Eutrema salsugineum , a species growing in highly saline habitats, is a good model for use in salt-stress research. However, its evolutionary migrations and genetic variations within and between disjunct regions from central Asia to northern China and North America remain largely unknown. We examined genetic variations and phylogeographic patterns of this species by sequencing ITS, 9 chloroplast (cp) DNA fragments (4379 bp) and 10 unlinked nuclear loci (6510 bp) of 24 populations across its distributional range. All markers suggested the high genetic poverty of this species and the limited number of genetic variations recovered was congruently partitioned between central Asia, northern China and North America. Further modelling of nuclear population-genetic data based on approximate bayesian computation (ABC) analyses indicated that the long-distance dispersals after the recent origin of E. salsugineum may have occurred from central Asia to the other two regions respectively within 20000 years. The fast demographic expansions should have occurred in northern China in a more recent past. Our study highlights the importance of using ABC analyses and nuclear population genetic data to trace evolutionary migrations of the disjunct distributions of the plants in the recent past. Phryma leptostachya were estimated to diverge anciently within the early to middle Pliocene and this species migrated to eastern Asia via the Bering land bridge after its origin in North America [Intercontinental disjunctions within the Northern Hemisphere have attracted much attentions of biogeography researchers –5. Most  America  although America ,7 probab America . More re America .rbcL and matK) whose genetic variations were used to construct phylogeny at the genus level, show no or fewer mutations within such species. Second, the previous approaches or methods are largely descriptive, without robust modeling and testing of alternative scenario. However, it should be noted that population genetic data based on sequencing multiple loci prove a powerful tool for overcoming these limitations and addressing related questions [However, it is difficult to trace evolutionary migrations of such plants with recent disjunctions for two reasons. First, the commonly used DNA fragments  is disjunctly distributed from central Asia to North America. This species as well as its two close relatives, E. halophilum  and E. botschantzevii (= Thellungiella botschantzevii) were together placed in Thellungiella, but now in Eutrema [Arabidopsis, seeds of these species are very small and probably dispersed mainly by wind [E. (Thellungiella) halophilum. In fact, they should be ascribed to E. salsugineum while the true E. halophilum occurs only in central Asia [E. halophilum is outcrossing with pinnate leaves and fewer seeds while E. salsugineum is self-compatible with entire leaves and more seeds [E. salsugineum occurs widely but disjunctly in saline habitats from central Asia to northern China and North America. The past collection records from the herbaria specimens suggest that the occurrence of this species in both central Asia and North America is infrequent and it is only limited to one and adjacent small localities. However, this species occurs commonly in northern China where it was collected from numerous localities. However, genetic diversity and phylogeographic history of this salt cress species remain largely unknown although similar studies on other model species, for example, A. thaliana as well as its close relatives, have received extensive attention and are clearly clarified (e.g. [ Eutrema –13. They Eutrema . Like Ar by wind . In addi by wind . In nume by wind –20). The by wind . Howeverral Asia . These ted (e.g. ).E. salsugineum across central Asia, northern China and North America at the population level. We firstly examined the nuclear ITS variation between all samples and no variation at this DNA fragment confirmed that these samples originated from a common ancestor and should be taxonomically placed within one single species. We then sequenced nine maternally inherited chloroplast DNA fragments , which are highly variable between intraspecific populations. We finally examined the sequence variation at 10 unlinked nuclear loci (around 6500 bp), all of which are known to be highly polymorphic within Arabidopsis thaliana . We . We E. sE. salsugineum species and its two close relatives, E. halophilum and E. botschantzevii that are not endangered, and these plants grow in public area where no permission for collection of leaves is needed in China, Russia, Kazakhstan and Canada.All leave samples employed in this study were collected from E. salsugineum, one from Xinjiang, three from North America, four from Russia and the others mainly from northern China. Around 15 individuals for each population growing at least 50 m apart were collected in the field. However, only three to six individuals  for each population were used for the final phylogeographic analyses because our initial scanning of these individuals using most different markers failed to recover any variation between sampled individuals of each population. All leaves were dried and stored in silica gel. The latitude, longitude, and altitude of each population were recorded using a GPS, and these data were noted on a map using ArcMap in ArcGIS9.2 . Sequencing reactions were conducted with the same PCR primers using an ABI Prism Bigdye Terminator version 3.1 Cycle Sequencing Kit. We conducted the following sequencing by an ABI 3730XL DNA Analyzer. All the sequences have been submitted to GenBank . All obtained sequences were aligned using CLUSTAL X version 1.81 [We extracted DNA using the modified cetylcrimethyl ammonium bromide (CTAB) procedure described by Doyle and Doyle . We amplna e.g. –24). The. TheA. tion 1.81  and doubS, the number of segregating sites; Nh, the number of haplotypes; He, the haplotype diversity; and the nucleotide diversity (π and θ) [We used DnaSP v. 5.00  to estimπ and θ) –31. Haplπ and θ) . Tajima’π and θ) , Fu and π and θ) , as wellπ and θ)  statistiK, where 1≤K≤10, was assessed and allowance made for the correlation of allele frequencies between clusters. Twenty runs were performed with a burn-in of 100,000 and then 1,000,000 iterations. The most likely number of clusters was estimated using the original method described by Pritchard et al. [K statistics given in Evanno et al. [We used STRUCTURE version 2.3.2  to assesd et al.  and the o et al. . Second,o et al. .P values calculated as the proportion of simulations that produced a larger SSD than the observed SSD. We calculated the raggedness index (RAG) and its significance to quantify the smoothness of the observed mismatch distribution. Estimation and testing were conducted using Arlequin version 3.0 [S. As suggested, this statistic is very sensitive