Saltonia incerta, once thought to be extinct, to determine the role of past climatological events in shaping the structure of the species. This species is restricted to salt crusts of intermittent or dry lakes, streams or rivers in the desert southwest, a region that was much wetter during glacial periods. We examine the distribution and genetic variability of populations to test whether there is recent dispersal throughout the range of the species. Analyses of mitochondrial and nuclear DNA indicate significant population structure, with one major clade comprising New Mexico localities and one comprising California-northern Baja California localities. Finer-scale structure is evident within the California clade, although not all of the subclades are reciprocally monophyletic. However, isolation with migration analysis suggests that migration is very low to non-existent. These results extend the known distribution of Saltonia, provide genetic evidence of strong isolation among localities within drainage basins and between drainage basins and provide a mechanistic understanding of population connectivity after the aridification of the American southwest. The implication is that although the species' distribution has been fragmented, populations have persisted throughout this area, suggesting that desert salt flats may have served as refugia for at least some terrestrial species.The deserts of southwestern North America have undergone dramatic changes over their recent geological history including large changes in size and connectivity during the Pleistocene glaciopluvial cycles. This study examines the population history of the rare spider  Throughout the Quaternary period, the entire ecosystem of southwestern North America changed dramatically in response to glacial climate change, volcanism and tectonic activity. One of the most extreme examples of change that occurred in this region is the transformation of wetter woodland and scrub habitats to some of the driest deserts on earth, including the iconic Death Valley. These geological and climatic shifts altered the distributional ranges of the region's biota Grismer . Today, In addition to overall changes in the environment, the climatic shifts have also altered corridors that previously connected populations across the landscape. For example, during the glaciopluvial periods of the Pleistocene, there were several large lakes in the Southwest that were periodically connected to each other by paleorivers or streams Fig.. Now, thSaltonia incerta Banks (Araneae: Dictynidae)  Fig.. This spdae) Fig.. Not surdae) Fig.. The typdae) Fig.. Thus, tdae) Fig.. HoweverThe aim of this study is to determine whether there is gene flow between populations of this species and how this informs our understanding of the evolutionary history of the species in the context of the hydrographic history of the desert Southwest. We focus on the distribution and population structure of the spiders from the salt crust habitats of dry lakebeds and shores  contained within six drainages  in southwestern North America Fig., Table1.Complete isolation (2) Dispersal during intermittent flooding, and (3) Aerial dispersal. The complete isolation scenario is based on the premise that Saltonia is dispersal limited, but suitable habitats were more extensive during wetter times. As desertification occurred, the spiders retreated to areas where moisture persisted, such as underneath the salt crust. This scenario would lead to high genetic drift and divergence among populations in isolated drainage basins and between salt flats within drainage basins. At the local scale we do not expect structure within individual salt flats because of gene flow within contiguous habitat. The second scenario, dispersal during intermittent flooding, assumes that the spiders would disperse rarely (during flooding) and passively along waterways during wetter times. If this is true, population structure between drainage basins will be higher than within a basin because salt flats within a single drainage basin are more likely to become connected during an episode of flooding than salt flats from different drainage basins. Also, a pattern of isolation by distance might be expected because the spiders may not be able to travel far during the short periods of time the landscape is flooded. Finally, the aerial dispersal scenario postulates that gene flow occurs between both lake basins and drainage basins via aerial movements  without the need for corridors. The expectation in this scenario is low genetic structure  both within and between basins, although prevailing winds and mountain barriers may lead to some directionality of movement.Three scenarios were examined regarding how populations may be structured following fragmentation caused by desertification across the entire region of study including (1) Amargosa River drainage, where specimens were collected in two localities, one near the town of Shoshone on ancient Lake Tecopa and two in Death Valley. (2) The Owens River drainage – China Lake. (3) The Mojave River drainage – Soda Dry Lake. (4) The Bristol drainage – four geographically proximate dry lakes – Bristol, Cadiz, Danby and Dale – which are included in a single drainage, although it is unclear whether they have ever been connected to each other or to lakes in other basins. Some evidence indicates that Bristol Lake was connected to Cadiz Lake  three sites on ancient Lake Otero in New Mexico – two in the White Sands Missile Range (WSMR) and one site from Lake Lucero in White Sands National Monument. Lake Otero is in the Tularosa Basin, into which the ancestral Rio Grande once drained, although the basin now only drains the surrounding mountains  basins (1–4 sites sampled per drainage) that fit the macro- and micro-habitat constraints of the species. These habitats all contain moderate to thick salt crusts and are in the arid Southwest of North America. Of 20 localities examined, the spiders were found at 15 localities in six drainages as follows  (Desidae) and Argyroneta aquatica (Clerck) (Cybaeidae). Outgroup selection was based on the placement of Saltonia incerta in a higher level molecular phylogenetic study of related spiders ; Hilden, Germany ) kit following the manufacturer's protocol. Because these populations were likely separated relatively recently, the fast-evolving cytochrome oxidase 1 gene (CO1) was chosen because previous studies  PCR kit buffer ‘F’ and the primers ˜1718 salt  and 2776 spiderA  from 83 individuals of PCR products were purified using Qiagen QIAquick (QIAGEN Inc.) spin columns or Exosap-IT (USB Industries) following the manufacturers' protocols and sequenced in both directions using the same primers that were used for PCR on an ABI 3730 capillary sequencer. DNA sequences were edited using Sequencher v4.6b2  and aligned by eye in MacClade 4.0  and hierarchical analyses of molecular variance , among populations within groups (FSC) and among individuals within populations (FST). Several distance methods were used , and the results were compared. Significance was tested using permutations of the data set described by Excoffier et al.  major clades-a New Mexico (NM) clade and a California/Northern Mexico (CA) clade. These clades are also supported in the analysis of H3, although H3 provided no within-clade resolution. The 50% majority rule consensus phylogram from the combined analysis is shown in Fig.A total of 41 unique CO1 haplotypes from 83 individuals plus two outgroup taxa were found. There are several subclades within the CA clade, indicating substantial population structure, although only four of them have high Bayesian posterior probability support. Only three localities  are monophyletic, and only one of these (Tecopa) is well supported, whereas a single mitochondrial haplotype is found in four geographically widespread salt flats  from two drainages (the Colorado River drainage and the Bristol drainage). Haplotype networks were constructed using TCS  ranged from zero at nearby sites to near fixation among basins within California (Tables S1 and S2). Large statistically significant differences in FST are evident between California and New Mexico sites. Although FST is large for all comparisons with China Lake, the values are not significant because of only a single sample from this site. The AMOVA indicates significant genetic variation at all levels, including among basins, salt flats within basins and individuals within salt flats  complete isolation, (2) dispersal during intermittent flooding and (3) aerial dispersal via ballooning. The data revealed significant genetic structure across multiple spatial scales and suggest that population structure may have been shaped by fragmentation with isolation following the drying of the southwest.Genetic data were collected from across the range of the spider FST values can be large within basins, for example ranging from 0.16 to 0.75 within the Bristol drainage.The results of the AMOVA reveal genetic structure both within and between drainage basins. The gene tree reveals shared polymorphism between some drainage basins, and the results of the IM analysis indicate that migration is uncommon, so the shared polymorphism could be because of incomplete sorting rather than gene flow. The high levels of genetic structure suggest that ballooning does not play a major role in these spiders' dispersal, and this is consistent with isolation caused by large areas of unsuitable habitat separating desert salt flats: If the spiders were dispersing aerially, low genetic structure and directionality of movement because of prevailing wind patterns would be expected; however, genetic structure is high, and the minimal value of estimated gene flow from Bristol drainage to the Colorado drainage is not sufficiently high to be consistent with directional aerial dispersal. The data also favor complete isolation over dispersal during intermittent flooding because we found high genetic divergence among populations from isolated drainage basins and salt flats, but generally low population genetic structure within individual salt flats. If the spiders dispersed along waterways during rare times of flooding, we would expect that genetic structure within drainage basins would be less than between, which, according to the data shown here, is not always the case. Pairwise Although most lineages within the California populations are not strongly supported by Bayesian posterior probabilities, the pattern of genetic divergence compliments natural geographical groupings. The Death Valley samples form a clade nested within the Soda Dry lake samples. This result is consistent with a historic connection that has been hypothesized to have linked Death Valley's ancient Lake Manly and Soda Dry Lake 186–120 kya Hooke . SimilarSaltonia to disperse more effectively across the region.In Laguna Salada in northern Baja California and El Doctór in Sonora (both within the Colorado drainage basin), in which admixture of haplotypes is evident, the pattern could be attributed to historical gene flow. During the last few thousand years, the Colorado River has flowed into the Salton basin instead of directly into the gulf Howard . This woSaltonia phylogram  The IM results do not support high migration rates. (2) This species has never been observed to balloon, and it resides under the salt crust with few elevated habitat features to ascend to initiate the activity. Moreover, most of the salt flats are in valleys surrounded by mountains, which might make it more difficult for spiders to disperse aerially. Additionally, ballooning might be risky for spiders residing in such a specialized habitat, because the chance of finding salt flats subsequent to ballooning is unlikely. (3) The direction of the prevailing winds at any one site is largely inconsistent with the molecular results.There are three reasons to consider that ballooning is an unlikely or uncommon means of dispersal for One way to examine the feasibility of ballooning without direct observation is to examine wind patterns, and whereas wind regime data from particular localities are not always available, most of the salt flats are associated with dunes and sand sheets, which can be used as a proxy for determining local wind pattern. In New Mexico, the dunes around Lake Lucero appear to be driven by winds from the west to southwest Smith , which iPrevailing winds in the Mojave Desert are typically from the west and the south , but low structure within individual salt flats because gene flow within salt flats is expected. These patterns are evident in the data. The exception to this pattern is in the Tularosa Basin in New Mexico, where proximate samples are genetically quite divergent, more so than on any of the salt flats to the west. However, rather than oppose the isolation scenario, the data from the New Mexico localities actually support it. The ancestral Rio Grande spilled into the Tularosa Basin via Fillmore Pass from the Pliocene to the Pleistocene  that rest on the assumption that waterways are a limiting factor for the spiders, we might expect them to show patterns similar to these aquatic organisms.To draw broader conclusions from the patterns in this dataset, the results can be considered in the context of other taxa inhabiting the same region and limited to similar habitats; in particular, the aquatic taxa, springsnails (Hershler and Sada Tryonia) from the Amargosa drainage are not monophyletic (i.e.