial isolation, reduced gene flow, or a combination of the two, perhaps due to more mountainous terrain. This higher mean Fst also suggests potentially lower gene flow and higher fragmentation among modern L. tumana populations compared to A. linda.Our interspecific comparisons of stonefly species also suggest reduced gene flow and higher fragmentation in P. californica were widely distributed across the western US, but nine were found within 50 km of each other in mountainous regions, conditions similar to those of our study. The mean Fst of these populations was 0.12, which was lower than the mean Fst value of 0.18 for all modern L. tumana populations (including the Grant Glacier basin population that is roughly 40 km south of the sampling core of this study). This suggests that genetic differentiation in L. tumana may be influenced by more than just terrain. These additional influences may include fragmentation associated with ice and snow loss and differing species vagility. Furthermore, although we only included three individuals from the Grant Glacier population, they carried two alleles not seen elsewhere, suggesting that over such scales L. tumana may be genetically divergent or diverging rapidly.The analyzed populations of L. tumana, which is indicative of low connectivity among populations. This suggests that the likelihood of recolonization and genetic rescue as a means to mitigate climate-change-induced threats is relatively limited in L. tumana. Our genetic data are also consistent with previous studies indicating that L. tumana will likely continue to experience increased fragmentation and isolation, and decreased population size, due to the ongoing loss of suitable cold water alpine streams driven by climate warming [Zapada glacier, a nemourid stonefly historically found throughout GNP in similar habitats to L. tumana. This species has experienced a dramatic reduction in its occupied range in recent years [Our data join a small but growing body of data from studies seeking to understand the genetic effects of climate-change-induced glacier and snow loss on rare alpine macroinvertebrates. We found relatively low diversity within populations and high differentiation between populations of  warming . Such prnt years .L. tumana , and regional climate change are relevant to current proposals to list L. tumana as endangered under the U.S. Endangered Species Act. If listing occurs, L. tumana could be the first invertebrate species listed as threatened or endangered due to climate change, and likely represents a guild of species facing similar threats in alpine headwaters worldwide.The link between our genetic data, the fundamental niche of