to recent demographic expansion for which large, negative values are typically obtained [To test for population expansion, based on nDNA sequences, mismatch distributions of the observed number of nucleotide differences between pairs of nDNA sequences were computed using Arlequin version 3.0 . We usedsion 3.0  with 1,0obtained . To asseobtained , a coaleE. salsugineum based on population genetic data from 10 loci. The assorted population genetic data were simulated under four hypothesized scenarios with population divergence, population size change and admixture [E. salsugineum as observed for all populations.ABC is as a powerful approach to select obtain the most suitable demographic history by statistically testing alternative hypotheses . We useddmixture . Three gTotal/KS × L, where L is the length of the locus, KTotal/KS is the ratio of the number of all substitutions per substitution site  to the number of synonymous substitutions per synonymous site (KS) and μCHS is the substitution rate per synonymous site per year of the CHS gene in Brassicaceae, estimated to 1.5 × 10–8 substitutions per site per year [–6 . We also used the mutation rate (μ = 5–10.0 × 10–9 per site per year) recorded for other genera of the same family [E. salsugineum-E. halophilum and E. botschantzevii [During the ABC analyses, we used the method of Ikeda et al.  to estimper year . The finyear [–6 , substite family  for ITS antzevii .E. salsugineum  [E. salsugineum—E. halophilum together diverged from E. botschantzevii between 240 and 480 thousand years ago.All ITS sequences showed no variation with the sampled 99 individuals of sugineum . In addiutations . Accordier year)  was adoppsbA-trnH, was found to be polymorphic  from central Asia Altai population and one mutation fixed in E. botschantzevii. The nucleotide diversities between all E. salsugineum individuals at the psbA-trnH locus and all cpDNA loci were estimated to be θ = 0.00345 and π = 0.00281, and θ = 0.00032 and π = 0.00026, respectively  and a total of ten non-coding mutations were recovered at four loci . Pairwise sequence diversity (θ) ranged from 0 to 0.00102 while π ranged from 0 to 0.00084  and ΔK were computed by means of STUCTURE analysis, using the runs with highest probability for each value of K. Bayesian clustering estimated the uppermost hierarchical level of structure at K = 10 groups, in the absence of a priori classification. However, the largest break in L(K) was located at K = 2 and K = 3 based on the modal value of ΔK as an indicator of the uppermost level of hierarchical structure [K = 2, two populations from central Asia (populations 17 and 18) (Group A) were separated from other populations  and 16 populations (1–16) from northern China while Group C consisted of one population (19) from central Asia and other four populations from Russia and North America (21–24).We further sequenced 10 unlinked nuclear loci  and the  0.00084 . The ave 0.00084 . The postructure  S2 Fig)CHS) and ulations . When K Analyses of both cpDNA haplotype distributions suggested distinct regional differentiation between central Asia, northern China and North America. AMOVA analyses also supported this inferences (results not shown). We further placed three populations from Russia into Group B  and Group C  as inferred from STUCTURE analyses and examined their genetic differences with the Group A from central Asia (two populations 17–18). We found that 76.82% of the nuclear variations were partitioned between these three groups, and 20.22% between populations .The mismatch analyses based on nuclear data for 17 populations for Group B from northern China plus Buriatia suggested a distinct population expansion . Further–6 substitutions per year per locus  of A. thaliana from 11 cpDNA segments was estimated to be 0.00169. Its nucleotide diversity (θ) observed from 334 nuclear loci has been estimated to be 0.00896 [RPS1 and PGIC loci have been estimated to be 0.0126 and 0.00380 for the mesic A. thaliana [E. salsugineum. In addition, we found no genetic variation in the other five loci    is similE. salsugineum compared with the mesic A. thaliana contrasts to species examined in comparative studies in Israel’s “Evolution Canyon” [E. salsugineum. In order to adapt to highly saline habitats, this species may have developed specific traits, with genetic diversity decreasing as all populations stabilized on these traits [The greatly lower genetic diversity in  Canyon” . In fact Canyon” . High ene traits . Any mute traits . In addie traits . In othee traits . Howevere traits . The cone traits . In addiE. salsugineum and E. halophilium have the same ITS sequence, which differs from another species E. botschantzevii with only three mutations. Based on the mutation rate recorded for other genera of the same family [E. salsugineum and E. halophilium together diverged from E. botschantzevii 240–480 thousand years ago (kya). The further divergence between E. salsugineum and E. halophilium should have occurred at a later stage. These divergences fall within the middle Pleistocene when central Asia became drier than before and desertification and salinization began to develop and expand [E. salsugineum and its divergence with the closely related species in central Asia [Since all sampled individuals of e family , E. salsd expand . This haral Asia . After iA. thaliana, are very small, it is highly likely that the frequent sandstorms since the late Pleistocene [E. salsugineum differs distinctly from the previous studies , b,63, bA. E. salsugineum originated and diverged from its two closely related species very recently. It started its long-distance dispersal to northern China and North America very recently. When this species arrived at the northern China, it may have expanded rapidly in the recent past. The recent origin and long-distance fast colonization together resulted in the low genetic diversity of this species despite the fact that this species seem to be distributed in a large scale from central Asia to North America. Such a genetic homogeneity across the total species’ range corroborate the idea that this species will prove a good model for salt-stress research (e.g. [Our results obviously suggested that ch e.g. –17) beca becaE. sS1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S1 Table(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.S3 Table(DOC)Click here for additional data file.S4 Table(DOC)Click here for additional data file.S5 Table(DOC)Click here for additional data file.S6 Table(DOC)Click here for additional data file.S7 Table(DOC)Click here for additional data file.S8 Table(DOC)Click here for additional data file.S9 Table(DOC)Click here for additional data file.S10 Table(DOC)Click here for additional data file.S11 Table(DOC)Click here for additional data file.S12 Table(DOC)Click here for additional data file.