-do not have a single geographic origin)  show a greater degree of genetic isolation than springsnails.Springsnails are dispersal limited, with moisture being the limiting factor. Therefore, little or no gene flow between localities is expected. Although some studies have shown that the snails may disperse via waterfowl . This could be because the snails, fish and beetles have diverged over longer periods and have had more time to accumulate differences or possibly because rates of morphological evolution are higher in these groups.Another terrestrial arthropod that justifies further study and occurs in the same salt flat habitats as Chandler , 1984. TChandler . InteresHabronattus pugillis on the basis that, as in Saltonia, closely related haplotypes were geographically separated.The patterns documented here are somewhat unusual in that most phylogeographic studies of non-ballooning spiders have found reciprocally monophyletic populations in geographically isolated sites . Other localities are used for salt mining  via evaporation. Whether or not this can harm populations is unknown, although typically only small portions of these dry lake beds are used for this purpose . Although mandates forbid the driving of cars and OHVs across the surface of the lake beds, violations are frequent and detrimental effects of traffic across sensitive habitat has been demonstrated in dune ecosystems (Van Dam and Van Dam High levels of divergence suggest that populations are isolated and should be managed at the level of drainage basin. Because of the small number of populations in each basin, the potential impacts from continued habitat loss could be detrimental to Saltonia incerta is much more broadly distributed in salt flat habitats in southwestern North America than previously thought. Based on genetic data, we reject the hypotheses that the population structure of the spider is dictated by dispersal via intermittent waterways or by aerial ballooning. Instead, the data support a model of fragmentation followed by isolation of a formerly more widespread species. The geological evidence also argues for a once broader distribution of the spider because there is little congruence in the patterns of diversification of Saltonia with known paleoriver and paleodrainage data. Our results contrast with those of others who have examined populations and species that are restricted to paleoriver drainages, although most other studies have examined aquatic taxa in which gene flow is necessarily dictated by aquatic connections. Wetter conditions in the Southwest during the Pleistocene most likely contributed to a larger former distribution, whereas the subsequent desertification left populations restricted to salt flat habitats. Accordingly, the data are consistent with salt flats serving as refuges for relict populations from previous climatic periods, a finding that has been proposed for mesic oases in desert habitats (e.g., Grismer et al. This study shows that


Macaca fascicularis fascicularis) populations distributed in Peninsular Malaysia in relation to other regions remain unknown. The aim of this study was to reveal the phylogeography and population genetics of Peninsular Malaysia's M. f. fascicularis based on the D-loop region of mitochondrial DNA. Sixty-five haplotypes were detected in all populations, with only Vietnam and Cambodia sharing four haplotypes. The minimum-spanning network projected a distant relationship between Peninsular Malaysian and insular populations. Genetic differentiation  results suggested that the gene flow among Peninsular Malaysian and the other populations is very low. Phylogenetic tree reconstructions indicated a monophyletic clade of Malaysia's population with continental populations . The results demonstrate that Peninsular Malaysia's M. f. fascicularis belonged to Indochinese populations as opposed to the previously claimed Sundaic populations. M. f. fascicularis groups are estimated to have colonized Peninsular Malaysia ~0.47 million years ago (MYA) directly from Indochina through seaways, by means of natural sea rafting, or through terrestrial radiation during continental shelf emersion. Here, the Isthmus of Kra played a central part as biogeographical barriers that then separated it from the remaining continental populations.The phylogenetic relationships of long-tailed macaque ( Macaca fascicularis [ kera in Malaysia [ M. fascicularis is probably the most successful nonhuman primate in Southeast Asia; long-tailed macaques are distributed in Malaysia, Brunei, Bangladesh, Cambodia, Nicobar Islands, Indonesia, Laos, Myanmar, the Philippines, Singapore, Thailand, Timor-Leste, and Vietnam [ M. fascicularis enables it to naturally inhabit a wide range of habitat, namely, primary and secondary forest, riverine, swamps, coastal areas, and mangrove forest from sea level up to an elevation of 2000 m [icularis , commonlMalaysia . M. fasc Vietnam . In addif 2000 m , 5. Furtf 2000 m . M. fascicularis [ M. fascicularis that are presently recognized based on their morphological characteristics are as follows ( M. f. atriceps (Kloss 1919), M. f. aurea (Geoffroy 1831), M. f. condorensis (Kloss 1926), M. f. fascicularis (Raffles 1821), M. f. fusca (Miller 1903), M. f. karimondjawae (Sody 1949), M. f. lasiae (Lyon 1916), M. f. philippinensis (Geoffroy 1843), M. f. tua (Kellog 1944), and M. f. umbrosa (Miller 1902) [ M. f. fascicularis [ M. f. fascicularis is found on the mainland peninsula and surrounding islands, such as Langkawi Island. Furthermore, M. fascicularis is at the center of human-wildlife conflict in this region, especially in secondary forest and palm oil plantations neighboring human settlements [Long-tailed macaques have been classified into as many as 50 subspecies and even several different species , but theicularis –8. The 1 follows : M. f. aKloss 192, M. f. f M. fascicularis using various molecular data such as allozymes [ M. fascicularis in Southeast Asia, but whether Peninsular Malaysia's M. fascicularis belong to continental or insular groups remains unknown. Roos et al. [ M. fascicularis cannot simply be defined as a continental group as in [Previous studies have extensively examined the molecular phylogeny and biogeography ofllozymes , mitochollozymes –20, and llozymes , 12, 14.llozymes  successfs et al.  proposeds et al. . Thus, Pup as in , becauseIn mammals, mtDNA is inherited as a haploid from the mother . This gi M. fascicularis in relation to other countries remain unknown. This may hold the key to revealing the biogeographic and radiation history of this species in Southeast Asia. Thus, the objectives of this study were to analyze the phylogeography and population genetics of Peninsular Malaysia's M. f. fascicularis through comparison with the available data on M. f. fascicularis from other countries using the D-loop region of mtDNA.Despite extensive studies on the molecular phylogeny of long-tailed macaques, the molecular data and phylogenetic position of Peninsular Malaysia's M. f. fascicularis were used in this study  following the manufacturer's protocol.A total of 23 fecal genetic samples ofis study . These wis study , while wicularis , 32–34.  M. f. fascicularis, we used representatives of all M. f. fascicularis sequences available in GenBank for the mtDNA control region (CR). There were 253 available sequences. Two were from Java [ M. f. fascicularis were randomly selected from these 253 sequences  across seven countries , extreme speed (extension times of 15 s/kb or less), and a very high yield in a reduced length of time. We designed our own primer for the PCR reactions in order to maximize the extent of the D-loop sequence and to avoid amplifying Numts. The primers used were LATIFF1638_F (5′-ACAGTCCTAGTATTAACCTGC-3′) and LATIFF1689_R (5′-CAAGGGGTGTTTAGTGAAGT-3′). The parameters for the PCR reaction were as follows: initial denaturation for 10 s at 98°C, followed by 30 cycles of denaturation for 1 s at 98°C, annealing for 30 s at 52°C, extension for 15 s at 72°C, and a final extension stage for 1 min at 72°C. Vivantis G-F1 PCR Clean-up Kits were used to purify the PCR product, and the samples were subsequently sent to 1st Base Sdn Bhd  for sequencing purposes.A 1,100 bp fragment of mtDNA D-loop was amplified through a polymerase chain reaction (PCR) using a Mastercycler Nexus . PCR reactions were generated using the Phusion Flash High-Fidelity PCR Master Mix , which has high accuracy (proofreading DNA polymerase with a fidelity of 25Xπ).D-loop sequences obtained after the sequencing process were edited using Bioedit Sequence Alignment Editor. To ensure the targeted species locus sequences were obtained, the edited sequences were validated using sequence similarity searches (GenBank BLASTn). The MEGA 5 ClustalW multiple alignment algorithm was used to align all 102 sequences . The ali M. fascicularis were reconstructed by assuming that at any given site, two randomly drawn haplotypes were unlikely to have arisen from more than one mutational step [FST), and number of migrants per generation (Nm) estimated using [Population expansion events were inferred by employing mismatch distribution analysis , 40 usinnal step . Networknal step , populated using  were cal M. f. fascicularis in Southeast Asia was examined by employing Tajima's test of neutrality, D [D* and F* [D test compares the average number of pairwise nucleotide differences (k) between haplotypes in a sample (M) expected from the number of segregating sites (K). Fu and Li's D* and F* and Fu's Fs were used to test for deviation of sequence variation from evolutionary neutrality. Fu's Fs is based on the probability of the observed number of haplotypes occurring under conditions of neutrality, whereas Fu and Li's D* and F* compare estimates of theta based on mutations in internal and external branches of a genealogy.The demographic history ofality, D , Fu and * and F* , and Fu'* and F* . Tajima'Phylogenetic trees were constructed using three distinct criteria, namely, distance-based (neighbor-joining (NJ) tree), character-based (maximum parsimony (MP) tree), and Bayesian inference. Different software programs were used to construct the phylogenetic tree, namely, MEGA 5 for the NJ tree , PAUP 4.P) of 0.006136 was obtained across two different runs of MrBayes. The first 10% of the trees obtained in the analysis were discarded as burn-in , and a majority-rule consensus for the remaining trees was constructed; the posterior probabilities (PP) were summarized for each branch.The best substitution model for D-loop sequences was selected using Modeltest version 3.7 software  by means M. fascicularis in this study were estimated using BEAST version 1.7.5 [ M. sylvanus. The uncorrelated lognormal relaxed-clock model [ M. sylvanus was used to root the ingroups and as the most recent common ancestor (TMRCA) or calibration point, estimated around 5.5 million years ago (MYA) based on fossil data [The divergence times ofon 1.7.5 . Two datck