Dispersal of pollen and seeds are essential functions of plant species, with far-reaching demographic, ecological and evolutionary consequences. Interest in plant dispersal has increased with concerns about the persistence of populations and species under global change. We argue here that advances in plant dispersal ecology research will be determined by our ability to surmount challenges of spatiotemporal scales and heterogeneities and ecosystem complexity. Based on this framework, we propose a selected set of research questions, for which we suggest some specific objectives and methodological approaches. Reviewed topics include multiple vector contributions to plant dispersal, landscape-dependent dispersal patterns, long-distance dispersal events, spatiotemporal variation in dispersal, and the consequences of dispersal for plant communities, populations under climate change, and anthropogenic landscapes.The online version of this article (doi:10.1186/s40462-014-0016-3) contains supplementary material, which is available to authorized users. Pollen and seed dispersal are essential functions of plants, with far-reaching consequences for reproduction, population and community dynamics, neutral and adaptive evolution and, ultimately, population and species persistence. Because an understanding of gene and individual movement capacities is critical to predicting the response of individuals, populations and species to ecosystem perturbation and climate change, the long-standing interest in plant dispersal has seen an upsurge in recent years. Extensive monographs have recently dealt with the ecology and evolution of dispersal of organisms in general  and of pHere, we pose eight general questions that we believe will define some of the research frontiers in plant movement ecology in the coming years. We do not attempt to answer these questions, or to exhaustively review the state-of-the-art in these areas, but rather offer our perspectives regarding a selection of important research topics, with an emphasis on specific empirical objectives and methods. The paper is oriented along three axes, representing three fundamental dimensions that challenge ecological inference and models in general, and dispersal ecology research in particular: space, time and complexity Figure . SpatialWithin this broad framework, we first argue that mechanistically accounting for the relative contribution of multiple vectors to dispersal of particular plant species constitutes an essential basis for explaining and predicting dispersal patterns in spatiotemporally changing ecosystems . Next, wIt is now acknowledged that for many if not most plant species multiple vectors contribute to dispersal : polychoThe relative contribution of multiple vectors to dispersal can be investigated using empirical and modelling approaches. Dispersal kernels  can be estimated empirically based on direct observation of propagule deposition patterns at a sample of settling locations (Eulerian methods), or by tracking individual propagules (Lagrangian methods), most frequently over short to intermediate scales e.g. ,14-19). -19. 14-1Mechanistic approaches are valuable for understanding landscape-dependent dispersal patterns  and the Historically, most studies of seed and pollen dispersal have described dispersal patterns exclusively in terms of the distribution of deposition distances from sources e.g. ,31). Cle. Cle31])In many systems, the probability of a seed ending up in a particular location depends on the type of substrate or habitat at that location – deposition is essentially habitat-specific . A speciet al. e.g. [95]Although historical LDD is interesting for biogeographic, population genetic and evolutionary studies, broad-scale patterns of dispersal under current (non-equilibrium) demographic and environmental conditions are becoming of greater concern. We anticipate growing emphasis on contemporary dispersal research spanning increasingly larger scales, using spatially and environmentally explicit approaches, and distinguishing effective dispersal (leading to successful establishment or reproduction) from basic dispersal (encompassing only propagule movement from source to deposition sites) . TrackinThe combination of mechanistic and genetic methods could be formalized within an ‘inverse problem’ framework: parameter estimates of the underlying mechanisms are retrieved from the (noisy) observation of resulting spatial patterns through mechanistic-statistical models  or stateFor wind-dispersed propagules, mechanistic models for among-population migration rates could embed regional wind data in the form of connectivity maps, describing the probability of basic seed or pollen dispersal along possible trajectories linking a set of locations ,112, as This combined genetic-mechanistic framework might also be applied to continuously distributed species, provided genetic assignment remained feasible. If significant clinal genetic variation were present over the spatial scale of the dispersal study, genotypic probabilities for dispersed propagules at any given location could be expressed as a continuous function of distance along the allele-frequency cline, potentially enabling the estimation of the LDD component along this direction. In the case of non-clinal (patchy) spatial genetic structure, allele frequency smoothing techniques may allow genetic assignment of propagules to a set of sampled and unsampled sources across the species range ,126, altPlant dispersal kernels are expected to be phenotype- and environment-dependent, given the number of intrinsic and extrinsic variables influencing the release, transport and settlement of seeds and pollen. Less evident is the relative weight of each variable, and how dispersal kernel variation is hierarchically distributed across individuals, populations and species, as well as over time. We deal with temporal dispersal variation in At a narrower spatial scale, dispersal kernel variation within populations is primarily caused by local-scale heterogeneity in phenotypic dispersal traits and/or by the effects of local environmental variation on dispersal vectors , drift (stochastic changes in species abundances), and speciation . The impSeveral types of empirical studies to date have provided insights into the role of seed dispersal in community patterns, but each has major shortcomings. Empirical analyses of species turnover in space (beta diversity) often invoke seed dispersal as the explanation for distance-dependent patterns not explained by environmental variation ; howeveThe lack of good empirical tests of theory on the importance of seed dispersal to communities in part reflects a mismatch between the simplistic way in which dispersal is generally represented in models and the more complex dispersal patterns observed in most real ecosystems. Most models set seed dispersal rates as identical for all species, when in the real world dispersal rates invariably vary greatly among species within communities. Further, a common approach is to model seed dispersal as a dichotomy of within-patch vs. between-patch dispersal using a metacommunity framework ,193. Fewet al. )The prospects are particularly worrying for animal-mediated pollen and seed dispersal, because plant-animal mutualisms tend to be negatively affected by most drivers of GEC . For exaGeneralizing the effects of multiple drivers of GEC on communities is challenging, because communities are interlinked by interactions of variable sign and strength, and because these effects are likely to be scale- and species-dependent  approaches that, in the unavoidable trade-off between spatial scope, sampling intensity and accuracy, should seriously assess expected statistical power and uncertainty for low-frequency  dispersal rates, model miss-specifications, and limited sampling. Temporal scale issues will pervade plant dispersal ecology studies, from a more meaningful characterization of average dispersal patterns given variation in dispersal within and among seasons, through the assessment of the consequences of such temporal dispersal variation for population and community dynamics, to long-term predictions about population and species persistence based on observed and modelled feedbacks between dispersal, demography and evolution in changing environments. Finally, sufficiently approximating the dynamic complexity of environments, ecological networks and communities will be essential for characterizing all relevant biotic and abiotic mechanisms driving plant dispersal and their sensitivity to global change, and for better understanding the ecological consequences of dispersal in changing environments. We will certainly need to increasingly pool data and expertise from multiple disciplines to meet these big challenges, for which we advocate not only further cooperative research efforts, but also the implementation, standardization and usage of open repositories of dispersal data and models.


Euphydryas aurinia individuals (46 European locations) for 17 allozyme loci, and sequencing a subset of 492 individuals (21 sites) for a 626 base pairs COI fragment. Genetic diversity indices, F statistics, hierarchical analyses of molecular variance, individual-based clustering, and networks were used to explore the phylogeographic patterns. The COI fragment represented 18 haplotypes showing a strong geographic structure. All but one allozyme loci analysed were polymorphic with a mean FST of 0.20, supporting a pronounced among population structure. Interpretation of both genetic marker systems, using several analytical tools, calls for the recognition of twelve genetic groups. These analyses consistently distinguished different groups in Iberia (2), Italy, Provence, Alps (3), Slovenia, Carpathian Basin, the lowlands of West and Central Europe as well as Estonia, often with considerable additional substructures. The genetic data strongly support the hypothesis that E. aurinia survived the last glaciation in Mediterranean, extra-Mediterranean and perialpine refugia. It is thus a rare example of a model organism that combines attributes of faunal elements from all three of these sources. The observed differences between allozymes and mtDNA most likely result from recent introgression of mtDNA into nuclear allozyme groups. Our results indicate discrepancies with the morphologically-based subspecies models, underlining the need to revise the current taxonomy.Ice ages within Europe forced many species to retreat to refugia, of which three major biogeographic basic types can be distinguished: "Mediterranean", "Continental" and "Alpine / Arctic" species. However, this classification often fails to explain the complex phylogeography of European species with a wide range of latitudinal and altitudinal distribution. Hence, we tested for the possibility that all three mentioned faunal elements are represented within one species. Our data was obtained by scoring 1,307  Climatic oscillations with ice ages and interglacial periods have had strong impacts on the distribution of European animal and plant species e.g. ,2). In t. In t2])“Mediterranean” species usually had important glacial differentiation centres at least in one of the three major Mediterranean peninsulas, but also in the Maghreb and Asia Minor ,5. Howev“Continental” species are supposed to have survived during ice ages in extra-Mediterranean refugia  with mor“Arctic” and/or “Alpine” species often survived ice ages in perialpine refugia and retreated to alpine and/or arctic areas when temperatures increased after their inter-/postglacial deglaciation ,10.Vipera berus [Arion fuscus [This classification is simplistic and often fails to explain the rather complex phylogeography of European species with a currently wide range of latitudinal and altitudinal distribution. Thus, a number of continental elements also had important glacial refugia in the Balkan Peninsula . Mediten fuscus . Severaln fuscus ,10. Furtn fuscus ,15. Thisn fuscus ,17. Varin fuscus , but theEuphydryas aurinia  . This species is a univoltine butterfly found from Mediterranean shrub-lands at sea level to alpine meadows, and from the European Atlantic coast throughout temperate Asia [Quercus woodlands in the Iberian Peninsula ) [E. aurinia aurinia Rottemburg, 1775) [E. aurinia debilis Oberthür, 1909) and the Alps  [Lonicera, Succisa, Scabiosa and Gentiana [Therefore, we selected a particularly widespread and ecologically diverse species as study object, the fritillary ate Asia . It colo, 1851)) ,20, dry g, 1775) –24 as wey, 1928) ,26. FurtGentiana ,28. Due Gentiana ; recent Gentiana , with siEuphydryas aurinia is highly threatened in Central Europe due to dramatic damage of its habitats during the last few decades [ decades –33 and h decades ,34–36. B decades ,37.E. aurinia may represent, within a single species, all three major biogeographic groups known for Europe . By appE. aurinia individuals (46 populations) distributed over major parts of Europe for allozyme electrophoresis  from southern Portugal was included as outgroup . The saSpecific permission for this study were granted by: Instituto da Conservacao da Natureza Lissabon for Portugal, Nature Protection Agency of the Province of Extremadura, Nature Protection Agency of the Province of Aragon, Nature Protection Agency of the Province of Catalonia, Directory of the National Parc of Majella , Naturschutzbehörde des Landes Salzburg, Obere Naturschutzbehörde des Saarlandes, Ministère de l'Environnement (Paris). Several samples were provided by local collectors or were collected together with them. No rights on private land were violated.2, 0.2 mM of each dNTP, 0.4 μM of each primer, 1U GoTaq Flexi DNA polymerase (Promega) and 50 ng template DNA. PCR cycling conditions were as follows: initial denaturation of 5 min at 95°C; followed by 40 cycles of 1 min at 95°C, 1 min at 51°C, 1 min at 72°C; and a final extension step of 10 min at 72°C. PCR products were purified by ethanol/sodium acetate precipitation. Sequencing was performed with the primer LCO1490 on an ABI 3130xl automated sequencer . Sequences were checked, manually edited and aligned using Geneious ver. 5.4 (http://www.geneious.com/). All sequences were deposited in GenBank (Accession Numbers: KT896753—KT897244).Total genomic DNA was extracted from butterfly legs using an adapted Chelex-100 (Bio-Rad) protocol . A partiE. aurinia individuals, the whole abdomen of each imago was homogenized with ultrasound in Pgm-buffer [For allozyme electrophoresis of 1,307 m-buffer  and centm-buffer ,44. A toDnaSP 5.10 [Arlequin 3.5 [jModelTest [FST estimator and Jost’s [Dest. Calculations were executed in the Diversity R package [Information on mtDNA haplotype diversity, nucleotide diversity and frequency of each haplotype was extracted using aSP 5.10  and Arlequin 3.5 . The besodelTest . AccordiodelTest , the TamodelTest  model fiodelTest , availabodelTest  that conodelTest . PairwisodelTest  FST estd Jost’s  Dest. C package .