model  was usedck model , which ssil data , 54. MCMD-loop sequences as low as 1,000 bp were successfully sequenced for all 23 genetic samples used in this study . All samA total of 395 bp D-loop sequences of the HVSII region were obtained for sequence, phylogenetic, and population genetic analyses. It was found that 291 (73.6%) out of 395 characters in the sequences were constant, leaving 104 (26.3%) variable characters. Eighty one (20.5%) characters were parsimony informative, while the 23 (5.8%) remaining characters were parsimony uninformative . The seq M. f. fascicularis (population as in the country they are distributed in) based on the Kimura 2-parameter model were also calculated  and the insular populations . The genetic distance between these two groups  was relatively high compared to distance within the groups. For instance, the genetic distance between Malaysia and Vietnam was only 0.033, but with the three insular populations , it was as high as 0.098. In contrast, the distances within the groups were only 0.026–0.050  and 0.026–0.031 (insular). SNP analysis conducted on the D-loop sequences revealed 104 SNPs throughout the sequences analyzed.Average pairwise distances amonglculated . The paiπ) and net nucleotide divergence (Da) among M. fascicularis populations revealed the same model of continental-insular relationships and separate Peninsular Malaysian populations from insular populations (π and 0.02025 (Mauritius-Philippines) for Da was obtained, similar to the continental group, with 0.03227 for π  and 0.03163 for Da . However, the analysis between the continental and insular groups showed a relatively high value for both π and Da, a minimum value of 0.03212  and 0.04562 (Indonesia-Vietnam), respectively. M. f. fascicularis populations in Peninsular Malaysia, Thailand, Cambodia, and Vietnam were included in the continental group. The Philippines, Mauritius, and Indonesia were included in the insular group for additional analysis and were found to exhibit 0.05101 for π values and 0.04549 for Da.Nucleotide diversity  values were calculated to further elucidate the relationships among M. f. fascicularis populations in seven different countries , except for Cambodia-Vietnam (0.001). The lowest FST value between continental-insular groups was 0.605 (Indonesia-Vietnam), in contrast to Mauritius populations, which showed the highest division from Thailand, with 0.992. Nst analysis can be used to estimate a population's subdivision at the nucleotide level [FST, with Cambodia-Vietnam exhibiting lowest Nst of 0.00, followed by Philippines-Indonesia populations with 0.31. The division of Mauritius populations from Thailand revealed the highest Nst, at 0.99. Theoretically, when the Nm value is <1, populations are expected to genetically diverge over time, and when Nm is >1, they are expected to retain gene flow. Thus, the Nm value will be inversely proportional to both FST and Nst. Here, Nm analysis validated both FST and Nst, as Cambodia-Vietnam had the highest Nm, with an astonishing value of 494.53 with the same population size (15 for each population). Nm describes the average number of individuals per generation migrating between populations; thus, there appear to be nearly 500 individuals migrating between Cambodia and Vietnam per generation, retaining their gene flow as a result. The Nm value for Mauritius-Thailand was the lowest, at 0.00, suggesting that the gene flow between these two populations was cut off over time.Genetic differentiation , as there are far fewer mutational steps between the populations as compared to insular populations. The analysis also portrayed Cambodia/Vietnam as the connecting point for both populations from Peninsular Malaysia and insular populations; the fewest mutational steps to their respective haplotypes were found.Sixty-five haplotypes with a size of 104 bp were defined from the seven populations oficularis  and obtaicularis . The net M. fascicularis populations in Southeast Asia using the seven populations in this study as a model. By assuming that the amount of difference between alleles depends directly on the length of time since they diverged, we could manipulate the whole distribution of sequence differences to observe the demographic expansion of M. fascicularis. Thus, mismatch distributions of continental  and insular  groups  expansion model [Mismatch distribution of pairwise nucleotide differences of the HVSII sequences was estimated to study the demographic history of) groups  were caron model , 58 and on model , 59). ThM. f. fascicularis), supported with a 79% bootstrap value. Two clades of M. fascicularis populations were further defined, namely, the continental groups  and insular groups , supported by 97% and 81% bootstrap values, respectively. The MP phylogenetic tree  and anthropological  factors may play a crucial role in separation ofrop raid , 61. M. fascicularis revealed a complete separation of the populations from six populations in other countries. While it showed a closer relationship to populations originating from Vietnam, Cambodia, and Thailand as compared to other insular populations, the results  suggest an almost cut-off gene flow concerning the rest of the continental populations. Not a single haplotype is shared between the Peninsular Malaysian population and the rest of the continental populations, and MSN analysis clearly showed the distant relationship between all the populations, particularly continental-insular groups. Furthermore, the network analysis  based on morphological and genetic traits [This southern transition also involves a distinctive change between perhumid evergreen rainforest and wet seasonal evergreen rainforest –72 but hc traits –12, 74. M. f. fascicularis populations that does not undergo introgression with M. mulatta because the Y-chromosome of M. mulatta detected in M. fascicularis north of the Isthmus of Kra is absent in the southern populations. The differences discovered in fossil records, the variety of vegetation, and the genetic and morphological traits of M. f. fascicularis have led to the assumption that the differences have arisen because they belong to distinct biogeographical regions. The southern groups are always assumed to be the Sundaic population, and these deviate from the characteristics of those in Indochinese region. The phylogenetic analysis  in this study indicated a definite separation of Peninsular Malaysia's M. f. fascicularis from insular populations and the monophyletic state of Peninsular Malaysia's population, which is more closely related to Indochinese populations.Tosi and Coke  proposed M. f. fascicularis populations in Malaysia indicates that the colonization in Peninsular Malaysia occurred roughly ~0.47 MYA, which is largely congruent with Tosi and Coke's [ M. f. fascicularis after the radiation of the species from Indochinese regions via terrestrial radiation during continental shelf emersion or natural sea rafting.The estimation of divergence time ofd Coke's  estimatid Coke's  study, w M. fascicularis in Southeast Asia.The biogeographical history of Southeast Asia is complex due to active tectonic plate movement, the rise and fall of sea level during the Pliocene and Pleistocene, drastic climate change, and the temporary formation of land bridges connecting mainland Southeast Asia to Sunda, Java, Sumatra, and Borneo –78. Thes M. f. fascicularis in Peninsular Malaysia were found to be monophyletic in all phylogenetic analyses, with the absence of shared haplotypes with the other populations in Southeast Asia. This demonstrates that the species should be treated as a single unique lineage of long-tailed macaques in Southeast Asia. Nevertheless, populations from Malaysia are much more closely related to populations in Indochinese regions compared to insular populations, thus supporting the view that Peninsular Malaysia's populations are continental populations belonging to the Indochinese biogeographical regions, as opposed to Sundaic populations. The results of this study are crucial in the field of biogeography, as in the case of M. f. fascicularis; the inclusion of Peninsular Malaysia in the Sundaic regions is unsuitable.Populations of


Coregonus albula) normally spawns in late autumn, but in a few lakes in Northern Europe sympatric autumn and spring- or winter-spawners have been described. So far, the evolutionary relationships and taxonomic status of these main life history forms have remained largely unclear. With microsatellites and mtDNA sequences, we analyzed extant and extinct spring- and autumn-spawners from a total of 23 Swedish localities, including sympatric populations. Published sequences from Baltic ciscoes in Germany and Finland, and Coregonus sardinella from North America were also included together with novel mtDNA sequences from Siberian C. sardinella. A clear genetic structure within Sweden was found that included two population assemblages markedly differentiated at microsatellites and apparently fixed for mtDNA haplotypes from two distinct clades. All sympatric Swedish populations belonged to the same assemblage, suggesting parallel evolution of spring-spawning rather than secondary contact. The pattern observed further suggests that postglacial immigration to Northern Europe occurred from at least two different refugia. Previous results showing that mtDNA in Baltic cisco is paraphyletic with respect to North American C. sardinella were confirmed. However, the inclusion of Siberian C. sardinella revealed a more complicated pattern, as these novel haplotypes were found within one of the two main C. albula clades and were clearly distinct from those in North American C. sardinella. The evolutionary history of Northern Hemisphere ciscoes thus seems to be more complex than previously recognized.Presence of sympatric populations may reflect local diversification or secondary contact of already distinct forms. The Baltic cisco ( Coregonus (whitefishes and ciscoes) is renowned for its elusive taxonomy and systematics, reflecting a complex mixture of past glacial events and subsequent evolution  is natively distributed in Northern Europe (Kottelat and Freyhof Coregonus sardinella). Vendace from England and Scotland (Coregonus vandensius) is often regarded as conspecific to C. albula  and C. fontanae  populations are known from four lakes in southern Sweden Svärdson  and two Svärdson , whereasIn the Swedish and German lakes with spring-spawning ciscoes, there is also a sympatric autumn-spawning population. In Finland, the situation is somewhat different with winter-spawning being the most common exception to the dominating autumn-spawning form, whereas spring-spawning has only been reported from two lakes , the C. albula group is less divergent. Resolving in further detail the diversity in this group could thus facilitate our understanding of the more complex diversification patterns observed in other coregonids. Inclusion of Swedish material is particularly needed to increase the amount of available genetic information, allowing different hypotheses to be tested regarding the present-day diversity in C. albula. So far, only two allozyme studies limited in scope have been carried out . Two specimens of C. maraena (Maraena whitefish) and one specimen of Prosopium cylindraceum (round whitefish) were included as outgroups. Apparently low mtDNA variation in the four Swedish target lakes with sympatric populations  together with time-consuming protocols for old scales prompted a sampling strategy focused on small sample sizes from a larger number of populations  fragments were amplified and sequenced, a part of the ND3 region and the D-loop of the control region (see supporting information for details). Our sequences were combined with already published ones from GenBank  was usedAn unrooted network was constructed for the concatenated dataset using the software TCS . A neighbor-joining tree  as implemented in DIYABC 2.0 . For dry scale specimens, the amplification success rate was lower (65%). The found haplotype distribution is summarized in Figure C. albula . The Siberian C. sardinella samples also yielded two novel combined haplotypes.We found previously undescribed haplotypes for both ND3 and D-loop, resulting in a total of 13 (16 with extended regions included) novel combined haplotypes for the Swedish material of Phylogenetic analysis of the mtDNA revealed two highly supported clades, from here on referred to as Clade I (further divided into subclades IA and IB) and Clade II Fig. . The topC. sardinella.Clade I has a wide geographical distribution in Sweden, Germany, and Northern America Fig. . SubcladC. sardinella from Siberia (Cosa–6 and Cosa–7) were placed within Clade II, in contrast to the North American C. sardinella haplotypes that are all in subclade IB  to 51 (BWF2), of which 27 (14 %) were private to a single sample. Bubble diagrams illustrating sample allele frequencies at each locus are shown in icrochecker did not detect evidence of stuttering or large allele dropouts, whereas presence of a putative null allele(s) was indicated at a single locus in sample Kalix (Sfo8), E L. Mälaren (Sfo23), L. Siljan (BWF1), and L. Åsunden (BWF1).A total of 199 alleles were found at the nine microsatellites, ranging from 4 , close to the proportion expected by chance alone.Statistically significant deviations from Hardy–Weinberg proportions, quantified as average FST and RST across all loci are given in P < 0.001) existed between the samples of sympatric spring- and autumn-spawners from L. Fegen, but without signs of a mutational component . In contrast, no significant allele frequency differences were found between the two samples from L. Mälaren (E vs. W) and L. Dellen , respectively.Pairwise sample estimates of vice versa  and allelic richness . Before testing, the genetically homogenous sample pairs from L. Mälaren and L. Dellen were lumped to avoid pseudoreplication.When comparing the two population groups defined by congruent microsatellite variation, Assemblage II was found to have overall higher levels of genetic variability  and nearly across all nine loci (P = 0.06). Of all microsatellite alleles observed, 18% and 20% were private to samples in Assemblage I and Assemblage II, respectively . However, the estimate was associated with a considerable degree of uncertainty . The estimated divergence time between the genetically distinct populations (P < 0.001) from Kalix and L. Vänern  ranged from c. 1700 to 4200 generations, or 6800–16,800 yBP .Mean, median, and modal ABC estimates of divergence time between the ancestors of the two population assemblages based on microsatellite data  ranged between C. sardinella, interpreted by them as past introgression into the C. albula complex. However, the inclusion of two additional samples of Siberian C. sardinella in this study together with a better supported phylogenetic tree  with Clade IA haplotypes (mtDNA) are geographically restricted to a limited area in south-central Sweden Fig. . This reCoregonus may have spread to this region by means of ice readvances and temporarily ice-dammed lakes, which was originally proposed by Högbom , the present-day distribution of Swedish populations, with Clade II mtDNA and microsatellite alleles belonging to Assemblage II, is restricted to water bodies at lower elevations associated with the Ancylus Lake (the Lake Vänern basin included). Thus, we suggest that the rapid lake level drop and marine influx in the Baltic basin c. 10,600 yBP may have suppressed the occurrence of the first arriving Group I in the Baltic basin, whereas a second immigration wave from another ice age refugium (Group II) arrived some 1000 years later in association with the formation of the Ancylus Lake. Depending on whether ciscoes remained in the Baltic basin or not, the later arriving Group II either recolonized all accessible areas or replaced  Group I.According to geological records, the three westernmost Swedish lakes studied herein, all with Clade II haplotypes . Thus, future genetic studies should focus on remaining autumn-spawners in those Finnish lakes to assess whether a genetic pattern similar to the one in Sweden could exists. Previous allozyme data , supporting a longer time of independent diversification. In line with these findings, the ABC estimate of divergence time among the two Swedish population assemblages suggested a split between their ancestors that coincides roughly with the onset of the last glaciation , although we note that this time estimate is associated with a wide credibility interval. The second ABC analysis, including L. Vänern and Kalix , suggested a much later population split close to the estimated geological date for the closure of the Närke strait separating the two basins . Although this second estimate is also uncertain, its correlation with independent geological data may indicate that the above ABC estimate of divergence time (between the two major population assemblages) could be of a correct order of magnitude.Some of the most variable microsatellites display a high proportion of private alleles within the two assemblages, and signs of a significant mutational component to population divergence were also obtained  extensive introgression occurred during the Pleistocene, involving populations of C. albula and C. sardinella as we recognize them today, or that (ii) the current mixture of haplotypes is a result of incomplete lineage sorting among ancestors to the two present species. The answer could also be a combination of these two processes, as their distinction is not always clear cut. Regardless, the present results reveal that C. albula and C. sardinella, in some way, have had extensive contact back in time.Focusing on the Stenodus in the Arctic and Caspian Sea . However, we note that Yakhnenko and Mamontov  that was more similar to Siberian C. sardinella haplotypes than it was to those typically found in C. albula. Hence, a comprehensive molecular and morphological study, including East European and Siberian material combined with expanded sampling from North (and Western) Europe appears warranted for a proper genetic and taxonomic distinction of C. sardinella vs. C. albula, including also doubtful taxa like C. ladogae (L. Ladoga) and C. kiletz (L. Onega), and for providing a better understanding of the group's complex evolutionary history.The taxonomic position of Mamontov  discusse Makhrov  found a C. trybomi as a distinct species (Svärdson With respect to spring-spawning ciscoes in Sweden, the present results do not support the previous recognition of Svärdson . The onlSvärdson  should b


Admixed populations arise when two or more previously isolated populations interbreed. Mapping asthma susceptibility loci in an admixed population using admixture mapping (AM) involves screening the genome of individuals of mixed ancestry for chromosomal regions that have a higher frequency of alleles from a parental population with higher asthma risk as compared with parental population with lower asthma risk. AM takes advantage of the admixture created in populations of mixed ancestry to identify genomic regions where an association exists between genetic ancestry and asthma (in contrast to between the genotype of the marker and asthma). The theory behind AM is that chromosomal segments of affected individuals contain a significantly higher-than-average proportion of alleles from the high-risk parental population and thus are more likely to harbor disease–associated loci. Criteria to evaluate the applicability of AM as a gene mapping approach include: (1) the prevalence of the disease differences in ancestral populations from which the admixed population was formed; (2) a measurable difference in disease-causing alleles between the parental populations; (3) reduced linkage disequilibrium (LD) between unlinked loci across chromosomes and strong LD between neighboring loci; (4) a set of markers with noticeable allele-frequency differences between parental populations that contributes to the admixed population (single nucleotide polymorphisms (SNPs) are the markers of choice because they are abundant, stable, relatively cheap to genotype, and informative with regard to the LD structure of chromosomal segments); and (5) there is an understanding of the extent of segmental chromosomal admixtures and their interactions with environmental factors. Although genome-wide association studies have contributed greatly to our understanding of the genetic components of asthma, the large and increasing degree of admixture in populations across the world create many challenges for further efforts to map disease-causing genes. This review, summarizes the historical context of admixed populations and AM, and considers current opportunities to use AM to map asthma genes. In addition, we provide an overview of the potential limitations and future directions of AM in biomedical research, including joint admixture and association mapping for asthma and asthma-related disorders. Asthma is the most common chronic illness affecting children in the United States CDC, . It is ahttp://cvp.ucsf.edu/docs/asthma_factsheet.pdf) . Variations in asthma prevalence among subpopulations suggest that asthma is a heterogeneous disease with varied risk profiles  and African Americans (14.6%) is higher than among European Americans 8.2%), Mexican Americans (4.8%), and Asian Americans (4.2%)  Figure , 2014. F Miller, . These p Miller, . The rea Miller, . Admixedadmixture refers to the process in which individuals from two or more geographically isolated populations with different allele frequencies mate and form a new mixed or “hybrid” population  is a gene mapping method used for persons of mixed ancestry to identify genomic segments in which the proportion of a particular ancestry is strikingly higher or lower than that seen elsewhere in their genomes  genetically separated populations that have been intermixed through several generations of mating to form an admixed population. The genetic structure of the new population is a mixture of the genetic materials of the founding populations. In the experimental genetics of inbred lines, phenotypically distinct strains are used. Using AM to correlate phenotypes to the originating populations, differentially selected traits may be identified in the two founding populations. There are important differences between mapping in an admixed population and mapping with crosses of inbred strains. Whereas, inbred strain genetic mapping is mainly experimental, human genetic mapping is observational .The concept of using admixed populations to localize disease-associated genes was proposed more than 50 years ago Rife, , but it Several variants associated with complex traits in admixed populations have been identified with the application of AM. These include risk variants associated with cardiovascular disease  select a panel of AIMs or high-density SNPs that differentiate well between the ancestral populations; (2) ancestry estimation. Genotype asthmatic and control subjects using the selected AIMs or a high-density SNPs panel followed by inferring admixture proportions; (3) admixture mapping using cases-only or cases and controls analysis. Ancestral estimates will be used to search for an aberration toward the ancestral population with a higher disease risk locus using the AM procedure; and (4) the prioritization of variants in AM peak region using step-wise regression and conditional analyses.ideal  to describe the alleles fixed in different populations. Chakraborty et al.  value of 1.0 .AIMs are used to estimate the geographical origins of an individual's ancestors, which is typically expressed as the proportion of ancestry from different continental regions  than a sparse panel of AIMs  are more cost effective and can estimate Although human genetic variations reflect differences at single alleles as well as at the haplotype level, most ancestry estimators use allele frequency (locus-by-locus) data between parental contributions along the chromosome and ignore the molecular information that is available in the ancestry-haplotype block structures in the genome. Individual mutations carry only weak signals as they apply to population ancestry. Inferring information about admixture proportions by combining information from across multiple loci that form haplotypes or haploblocks is quite valuable. By adding information across the whole genome at the haplotype