-stat [A, expected heterozygosity He, observed heterozygosity Ho, total percentage of polymorphic loci Ptot and percentage of polymorphic loci with the most common allele not exceeding 95% P95). We calculated allozyme allelic richness (AR) with Fstat [N < 7). Allozyme locus-by-locus analyses of molecular variance (Amova), hierarchical genetic variance analyses, test of Hardy-Weinberg equilibrium and linkage disequilibrium were performed with Arlequin 3.5 [Phylip 3.5.c [Allozyme alleles were labelled according to their relative mobility during electrophoresis. We used G-stat  to computh Fstat  to allowquin 3.5 . Nei´s squin 3.5 , neighboquin 3.5  and bootip 3.5.c .SAShA) method [Geneland 2.0 [K was set from 1 to 21 clusters on a correlated frequency model. We inferred the number of clusters from the modal value of K with the highest posterior probability.Spatial genetic structure was assessed using two different approaches. First, we used the Spatial Analysis of Shared Alleles  method  to establand 2.0  producedE. aurinia from 21 sites in Europe for 626 bp of the COI gene. No pseudogenes were present as evidenced by the absence of stop codons, the prevalence of synonymous substitutions or low pairwise divergence. A total of 18 haplotypes were identified, including two non-synonymous and 17 parsimony informative sites  (Dest and in 159 (84%) when using FST  . Pairwising FST .Geneland analyses detected nine major clusters (mt1 –mt9) that correspond to Iberia (mt1), Pyrenees (mt2), Provence (mt3), Alps  (mt4), Brittany (mt5), Europe north of the Alps and the Pyrenees including Northern Europe (mt6), northern Apennines (mt7), Slovenia (mt8) and eastern Carpathian Basin (mt9) (Dest and FST pairwise comparisons between clusters mt4 and mt6 were not significant (in (mt9) . Howevernificant .E. desfontainii, as all E. aurinia populations were monomorphic for this locus. The number of alleles per locus ranged from two (Mdh1) to ten  (mean: 5.4 ± 2.7 SD). Allele frequencies can be obtained from the authors on personal request.All 17 loci analysed were polymorphic and showed banding pattern consistent with known quaternary structures . The polA) ranged from 1.41 to 2.53, with a mean of 2.00 (± 0.26 SD), while allelic richness (AR) ranged from 1.30 to 1.76 with a mean of 1.51 (± 0.10 SD) . The highest values for allelic richness were found in populations from Provence, the western Alps and the Carpathian Basin. For the polymorphic loci with the most common allele not exceeding 95% (P95), frequencies ranged from 23.5% to 52.9%, mean: 42.1% (± 8.5% SD), while the total percentage of polymorphic loci (Ptot) ranged from 35.3% to 82.4% with a mean of 57.9% (± 10.2% SD). The mean expected heterozygosity (He) was 14.8% (± 2.7% SD), ranging from 9.1% to 22.9%, and the mean observed heterozygosity (Ho) was estimated at 14.6% (± 2.6% SD), varying from 9.5% to 22.4%. The genetic diversity of the outgroup population E. desfontainii was considerably lower than the average of E. aurinia.The mean number of alleles per locus (0.10 SD) . ExcludiNone of the loci/population combinations showed any significant deviation from Hardy-Weinberg equilibrium, after Benjamini-Hochberg procedure for multiple testing ,63. TherE. aurinia populations was 1.585 with 0.326 genetic variance among populations  and 0.020 genetic variance among individuals within populations  (E. desfontainii as outgroup).The total genetic variance of all 46 < 0.005) . Excludip = 0.001)  . HoweverE. aurinia  within E. aurinia  and the Italian group   showed remarkable within-group differentiation, while five other groups had low FST values ranging from not significant to 0.0246  showed the strongest differentiation among populations . The mean genetic distance [Hierarchical variance analyses showed a strong differentiation between the samples of distance  between E. aurinia of the Ponto-Mediterranean refugium with its putative substructures , Italy, Provence, Alps (3), Slovenia, Carpathian Basin, lowlands of West and Central Europe as well as Estonia, often with considerable additional substructures. However, despite the mostly consistent genetic pattern found between allozymes and mtDNA, differences were detected in peninsular Italy, the Alps, West and Central Europe as well as Estonia. In spite of the wide geographic sampling undertaken in the present study, the main caveat of this publication is the lack of samples from the Balkan Peninsula. This prevents us clarifying the importance for , Italy, FST values obtained here for E. aurinia are higher than those given previously for this species in nationwide surveys [FST = 0.0246) is much lower than that given in a previous study of this area with more samples  and  and FSTae e.g. ,71,72). ,72. FSTatyrinae –75 or Lyatyrinae ,77 and imintheus  and in tE. aurinia  and its Italian populations . The name E. aurinia aurunca Turati, 1910 [E. aurinia glaciegenita according to their distribution, while the clustering shows that al4 and al5 together belong to a different cluster than al6, which positions itself within E. aurinia aurinia groups; subspecies E. aurinia glaciegenita therefore does not correspond to a single cluster. Its eastern component, group al6 from high altitude Austria, may have had genetic exchange with lower altitude populations, without substantial changes in its phenotype  and ecological requirements (i.e. it feeds on Gentiana), which are under strong selection in Alpine habitats. Our results clearly show that the Alpine populations sometimes known as Euphydryas glaciegenita belong to Euphydryas aurinia, and should not be given specific status [The clustering of the studied samples strongly suggests that recognised subspecies do not correspond to phylogenetic groups. ti, 1910  may applc status .E. aurinia within Europe argues for the existence of several refugia during the last glaciation, if not before this. A similar assumption was made by Varga [E. aurinia a polycentric species with holo-Palaearctic distribution. However, this author provided no details about the number and locations of refugia.The high degree of genetic differentiation of by Varga  who consE. aurinia. Our analyses revealed independent genetic groups in south-western Europe  and Italy  which respectively indicate an atlanto-Mediterranean and an adriato-Mediterranean glacial refugium for this species. There might also be several subrefugia in Iberia and Central Italy  and south of the Pyrenees    during tructures ,20, and ulations –24, is tE. aurinia in Italy is supported by phylogeographic structures of many other species showing often numerous genetic groups and hence core areas scattered over Italy [Similar events might have taken place in Central Italy , although no clear conclusions can be drawn concerning the location of different subrefugia in this area because only four populations were analysed. However, polycentricity of er Italy –102.Furthermore, the more southern Italian population IT3 shares its haplotype with populations from the Alps and has no haplotypes endemic to peninsular Italy, unlike the more northern population IT2. This discrepancy between nuclear and mitochondrial genes is most likely due to mitochondrial introgression from a more northern perialpine centre of survival (see below) along the Apennines to the South.E. aurinia could not currently be verified due to lack of samples. However, the descriptions of independent subspecies from the Balkans  are evidence for further centres of genetic differentiation in this area. Furthermore, refugia