level, these admixture events can be reconstructed more accurately  levels. Theoretically, it is possible to measure every point in the genome and determine ancestry. However, recombination events at each intermixing stage cannot be observed directly  were inherited from prespecified ancestral populations  of ancestral blocks from each contributing population across the markers of interest. Several tools are used to infer global ancestry. LAMP-LD uses high-density SNP data and incorporates LD information when estimating local ancestry from two or more ancestral populations  and πc(θ) be the proportions of alleles that are from ancestral population X among cases and controls in the current admixed population (c), respectively, t represent the chromosome location, and θ represents the genetic distance between the disease location and the candidate marker. The null hypothesis is θ = 0.5 between a marker locus and a disease locus (the marker is unlinked to the disease risk). In both models, regions with statistically significant regression coefficients for local ancestry are inferred to harbor disease modifying genes. The case-only or case-control admixture tests are not affected by population structure, because the excess of ancestry is being tested at a marker position   Zhu, . Estimatue) Zhu, . To dete−6   and |Z| > 4.0 (significant)  are used in asthma to identify genomic regions that differ significantly between ancestries  +β2 +β3 +β4(covariates). The generalized linear models are flexible, allowing for multiple types phenotypes  and covariates to be included  ~ βgenetic confounding caused by population stratification   and the AncestrySNPminer  in combination with the Greater Cincinnati Pediatric Clinic Repository cohort  and INSIG2 (in populations of African American ancestry)—that were associated with asthma  and PhenoGram visualization software  is the link function, Aij is the local ancestry for the ith individual at the jth marker , forced expiratory volume in 1 s (a standard measure of lung function), and the ratio of forced expiratory volume in 1 s to forced vital capacity could be used to explain differences in disease prevalence among racial groups  or African American (n = 385) were studied. Self-identified African American women were more likely to be sensitized to at least one allergen  as compared with white women [odds ration (OR) = 2.19]. However, genetic ancestry based on AIMs was not significantly associated with allergic sensitization (OR = 1.34) after adjusting for location of residence (urban vs. suburban). Similarly, Keet et al.  cohort, 601 WHEALS mothers who identified themselves as white  strategies are now being used to find causal variants associated with asthma . As a result, a shared or unique allele might reflect similar or different environmental exposures rather than shared or unique ancestries  that is unrelated to asthma risk Admixture is an error containing proxy measurement of ancestry, i.e., Admixturei = Ancestryi + Errori. The error could be due to measurement (incomplete coverage of genome), missing data , biological variation (meiosis/recombination) and other errors such as genotyping, information content of markers  The history of human admixture is not under experimental control or even necessarily known. The number of generations is usually estimated. (2) Ancestral populations are not available for study. Ancestry-specific allele frequency is re-estimated within the admixed population, with prior estimates based on sampling unadmixed modern descendants. However, the true number of ancestral populations in an admixed population is not known. (3) The exact mix of ancestral populations that contributes to the admixed gene pool cannot be sampled. (4) Human racial groups are not inbred strains, and markers with 100% frequency differentials are rare. Thus, we cannot unequivocally infer ancestry at a specific locus from a marker genotype. (5) Despite recent interest in applying admixture methods to diverse populations  have distinguishable ancestral origins and provides valuable information for AM. Studies have shown that the frequency of alleles associated with asthma differ by race. Differences among individuals of African and European descent, for example, and the admixture nature of the African American population create an ideal opportunity of AM to map ancestry-specific variants. Ancestry-based gene-mapping approaches in admixed groups can be expected to play a great role in the mapping disease susceptibility loci, and to elucidate the genetic basis of inter-racial differences. Furthermore, they will provide valuable opportunities to study the interactions of race, genetics, culture, and environments.Most genomic studies in admixed populations use commercial genotyping arrays that are developed based on reference panel from relatively homogeneous European ancestry population, and may not adequately tag relevant variations in admixed populations. Increasing availability of polymorphic molecular markers from directly sequencing multiple individuals/populations using NGS , allows admixture proportions to be accurately estimated at unprecedented local detail. Consequently, researchers are able to distinguish between closely related individuals of variable with varying ancestry and to determine the ancestry of genomic segments at a fine scale across an individual's genome. Although recent technologies make it possible to sequence and generate millions of high-density variants to identify ancestry-specific markers, well-characterized and standardized phenotyping still lags behind.Until now, AM approaches for asthma have focused on single-locus ancestry effects, whereas comprehensive analyses of haplotype–ancestry blocks from multiple loci or ancestry–environment interactive effects in the context of disease genetics have not yet been incorporated. Studies focusing on accurate and stringent phenotyping and genotyping at multilocus ancestry markers—including other types of variants  and omics data —and that take into account potential population stratification due to intrinsic and extrinsic environmental stimuli will facilitate ancestry-based asthma gene mapping. Taken together, investigating the genetic architecture of asthma in diverse ancestral and admixed populations will help to understand its etiology and perhaps shed light on the disparities seen in childhood asthma risk between races.TM conceived and wrote the manuscript.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Paraisometrum mileense ( = Oreocharis mileensis), was presumed extinct for 100 years. After its re-discovery, the species has become one of five key plants selected by the Chinese forestry government to establish a new conservation category for plants with extremely small populations. For conservation purposes, we studied the phylogenetic and population genetic status of P. mileense at the three only known localities in Guangxi, Guizhou and Yunnan.The karst regions in South China have an abundance of endemic plants that face high extinction risks. The Chinese Gesneriaceae endemic Oreocharis and 8 samples from three provinces of P. mileense and generated molecular phylogenies, and inferred that P. mileense represents a relatively isolated and derived taxonomic unit within Oreocharis. Phylogeographic results of 104 samples of 12 populations of P. mileense indicated that the populations in Yunnan have derived from those in Guangxi and Guizhou. Based on AFLP data, the populations were found to harbor low levels of genetic diversity (He = 0.118), with no apparent gradient across the species’ range, a restricted gene flow and significant isolation-by-distance with limited genetic differentiation among the populations across the three provinces . The 10 populations in Yunnan were found to represent two distinct lineages residing at different altitudes and distances from villages.We collected 64 samples (52 species) of P. mileense are perhaps a consequence of severe bottlenecks in the recent past. The distribution of the genetic diversity suggests that all populations are significant for conservation. Current in situ and ex situ measures are discussed. Further conservation actions are apparently needed to fully safeguard this conservation flagship species. Our work provides a model of an integrated study for the numerous endemic species in the karst regions with extremely small populations.The low levels of genetic diversity found in  Paraisometrum mileense W.T.Wang was until relatively recently regarded as extinct in the wild because it had not been recollected for more than 100 years since its type specimen collection in 1906 P. mileense grows  Mich.Möller & A.Weber Until 2011, P. mileense has been included in phylogenetic analyses previously, its exact phylogenetic affinities in Oreocharis are still unclear. While it was clearly shown in the phylogenetic analyses that the species has evolved from within the enlarged Oreocharis, it fell on a polytomy with species of the hitherto genera Ancylostemon Craib, Briggsia Craib, Isometrum Craib, Opithandra B.L.Burtt and Tremacron Craib Paraisometrum are the presence of four upper corolla lobes, and one lower lobe of the pentamerous flowers Oreocharis (e.g. O. saxatilis (Hemsl.) Mich.Möller & A.Weber = Ancylostemon saxatilis Hemsl.) P. mileense. Furthermore, the plants in Guangxi and Guizhou appear to possess some floral features different from those in Yunnan (pers. obs. YMS and WHC). However, only two samples of P. mileense, one from Yunnan and one from Guangxi, were included in the most comprehensive study of Oreocharis to date Oreocharis.To be able to devise meaningful conservation strategies, knowledge of the taxonomic status, closest congeners and the level and distribution of genetic diversity within a taxon is essential Primulina tabacum Hance. This species occurs in only four populations distributed in Guangdong and Hunan with less than 1,000 plants in each P. tabacum was seen as ‘a window of opportunity’ to preserve a high level of extant genetic variation in the species. To determine whether a similar situation is present in P. mileense, we generated population genetic data using amplified fragment length polymorphisms (AFLPs) for individuals from the three localities in Guangxi, Guizhou and Yunnan. AFLP is a powerful tool for generating data from multiple loci for the detection of genetic variation without the need for pre-existing knowledge of genomic sequences There are many Gesneriaceae species endemic to the karst region in China, typically with small populations trnLF, and the nuclear ribosomal internal transcribed spacer regions (nrITS), to a) test the taxonomic status of the species, b) to determine the phylogenetic position and relationships of P. mileense within the enlarged Oreocharis, c) to reconstruct the phylogeographic history of the P. mileense populations using trnLF, matK, and ITS sequence data for individuals from 12 populations (or sub-populations) from the only three localities in Guangxi, Guizhou and Yunnan, and to acquire and analyse AFLP data for population samples to d) determine the levels of genetic diversity and differentiation of this species to assess its conservation requirements.Thus, our main aims were fourfold, to phylogenetically analyse sequence data, from the chloroplast intron–spacer sequences of Paraisometrum mileense is listed as one of five selected key plants to be used to establish a new category of protected areas in China for plant species with extremely small populations (PSESP). Approvals and permission for field studies were obtained from the Yunnan Forestry Bureau, China (permit no. [2011]–115). The GPS data are sensitive and are not provided for the protection of the plants according to the Yunnan forestry administrative request in the permit. However, we gave their approximate location in Of all sampled species, the target species Oreocharis species plus one variety (41 samples) P. mileense, we included three samples each from Guangxi and Guizhou and two samples from Yunnan (Oreocharis ingroup sampling covered about 2/3 of the genus (53 out of about 80 species) sensuDidymocarpus Wall. samples to root the trees For the phylogenetic study, we expanded the ingroup sampling from 38 m Yunnan . Thus, tParaisometrum materials for the phylogeographic analyses included 43 individuals from Yunnan (from 10 populations or sub-populations), 15 from the Guangxi population and 15 from the Guizhou population, and for the AFLP analysis 68 individuals from Yunnan , 19 from the Guangxi population and 17 from the Guizhou population , 100 mM KCl, 3 mM MgCl2) and ddH2O to make up 20 µl. PCR amplifications were conducted under the following profile: 95°C for 3 min followed by 35 cycles at 94°C for 30 s, at the annealing temperature specific for each primer pair for 30 s , 72°C for 1 min, and a final extension step at 72°C for 5 min. After PCR amplicon purification, Sanger sequencing was carried out in 6 µl reactions containing 0.5 µl PCR product, 0.3 µl primer (5 µM/µl), 1.05 µl SeqBuffer, 0.3 µl BigDye Terminator Mix  and 3.85 µl distilled H2O. Sequencing reactions were cycled under the following conditions: 32 cycles at 96°C for 10 s, 50°C for 5 s, and 60°C for 3 min, and the products analysed on an ABI 3730×l sequencer . The newly acquired sequences have been submitted to GenBank.Genomic DNA was extracted from silica-gel-dried leaves collected in the field following a modified CTAB method PstI/MseI, 2.5 µl 10× reaction buffer, 2.5 µl ATP (10 mM), 1 µl T4 Ligase, 7 µl distilled H2O. The solution was centrifuged for a few minutes after stirring, and incubated at 37°C for 5 h, then at 8°C for 4 h and then at 4°C overnight. Pre-amplification reactions were performed containing 2 µl template DNA, 1 µl Pre-ampmix, 0.5 µl dNTPs . The amplified fragments were separated and detected with an ABI Prism 377 sequencer . Due to ambiguous banding patterns or obvious PCR failures, 19 additional samples (GX11a-s) collected exclusively from Guangxi province were included  and the ITS and trnLF data sets were analysed combined. Parsimony was implemented on unordered and unweighted characters and through heuristic tree searches on 10,000 random starting trees with both TBR swapping and MulTrees on. Clade support was obtained as bootstrap indices with 10,000 heuristic replicates of random additions with TBR on and MulTrees off.The partition-homogeneity test in PAUP*  were discarded as burn-in , and the posterior probabilities (PP) obtained from 50% majority rule consensus trees obtained using the ‘sumt’ command in MrBayes. A low value of the average standard deviation of split frequencies (0.009025), the high correlation of the PP support values and the parallel distribution of variance values found between the two parallel runs of the Bayesian analysis, and the level runs of the split posteriors indicated a good convergence of the MCMC runs . Phylogeographic networks were reconstructed with NETWORK 4.6.1.1 (available at http://www.fluxus-engineering.com). Ancylostemon hekouensis Y.M.Shui & W.H.Chen ( = Oreocharis hekouensis (Y.M.Shui & W.H.Chen) Mich.Möller & A.Weber) was used to root the networks, based on finding of the phylogenetic analyses here.Chloroplast haplotypes were determined using combined chloroplast P), number of different alleles (Na), number of effective alleles (Ne), Shannon’s Information index (SI), expected heterozygosity (He) and unbiased expected heterozygosity (uHe). To investigate for patterns among the Paraisometrum populations, we used STRUCTURE 2.3.4. K) from K1 to K10. Each run of 100,000 iterations was preceded by 10,000 iterations as burn-in when convergence was achieved. We plotted the mean likelihood for each cluster L(K) against the cluster number (K). To establish the optimal number of clusters, the relationship between K and ΔK, the second order rate of change of the likelihoods, was plotted The ABI trace files were analysed in GeneScan 3.1 (Applied Biosystems), and only intensive bands between 70 and 500 base pairs in size converted to a binary matrix  . Geneticln geographic distance versus Nei’s genetic distance (D) in GenAlEx. For a 3D illustration of spatial relationships of the populations, a principal coordinate analysis (PCoA) was performed using the Jaccard distance in R-pack Le Progiciel R.4.0d10 To test an isolation-by-distance scenario, a Mantel test was performed on the geographic and Oreocharis in its new definition formed a strongly supported clade in the strict consensus tree (BS = 100%). Paraisometrum was nested deeply within this clade in a derived position with the P. mileense samples forming a strongly supported clade (BS = 100%)  . Sister Paraisometrum and A. hekouensis samples  and significant genetic differences existed among populations   and among populations  (The AMOVA indicated that most genetic diversity resided within the populations of P<0.001) . When stP<0.001) .K = 4  and ln geographic distance .The optimal number of clusters for the STRUCTURE analysis was determined as K = 4 . The anaK = 4 . The Guast axis 12% variance; 2nd axis = 7.7%; 3rd axis = 6.6%) separated the samples of the three regions strongly in the first axis, including the Yunnan populations 1–4 and 5–10 , and a derived Yunnan population  , Table 3Paraisometrum being pollinated by bees, which have a relatively limited flying distance , are not dust-like, as in orchids Paraisometrum localities. Taking the average rate of evolution of the ITS spacers of 11 herbaceous plants (4.13×10−9 substitutions per site per year) The significance in the Mantel test on the AFLP data indicates an isolation-by-distance scenario nce e.g. , and thell ca. 0. mm long,Our study provides a first insight into the history of a plant considered extinct in the wild. To fully address the evolution of the species, and to untangle historic from contemporary events, an increased sampling is required, additional markers employed such as microsatellites, combined with population demographic analyses e.g. , and, wiParaisometrum populations; firstly, that the Yunnan populations represented two distinct lineages with at least three gene pools and can be divided into two main populations, populations 1–4, and populations 5–10 with some admixture , and much lower compared to Primulina tabacum (He = 0.339), another herbaceous Gesneriaceae species from China Primulina tabacum and P. mileense have similar distribution sizes though in different provinces , but both occur in limestone karst areas, though at different altitudes  and in different aspects . The high genetic diversity levels found in P. tabacum populations were explained by their refugial history and/or breeding system P. tabacum, as opposed to the more open forest habitats of Paraisometrum could be a factor that may have allowed a more continuous distribution and genetic connectivity of Paraisometrum populations. However, it is more likely that the greater population sizes of P. tabacum  has allowed P. tabacum to retain higher diversity levels. The similarly low levels of AFLP diversity compared to Paraisometrum found in other herbaceous perennials, such as Trollius europaeus L. (He = 0.158–0.229) Silene otitis St.-Lag. (He = 0.167–0.240) Draba aizoides L. (He = 0.07–0.15) Paraisometrum is likely a result of a combination of fragmentation and small population size. Intriguingly though, in Draba L., the low genetic diversity levels were not correlated with a limited reproductive fitness, as suggested by their high germination rates. This may have important conservation implications and could be a field of study in Paraisometrum in the future.The genetic diversity found at the population level was very low, although this might have been a consequence of the low number of individuals (often 5) included for analysis of the Yunnan populations. However, even when calculated within the three regions, the AFLP diversity levels were relatively low, with no marked difference between the regions . Even atParaisometrum are isolated in different limestone forests, and the occurrence nearby villages is not unusual for endemic plants of the karst region Our work provides an example of an integrated study for endemic species in the karst regions in South China with extremely small populations. It is well known that the karst region is characterized by a limestone topography with an abundance of endemic plants with extremely small populations The villages are commonly closely associated with characteristic forests on the limestone hills, the Feng Shui Forests. These are important for the water supply of the villages, but are recently influenced by activities of local people, such as free range poultry and goat keeping Paraisometrum, the only three distribution points known to date appear to contain a significant amount of diversity within their populations with only a limited level of differentiation. Though, analysing a larger number of individuals per population might show some more resolution. Furthermore, the STRUCTURE analysis shows that they seem to have experienced some gene flow between Yunnan and Guizhou populations and are thus not genetically greatly isolated  R. M. King & H. Robinson, an invasive plant alien to China in situ measures may also involve forest restoration ex situ conservation measures are required alongside in situ measures in the light of the observed strong detrimental effects of recent rainfall variation on the populations. This may, for the moment preclude transfer of plants from threatened habitats to new localities in the wild. Though, the plants are easily cultivated and propagated vegetatively and through seeds. Currently, about 56 plants are cultivated at the Kunming Botanical Garden, Yunnan, China, and seeds of three populations have been deposited at the Germplasm Bank of Wild Species, Plant Germplasm and Genomics Center, Kunming Institute of Botany, Yunnan, China. Thus, the immediate and medium term survival of the species seems ensured. However, seeds in germplasm banks are prone to genetic erosion during seed rejuvenation cycles even of inbreeding species ex situ strategy for Paraisometrum mileense.Paraisometrum mileense (now Oreocharis mileensis) represents an isolated and independent taxonomic unit, and appears closest related to the erstwhile Ancylostemon hekouensis (now Oreocharis hekouensis). The populations in Yunnan seem to have derived from those in Guangxi and Guizhou with some evidence of limited gene transfer between Yunnan and Guizhou. The occurrence points in Yunnan were divided into two quite distinct lineages, while the population in Guangxi is relatively homogeneous. Overall, the populations of P. mileense contain relatively low levels of genetic diversity with no apparent gradient across the species’ range. Several conservation measures are currently implemented, but additional actions are needed. From a conservation genetic perspective, all populations seem equally important for in situ and ex situ protection.The erstwhile monotypic genus Figure S1Comparison of posterior probability values between run 1 versus run 2 (10% burn-in) of the Bayesian inference analysis.(TIF)Click here for additional data file.Figure S2Posterior probabilities of splits at selected increments over MCMC run1 (A) and 2 (B) of the Bayesian inference analysis.(TIF)Click here for additional data file.Figure S3Comparisons of topological differences within and among MCMC runs of the Bayesian inference analysis.(TIF)Click here for additional data file.Figure S4Maximum parsimony strict consensus tree of 12 parsimonious trees of 1538 steps, based on combined ITS and trnLF sequence data . Numbers above branches are bootstrap values (x denotes branches receiving <50% support).(TIF)Click here for additional data file.Figure S5PCoA scatter plot based on AFLP data using the Jaccard distance on 12 populations of Paraisometrum mileense. Axes 1 (x) and 3 (z).(TIF)Click here for additional data file.Table S1Collection details and GenBank numbers of Oreocharis (including Paraisometrum mileense) and outgroup samples used for the phylogenetic analyses.(DOC)Click here for additional data file.Table S2Characteristics of the Bayesian inference analysis of 86 samples of combined ITS and trnLF data.(DOC)Click here for additional data file.Table S3Binary AFLP data for 12 populations of Paraisometrum mileense from Yunnan (Yu), Guizhou (Gz) and Guangxi (GX).(XLSX)Click here for additional data file.