in the Balkan Peninsula are the rule for widespread species of Mediterranean origin [E. aurinia.A ponto-Mediterranean refugium of n origin , thus maE. aurinia due to the remarkable genetic differentiation of the populations from Provence for both genetic marker sets   . The mucpolyxena , Carabusronitens , Natrix x natrix , Vipera ra aspis , Quercusus suber  and Faguylvatica .E. aurinia in West and Central Europe. We suggest that the origin of the western group  lies in an extra-Mediterranean refugium in Central France  might have had its origin east/south-east of the Alps, also an important area of extra-Mediterranean glacial refugia for many animal and plant species  and Bohemia (western Czech Republic). This refugium may have been in close contact with a Slovenian refugium, which might have been just a subrefugium of the former, in which stronger postglacial introgression processes led to its populations’ uniqueness.The Central European group of es e.g. ,107,108),108E. auGenetic differentiation at the nuclear  and mitochondrial (mt9) level speaks for an independent extra-Mediterranean glacial refugium in the Carpathian Basin. The extraordinarily high genetic diversities of the here analysed populations from Transylvania support the high importance of this Basin as a centre for extra-Mediterranean survival with particularly favourable conditions , as alsoCoenonympha arcania [E. aurinia might have their origin in the East  seem unral, cf. ). HoweveGentiana species) and do not show protandry like lowland populations do [All populations from the Alps belong to the distinct mtDNA group mt4. These Alpine populations consistently show a different phenotype, which is considerably smaller than all ordinary lowland populations, and the general colouring of the wings is much darker; furthermore, these individuals have a different mode of flight , use different larval food plants , so that three Alpine genetic groups can be distinguished  (Tables FCT = 0.0663), indicating that both groups arose by allopatric differentiation during presumably one ice age. This relatively short time of divergence accords with the lack of mtDNA differentiation among the Alpine populations. The respective refugia might have been located near to the foot of the Cottian Alps  and in the area south of the Upper Italian lakes   Tables . This caes  . Especiaelongata .E. aurinia in the Hohe Tauern  show strong genetic differentiation from those of the western and central Alps (FCT = 0.1530). This result indicates a longer isolation of the populations of the eastern Alps (but note the missing differentiation at the mtDNA level) and an independent refugium in this region. Especially the area south of the Carnian and Julian Alps apparently offered suitable environmental conditions for many species, often resulting in higher genetic diversities of the genetic groups in the eastern Alps [E. aurinia. Postglacial expansion might have been mainly in a northern direction into the higher Alps, as in the Slovenian populations , but with the latter showing some introgression from the eastern Alps and the Apennines group at the mtDNA level.The populations of ern Alps ,122–124.Euphydryas aurinia shows a large variety of morphological as well as ecological adaptations within Europe, which have been the cause of continuous taxonomic debate. However, the results of the present study revealed that the ecological adaptations are most likely the result of strong intraspecific differentiation processes and not a result of the existence of a super-species complex. Hence, E. aurinia represents a rare model organism that combines attributes of Mediterranean, continental as well as alpine faunal elements within one single species. The complex phylogeography of E. aurinia might therefore be an example of more complicated patterns of differentiation in species showing adaptations to various climatic and altitudinal conditions, as well as host plants, across the western Palaearctic.S1 TableSite abbreviations defined in (PDF)Click here for additional data file.S2 TableGroups defined in (PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.


Pinus engelmannii Carr. is one of the nine most common pine species in the pine-oak forest ecoregion in the state of Durango, Mexico. This species is widely harvested for lumber and is also used in reforestation programmes. Interspecific hybrids between P.engelmannii and Pinus arizonica Engelm. have been detected by morphological analysis. The presence of hybrids in P. engelmannii seed stands may affect seed quality and reforestation success. Therefore, the goals of this research were to identify introgressive hybridization between P. engelmannii and other pine species in eight seed stands of this species in Durango, Mexico, and to examine how hybrid proportion is related to mean genetic dissimilarity between trees in these stands, using Amplified Fragment Length Polymorphism (AFLP) markers and morphological traits. Differences in the average current annual increment of putative hybrids and pure trees were also tested for statistical significance. Morphological and genetic analyses of 280 adult trees were carried out. Putative hybrids were found in all the seed stands studied. The hybrids did not differ from the pure trees in vigour or robustness. All stands with putative P. engelmannii hybrids detected by both AFLPs and morphological traits showed the highest average values of the Tanimoto distance, which indicates: i) more heterogeneous genetic material, ii) higher genetic variation and therefore iii) the higher evolutionary potential of these stands, and iv) that the morphological differentiation (hybrid/not hybrid) is strongly associated with the Tanimoto distance per stand. We conclude that natural pairwise hybrids are very common in the studied stands. Both morphological and molecular approaches are necessary to confirm the genetic identity of forest reproductive material.Hybridization is an important evolutionary force, because interspecific gene transfer can introduce more new genetic material than is directly generated by mutations.  