Within a population, the differences of pharmacogenomic variant frequencies may produce diversities in drug efficacy, safety, and the risk associated with adverse drug reactions. With the development of pharmacogenomics, widespread genetic research on drug metabolism has been conducted on major populations, but less is known about minorities.2 tests to identify significantly different loci between these populations. We downloaded SNP allele frequencies from the ALlele FREquency Database to observe the global genetic variation distribution for these specific loci. And then we used Structure software to perform the genetic structure analysis of 12 populations.In this study, we recruited 100 unrelated, healthy Mongol adults from Xinjiang and genotyped 85 VIP variants from the PharmGKB database. We compared our data with eleven populations listed in 1000 genomes project and HapMap database. We used χOur results demonstrated that different polymorphic allele frequencies exist between different nationalities,and indicated Mongol is most similar to Chinese populations, followed by JPT. This information on the Mongol population complements the existing pharmacogenomic data and provides a theoretical basis for screening and therapy in the different ethnic groups within Xinjiang. It is well known that different individuals have different reactions to the same medications. Pharmacogenomics seeks to identify genetic markers that may influence a person’s response to pharmaceuticals. It will undoubtedly become an indispensable part of medical care in the future , 2. Pharhttp://www.pharmgkb.org) is the most comprehensive database and dedicates to propagating primary pharmacogenomic data and knowledge. They have extensively annotated the vital drug response genes and presented this information in VIP summaries, pathway diagrams, and curated literature [Numerous studies had shown that certain important genes and genetic variations affect critical functions during the drug reaction process. These genetic variations are called very important pharmacogenetic (VIP) variants and listed in the pharmacogenomics databases such as the Pharmacogenomics Knowledge Base (PharmGKB), the Pharmacogenetics of Membrane Transporters (PMT) database, and PharmaADME –8. Curreterature .In China, there are 56 different nationalities. Besides Han, the others account for approximately 100 million people. Due to the different genetic backgrounds and diverse environments of these minor populations, we distinguish them easily from the Han ethnicity. The Mongolian population represents one of the fifteen largest ethnic minorities in China , so the In this study, we random selected and genotyped 85 VIP variants from the PharmGKB VIP database in 100 Mongols from Xinjiang. We designed primers using MassARRAY Assay Design 3.0 Software . We compBlood samples and signed informed consent forms were obtained from all enrolls. All participants were informed both verbally and in writing of the procedures and purpose of the study, and signed informed consent documents. The clinical protocol was approved by the Clinical Research Ethics of Xizang Minzu University and Northwest University, and it is in compliance with Department of Health and Human Services (DHHS) regulations for human research subject protection.We recruited 100 random unrelated Mongol adults  from the Xinjiang Region of China and collected blood samples. The detailed recruitment criteria are the sample have good health body and had at least three generations of exclusive ethnic ancestries. They rarely communicate with other ethnics in Xinjiang because they are still nomads which living on relatively limited pasture. They were determined to be a representative Mongol population sample with regard to both ancestry and environmental exposure.Using the PharmGKB database, we screened published genetic polymorphisms associated with VIP variants, and finally 85 genetic variant loci from 37 genes were randomly selected for our investigation. We extracted genomic DNA from whole blood using a GoldMag-Mini Whole Blood Genomic DNA Purification Kit  according to the manufacturer’s protocol. The genomic DNA concentration was measured by absorbance at 260 nm using a NanoDrop 2000C . We used the Sequenom MassARRAY Assay Design 3.0 software  to design multiplexed SNP MassEXTEND arrays . We util2 tests. All p values calculated were two-sided and Bonferroni’s multiple adjustment was used to correction. The values were considered statistically significant when p < 0.05 and p < 0.05/(85 × 11), respectively [2) among the minor different population, then compared and calculated the selected SNP’s variant frequencies between the Mongol people and eleven HapMap populations (data from the second phase of HapMap: http://hapmap.ncbi.nlm.nih.gov) using a χ2 test. Afterwards, we downloaded the SNP allele frequencies of selected loci from the ALlele FREquency Database  and analyzed the global genetic variation patterns. We used Haploview software package (4.2) to perform the linkage disequilibrium (LD) analysis constructed haplotype, and genetic association of significant polymorphism loci.We used Microsoft Excel and the SPSS 19.0 statistical package  to perform a Hardy–Weinberg Equilibrium (HWE) analysis and χectively . We analhttp://pritchardlab.stanford.edu/structure.html) which based on the Bayesian clustering algorithmto assign the samples within a hypothetical K number of populations hypothesized by Pritchard et al [There are studies proved that the center of study which research human origins, DNA forensics and complex diseases is population genetic structure. It is also important to our study as a pharmacogenomics population study. Structure analysis is common in population genetic study. To further investigate variation at the VIP locus in terms of population structure we used STRUCTURE ver. 2.3.1 . We found that there are some loci have significantly different between them. In ASW population, there are 22 loci exist different with Mongol. The results of other groups are as follows: CDX, 14; CEU, 19; CHB,15; GIH,15; JPT,15; LWK,18; MXL,18; PUR, 22; TSI, 18; YRI, 18. There were a number of loci had significantly different distribution frequencies among Mongols and the 11 HapMap populations that listed in Table We used χVDR , showed the greatest number of significant differences between Mongol and 11 HapMap populations; the SNP rs776746 (12083G > A) is a SNP of CYP3A5 which located in an intron region and a significant locus that observed in these populations except TSI. After Bonferroni’s multiple adjustment (p < 0.05/(85 × 11)), the number of HapMap populations with a significantly different rs1540339 locus changed very large which included CEU, CHB, CHD, JPT, MEX and TRI. The rs776746 locus also changed very large which except TSI added CEU, CHD, GIH, JPT and MEX.When p < 0.05, rs1540339 locus (46489G > A) which located in an intron region of Of the 85 variants analyzed, 74 could be classified as part of a superfamily. When the gene superfamily categories were tallied, the number of the associated variants with significantly different frequencies between the Mongols and the eleven HapMap populations were as follows: ASW, 10; CEU, 9; CHB, 5; CHD, 1; GIH, 5; JPT, 4; LWK, 14; MEX, 1; MKK, 14; TSI, 4; and YRI, 21 . The results are indicated by K = 3–5  gene. MTHFR is located on chromosome 1p36.3 in human which is an important regulatory enzyme that involved in the folate pathway. It catalyzes the conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate [Two of the variants were identified, rs1801133 (C677T) and rs1801131 (A1298C), included one of the least significant locus in our data, they are located in the same gene -- methylenetetrahydrofolate reductase , which is located in exon 12 of SCN5A . SCN5A ele cells , 29. SCNle cells . Amountsle cells –31. SCN5le cells , 32 and le cells . Studiesle cells  We can cAmong Mongols and others global populations, numerous important genetic variants play critical roles in drug response and this information should directly applied to clinical guidelines. For instance rs1540339 (46489G > A), the most significant locus in our data, is associated with bronchodilator responsiveness . StudiesBeyond the genetic factor, we also determined that long-term survival in different environments affects genetic adaption. Environmental pressures shape genotype distributions towards specific functions, particularly in pharmacogenetic genes. Studies by Janha et al., Sabbagh et al., and Fuselli et al. directly demonstrated that the different genotype frequencies of CYP2C19, NAT2, and CYP2D6 significantly differed between populations race, subsistence modes, and dietary habits also play a role in the evolutionary trajectory –40.Different populations exists different genetic distribute frequencies. The drug dosage and usage of different genotype carriers is difference. Identifying genotype distribution and VIP variant frequencies in different populations to determine what medications might be most effective may provide a theoretical foundation for safe drug administration and improved curative effects. Besides, we figured out the minimum allele difference between Mongol and CDX. We also preliminary supplemented the pharmacogenomic data on the Mongol ethnic group and illustrated the differences between Mongols and other populations, and finally found Mongol and Chinese populations have the minimum difference. To the study, the sample size is relatively small and further investigation using a larger cohort of Mongols is needed to verify the generalizability of our results, and would be help us to establish a more reasonable and effective individualized treatment plan.ALFRED, the ALlele FREquency Database; ASW, a population of African ancestry in the southwestern USA; CEU, a northwestern European population; CHB, the Han Chinese in Beijing, China; CHD, the population of metropolitan Denver, Colorado, USA; GIH, the Gujarati Indians in Houston, Texas, USA; HWE, Hardy–Weinberg Equilibrium; JPT, the Japanese population in Tokyo, Japan; LWK, the Chinese living in Luhya in Webuye, Kenya; MEX, people of Mexican ancestry living in Los Angeles, California, USA; MKK, the Maasai people in Kinyawa, Kenya; MTHFR, methylenetetrahydrofolate reductase; PharmGKB, the Pharmacogenomics Knowledge Base; PMT, the Pharmacogenetics of Membrane Transporters database; PUR, a population of Puerto Ricans from Puerto Rico; TSI, the Tuscan people of Italy; VIP, very important pharmacogenetic; YRI, the Yoruba in Ibadan, Nigeria


Phytophthora infestans isolates sampled from seven geographic locations in China. Sensitivity to azoxystrobin and its genetic variation in the pathogen populations was measured by the relative growth rate (RGR) at four fungicide concentrations and determination of the effective concentration for 50% inhibition (EC50). We found that all isolates in the current study were sensitive to azoxystrobin and their EC50 was similar to that detected from a European population about 20 years ago, suggesting the risk of developing azoxystrobin resistance in P. infestans populations is low. Further analyses indicate that reduced genetic variation and high fitness cost in resistant mutations are the likely causes for the low evolutionary likelihood of developing azoxystrobin resistance in the pathogen. We also found a negative correlation between azoxystrobin tolerance in P. infestans populations and the mean annual temperature of collection sites, suggesting that global warming may increase the efficiency of using the fungicide to control the late blight.Knowledge of the evolution of fungicide resistance is important in securing sustainable disease management in agricultural systems. In this study, we analyzed and compared the spatial distribution of genetic variation in azoxystrobin sensitivity and SSR markers in 140  Synthetic fungicides have played an important role in securing food production, improving social economics and human health1P. infestans in 1980Pseudoperonospora cubensis, Plasmopara halstedii and Phytophthora erythosepitica8Synthetic fungicides inhibit or reduce disease development in plants by damaging pathogen cell membranes, inactivating critical enzymes or proteins required for growth and reproduction, interfering with key life processes such as energy production, affecting metabolic pathways such as the formation of sterols and chitin, or by triggering immunity responses in host plants34Knowledge of the evolution of fungicide resistance in pathogen populations is important for planning strategies to increase the effective use of fungicides and reduce the costs of plant disease management. Important factors affecting the emergence of fungicide resistance include the mode of action in fungicide itself and the biology and evolutionary ecology of pathogens such as their mating systems, dispersal mechanisms, the genetic variation and interaction with other species101314161820bc1 enzyme complex (Complex III), thereby blocking electron transfer and halting ATP synthesisBlumeria graminis collected from northern GermanyAzoxystrobin (Syngenta) is a fungicide which has been commonly used around the world to protect field crops, fruits and vegetables. It is the leading synthetic fungicide in the strobilurin family. The fungicide inhibits mitochondrial respiration of pathogens by binding its active compound to Qo in the cytochrome Phytophthora infestans (Mont) de Bary is among the most important plant diseases in the world. It is infamous as the under-lying cause of the Irish potato famine of the 1840s but still is the most devastating disease globally26Potato late blight caused by P. infestans has been widely documented in phenylamide fungicides and is also a concern of potato growers and agricultural chemical manufacturers for other types of fungicides such as azoxystrobin. Numerous transposable elements distributed around functional genes in the P. infestans genome28Potato late blight is mainly controlled by repeated applications of synthetic fungicides including azoxystrobin, together with the deployment of host resistances. In some developed countries, a successful potato harvest may rely on 10–15 fungicide applications each season. However due to its high evolutionary potential, development of resistance in P. infestans occurring in the main Chinese potato cropping regions; ii) investigate the relative role of genetics and plasticity in determining azoxystrobin resistance; and iii) infer the main evolutionary forces driving the evolution of azoxystrobin resistance in P. infestans by comparing spatial distributions of genetic variation in SSR marker loci and azoxystrobin sensitivity.The objectives of this study were to: i) monitor the spatial distribution of azoxystrobin sensitivity in field populations of P. infestans  sampled from seven locations across China were tested for sensitivity to four concentrations of azoxystrobin by calculating the relative growth rate (RGR) of the pathogen in the presence and absence of the fungicide. In all four azoxystrobin treatments, RGR displayed a continuous and unimodal distribution in all but one (Fuzhou) field populations of nfestans . More th15 μg/ml . RGR als15 μg/ml  and the 50) was calculated for each isolate using its RGR in four azoxystrobin concentrations. Though peaking at different levels, EC50 also displayed a continuous and unimodal distribution in both individual field  in azoxystrobin sensitivity was estimated in a way similar to the population differentiation in SSR marker loci (FST) by calculating the proportion of total quantitative genetic variation attributable to among population variation. The overall population differentiations in RGR, EC50 and SSR across the seven pathogen populations were 0.078, 0.072 and 0.116, respectively. The overall QST in RGR and EC50 was significantly lower than FST for SSR marker loci. The correlation between FST in SSR marker loci and QST in RGR and EC50 were 0.16 and 0.11, respectively, but none were significant.Population differentiation P. infestans to the fungicide. Since its introduction, azoxystrobin has been widely used to control plant pathogens included P. infestans in the geographic areas covered by the current study. No major change in sensitivity profile between the studies conducted ~20 years apart suggests that the risk of developing azoxystrobin resistance in the field populations of P. infestans may be low.Mutants with reduced sensitivity have been observed in laboratories and fields in many plant pathogensP. infestans populations is low. Variance analysis indicates that most of the variation in azoxystrobin tolerance is caused by environmental error and plasticity  the same charactersP. infestans isolates (>45%) grew better on the agar supplemented with than without azoxystrobin, under low fungicide doses (0.01 μg/ml). A similar scenario of increasing pathogen growth under low fungicide doses has been observed in many other pathogen-fungicide interactionsWe found that some P. infestans populations from warmer locations are more sensitive to azoxystrobin than those from cooler locations. Temperature is one of main factors regulating the chemical features of molecular compounds. Numerous studies have shown that mortality in animals increases when they are treated with pesticides under elevated temperatures18P. infestans may not be due to toxicity changes in the fungicide.It is interesting to find a negative association between fungicide tolerance and local temperature , suggestP. infestans and mean annual temperature suggests that global warming may increase the sensitivity of P. infestans to the synthetic fungicide, thereby increasing fungicide efficiency and reducing its applications needed. Though our results suggest that the risk of developing azoxystrobin resistance in P. infestans populations is low, further study with the combination of an experimental evolution approach and molecular analysis of target genes is required to confirm the conclusion.It is projected that average temperatures may increase a few of degrees in the next decades40P. infestans were sampled from seven fields located in Fuzhou, Gansu, Guangxi, Guizhou, Ningxia, Xiapu and Yunnan during the 2010 and 2011 growing seasons . The lyophilized mycelia were ground to a powder with a mixer mill . Total DNA was extracted using a Plant gDNA Miniprep Kit  according to the manufacturer’s instructions. The genomic DNA was suspended in 200 μL of ultrapure water and stored at −20 °C.Mycelia (~100 mg) were obtained by culturing P. infestans isolates was amplified with eight pairs of SSR primers42P. infestans genomic DNA (~20 ng), 12.5 μL of 2× PCR Buffer Mix , 1.0 μM each of forward and reverse primers in a 2720 thermal cycler  with the following conditions: initiated with a cycle of 2 min at 94 °C, followed by 35 cycles of 30 s at 94 °C, 25 s at 56–58 °C (dependent on the primers) and 60 s at 72 °C, and finished with an elongation cycle of 5 min at 72 °C. PCR products were loaded into 96-well plates and sent to Ruiboxingke Biotechn. Co. Ltd. (Beijing) to determine fragment sizes using an ABI 3730XL automated DNA sequencer  in which a DNA size ladder was included in each of the samplesGenomic DNA from each of the P. infestans populations) were selected to test for azoxystrobin sensitivity according to a Common Garden design3844P. infestans isolates from long-term storage were revived on rye B agar at 18 °C for 10 days. Mycelia plugs (3 mm in diameter) were taken from the margin of each revived colony and inoculated onto new rye B plates supplemented either with (treatments) or without (controls) azoxystrobin . Azoxystrobin concentrations used in the experiment were 0.01, 0.05, 0.10 and 0.15 μg/ml. Preliminary experiments indicate these doses yielded the best result in differentiating azoxystrobin sensitivity among strains. Many isolates did not grow when a higher dose was used while growth rates in many isolates were not significantly changed when a lower dose was used. The azoxystrobin was first dissolved in dimethyl sulfoxide to make a stock solution and then diluted with double distilled water to the required concentrations. Inoculated plates were kept in the dark at 18 °C and resultant colonies were photographed 3, 5, 7, 8 and 9 days after inoculation. Colony sizes were measured with the image analysis software Assess. All treatments including controls had three replicates.A total of 140 clonal lineages  and effective concentration for 50% inhibition (EC50). RGR of isolates were calculated by dividing the growth rate of an isolate in the presence of azoxystrobin with that in the absence of the fungicide. EC50 was calculated as described previouslyGrowth rates of Gene diversityripc, M, P, I(P), I*C,P*C Eript refer to the mean RGR of replicate r for isolate i in population p at concentration c, the overall population mean, genetic variance among populations, genetic variance within populations, variance due to genotype x concentration interaction, responses of populations to dose effect and the variance among replicates, respectively. In common garden experiments with asexually reproducing species, any among-replicate variation in the phenotypic value of an isolate can be treated as environmental effect. Therefore, variance among replicates in this case is equivalent to the environmental variance of RGR3844where Y50, the model is reduced to:For ECrip is the mean EC50 of replicate r for isolate i in population p. M, I(P), P and Erip indicate the overall mean, genetic variance within populations, genetic variance among populations and variance between replicates, respectively.where YHeritability was estimated by dividing genetic variance within populations with total phenotypic variancePopulation differentiation in RGR was estimated with following formula38452AP, δ2WP, δ2PC, δ2P·E and n are among population variance, within population variance, the variance in population x concentration interaction and the number of environments (concentrations), respectively. For EC50, QST was calculated using the following formula38where δ2AP is the genetic variance in EC50 attributed to among population variation and δ2WP is the genetic variance in EC50 attributed to within population variation. Like FST, QST for RGR and EC50 was also calculated for all possible pairs of populations as well as across all populations.where δFST in SSR loci and overall QST in azoxystrobin sensitivity was evaluated using the standard deviation of QST constructed from 100 resampling of original data as described previously50 among P. infestans populations sampled from different locations. Temperature data for each collection site was downloaded from World Climate (http://www.worldclimate.com/). Annual temperature at each location was estimated based on the mean temperature for each month. Pearson correlationStatistical differences between the overall How to cite this article: Qin, C.-F. et al. Comparative analyses of fungicide sensitivity and SSR marker variations indicate a low risk of developing azoxystrobin resistance in Phytophthora infestans. Sci. Rep.6, 20483; doi: 10.1038/srep20483 (2016).