Hybridization and backcrossing to one or both of the parental types can lead to incorporation of alleles from one taxon into the gene pool of the other . InteracPlant hybridization often leads to the formation of hybrid zones . As narrF1) hybrids, particularly between geographic races or closely related species, tend to exceed their parents in vegetative vigour or robustness ..F2) and Although most studies concerning hybridization are based on morphological evidence, introgression or hybridization are not necessarily indicated by the phenotypic expression of characters of one taxon in another. Identical characters in species may occur as a result of phenotypic plasticity, convergent evolution, or simply common ancestry . FurtherThe use of molecular markers has shown that interspecific hybridization is even more common than assessed by morphological and cytogenetic evidence alone. The hybrid nature of many species has indeed been confirmed by molecular studies  15] [16 [1615] , ii) lacPinus section Trifoliae with subsections Australes, Contortae, and Ponderosae)  [28 [2827] ). Recentderosae) . Pinus sderstood  32] [33 [33Pinusderstood  35] [30 [30Pinuspossible 37]38][[38][Pinu[[38] . Th. Th55]. Pe, Pco and Pdu are very closely related taxa [Because ted taxa  34]    and and60] aP. engelmannii stand includes hybrid trees, these should possess a genome that is a blend of alleles derived from both P. engelmannii and other pine species, caused by gene flow. These hybrids are detectable by genetic and/or stable morphological traits (whose expression is under exclusive genetic control), as stated in P. engelmP. engelmannii and other pine species, 280 P. engelmannii trees were screened. The alternative hypothesis was that there is no hybridization among the studied species.In order to test the hypothesis that genetic introgression exists between P. engelmannii to P. arizonica var. cooperi, ii) P. engelmannii to P. durangensis separately for the western and the eastern stands.In order to determine the degree of hybridism, 204 AFLP markers were used in four separate STRUCTURE (version 2.3.4) analyses  65] to  to 65] tK) = 2 was used for this purpose. If the probability of P. engelmannii affiliation of a putative P. engelmannii tree was less than 95% according to STRUCTURE, then that individual was recorded as a candidate hybrid. The affiliation probability was measured by the proportion of the dominant STRUCTURE populations in the eight P. engelmannii studied stands. Individuals were identified as first-generation (F1) hybrids when the probability of P. engelmannii affiliation of a putative P. engelmannii tree was in the range 45–55%.The Bayesian clustering method, as implemented in STRUCTURE was used to test whether affiliation of individuals to species on the basis of morphology was congruent with the assignment based on the AFLP markers. The population number (P. engelmannii are shown in parenthesis: crown apex (rounded), crown density (open), twig diameter (1–3 cm), needle length (20–43 cm), needle width (1–2 mm), needle thickness (ca 1 mm), needle colour , number of stomata on dorsal face of needle (6–23), needle position (spreading-ascending to slightly drooping), young sheath length (2–4 cm); mature cone shape (ovoid to oblong), mature cone length (7–16 cm), mature cone width (6–12 cm), cone apophysis . Characters not useful for distinguishing P. engelmannii from other related species (e.g. number of needles per fascicle) were not considered in this study.Then, in order to detect the degree of hybridization by morphological traits, vegetative and reproductive characters were assessed by using 14 qualitative and quantitative traits. Any tree displaying differences in these traits according to  and 50]50] was cfhyp) in each P. engelmannii stand, the AFLP data were also used to calculate the mean genetic dissimilarity between the binary vectors of two individuals in each stand, a and b, using the Tanimoto distance (TD)  were only produced by random events, rather than directed forces, a one-sided permutation test was performed  < 0.05) ). M). MP. en also by  for Sibe. Re. ReArbuted areas  75] ind indArbuted areas . The coned areas . New resP. engelmannii stands under study and that these hybrids are sometimes not morphologically obvious. Considering that the AFLPs results are less biased in the estimation of hybrids, morphological traits are not a good proxy to estimate hybridization in this species.Thus, both morphological and molecular approaches are necessary to confirm the genetic identity of forest reproductive material. Molecular approaches should involve a combination of plastid data, nuclear and mitochondrial DNA sequences and crossing experiments. The genetic identity of forest reproductive material is essential for a complete understanding of tree species phylogeny, for developing effective breeding programs, and for seed quality and reforestation success [We conclude that natural pairwise hybrids are very common in the  success  44] [77 [77P. enS1 Dataset(XLSX)Click here for additional data file.


Yersinia pestis was introduced to North America around 1900 and leads to nearly 100% mortality in prairie dog (Cynomys spp.) colonies during epizootic events, which suggests this pathogen may exert a strong selective force. We characterized genetic diversity at an MHC class II locus (DRB1) in Gunnison's prairie dog (C. gunnisoni) and quantified population genetic structure at the DRB1 versus 12 microsatellite loci in three large Arizona colonies. Two colonies, Seligman (SE) and Espee Ranch (ES), have experienced multiple plague‐related die‐offs in recent years, whereas plague has never been documented at Aubrey Valley (AV). We found fairly low allelic diversity at the DRB1 locus, with one allele (DRB1*01) at high frequency (0.67–0.87) in all colonies. Two other DRB1 alleles appear to be trans‐species polymorphisms shared with the black‐tailed prairie dog (C. ludovicianus), indicating that these alleles have been maintained across evolutionary time frames. Estimates of genetic differentiation were generally lower at the MHC locus (FST = 0.033) than at microsatellite markers (FST = 0.098). The reduced differentiation at DRB1 may indicate that selection has been important for shaping variation at MHC loci, regardless of the presence or absence of plague in recent decades. However, genetic drift has probably also influenced the DRB1 locus because its level of differentiation was not different from that of microsatellites in an FST outlier analysis. We then compared specific MHC alleles to plague survivorship in 60 C. gunnisoni that had been experimentally infected with Y. pestis. We found that survival was greater in individuals that carried at least one copy of the most common allele (DRB1*01) compared to those that did not (60% vs. 20%). Although the sample sizes of these two groups were unbalanced, this result suggests the possibility that this MHC class II locus, or a nearby linked gene, could play a role in plague survival. Infectious diseases can play an important role in shaping genetic variation and fitness in free‐living populations  in the 1930s and led to major population reductions in the five species found in western North America  because they are highly specialized predators of prairie dogs that are completely dependent on large persistent colonies for survival  (Cully C. parvidens) (Cully and Williams C. ludovicianus)  colonies seem to experience a lower mortality rate  Cully , Utah prC. gunnisoni and C. ludovicianus suggest that some resistance to Y. pestis may exist  was much higher during exposure to the fully virulent Y. pestis strain CO92 than individuals originating from a colony in western South Dakota  that had not previously experienced plague  . In contrast, the AV population has not experienced any observable mortality from plague, even during years when plague epizootics affected nearby colonies  and Espee Ranch (ES) populations are closely monitored because they are release sites for black‐footed ferrets Fig. . Plague Van Pelt . In 1996Van Pelt , which sRattus rattus) populations that are regularly exposed to endemic Y. pestis  when it is present, we hypothesized that this MHC locus would exhibit appreciably lower levels of genetic differentiation (FST) than observed at microsatellite markers. Neutral genetic markers should show greater levels of genetic structure due to population fluctuations and the increased strength of genetic drift on small populations. Likewise, we predicted that the AV population will have greater variation at the MHC locus, because this population has not been subjected to the multiple plague events that could result in reduced diversity at other colonies (ES and SE). We also generated MHC genotypes of all C. gunnisoni individuals from the previous experimental plague challenge study  and a neighboring colony at Seligman, Arizona . In the second session (2009), prairie dogs were live‐trapped from two colonies where black‐footed ferrets have been released in Arizona: AV (n = 30) and Espee Ranch (ES) (n = 30). The 60 animals from 2009 are designated as AVC or ESC in this study because they were taken into captivity and used in a plague challenge experiment for C. gunnisoni  for these 60 individuals are provided in Appendix C. ludovicianus) live‐trapped in Phillips County, Montana after a plague epizootic in 2008 . The C. ludovicianus samples were used as a species comparison for our initial description of MHC DRB1 alleles from C. gunnisoni. All work was approved by the Northern Arizona University Institutional Animal Care and Use Committee under protocol 05‐005.We sampled three wild populations of Appendix  using anC. gunnisoni. Tissues were disrupted with bead milling for 20 sec following a previous protocol  from DRB1 gene, which has not yet been described for any prairie dog species. First, we used a traditional approach to amplify, clone, and sequence the exon 2 fragment of the DRB1 locus using HLA primers GH46F/GH50R : 20–40 ng of gDNA template, 1X PCR buffer, 2 mM MgCl2, 0.2 mM dNTPs, 2 U Platinum®Taq DNA polymerase , and 0.2 μM of each primer. PCRs were thermocycled according to the following conditions: 95°C for 10 min to release the Plat Taq® antibody, followed by 35 cycles of 94°C for 15 sec, 65°C for 20 sec, 72°C for 30 sec, and a final extension step of 72°C for 5 min. PCR products were electrophoresed on a 2% agarose gel for 1 h at 100V. If amplification was faint for an individual, additional PCRs were obtained before cleaning with a QiaQuick PCR Purification Kit .We used two sequencing‐based approaches to initially isolate and characterize the MHC class II DRB1 alleles present in heterozygous individuals, purified PCR product was ligated into the pGEM‐T vector  and transformed into Escherichia coli JM109 cells. Cells were plated onto ampicillin/IPTG/X‐Gal agar and cultured at 37°C for 18 h before picking positive clones (white or light blue in color). PCR screens were performed on 24 colonies using the same conditions as for the initial PCR, except that primers M13F and R were used at an annealing temperature of 55°C. Eighteen positive clones from each sample were sequenced to ensure detection of each individual allele , and the products were separated on an ABI3130 automated sequencer. Sequences were aligned using the Lasergene software package .To determine the specific le Babik . SequencC. gunnisoni used a Roche 454 platform to pyrosequence PCR amplicons using GH46F/GH50R primers modified with sequencing adaptors and bar codes  by 0.4°C per cycle (decrement from 66°C to 62.4°C). The Ta was then kept at 62°C for the remaining 17 cycles , with a final extension of 72°C for 10 min. We pooled equimolar amounts of these PCR products and shipped them to the Genome Resource Center at the University of Maryland for 454 sequencing. Raw 454 sequence reads for each prairie dog were sorted by bar code and clustered into unique alleles. These allele clusters were aligned in SeqMan Pro  to generate consensus sequences for each prairie dog.Our second approach to MHC class II gene discovery in Appendix . These tDRB1 alleles, especially in nonmodel organisms. Therefore, we used a Total RNA Purification Kit  to extract RNA from approximately 40 mg of spleen tissue from a subset of C. gunnisoni (n = 10); these ten validation individuals are highlighted in the first column of Appendix V” notation. We treated the total RNA with amplification grade DNase I  to degrade possible residual gDNA that may have been present in the RNA extractions. To confirm the absence of gDNA, we diluted the RNA 1/10 with molecular grade H2O for PCR and used the exon 2 primers GH46F/GH50R  and two oligos: 3′ SMART CDS Primer II (5′‐AAGCAGTGGTATCAACGCAgtacTTTTTTTTTTTT TTTTTTTTTTTTTTTTTTVN‐3′) and SMART II G‐linker (5′‐AAGCAGTGGTATCAAC GCAgtacGCGGG‐3′). The incubation time for first strand synthesis was extended to 90 min, and the reaction product was diluted with 40‐μL molecular grade H2O. Resulting cDNA was quantified on a NanoDrop 8000 spectrophotometer  and diluted to 20–40 ng/μL for PCR. Successful cDNA synthesis was confirmed using the exon 2 primer PD1DRB_F (5′‐CGTTTCCTGGAGCAAGTTTCACATG‐3′) and a reverse primer anchored in exon 3, DRBexon3‐R2 (5′‐AGACCAGGAGGTTGTGGTGCTG‐3′) : 20–40 ng of cDNA template, 1X PCR buffer, 2 mM MgCl2, 0.2 mM dNTPs, 0.2 μM of each primer, 1 U Platinum®Taq DNA polymerase, and 0.2 μM of each primer. PCRs were thermocycled according to the following conditions: 95°C for 10 min to release the Plat Taq® antibody, followed by 35 cycles of 94°C for 15 sec, 65°C for 20 sec, and 72°C for 30 sec, with a final extension step of 72°C for 10 min. PCR products were electrophoresed on a 2% agarose gel for 1 h at 100V. Amplicons were cloned into the pGEM‐T vector (Promega) and sequenced as described above. Each unique allele that was validated at this stage was named according to conventions for nonmodel species   had accumulated multiple single nucleotide polymorphisms (SNPs) in the exon 2 priming site for GH50R, which was originally designed for human DRB sequences (Appendix CyguDRB1_exon2_R1: 5′‐CTCTCCTCTCCACAGTGAAGCTCTCA‐3′ and CyguDRB1_exon2_R2: 5′‐CTCTCCGCTCCACAGCGAAGCTCTCA‐3′) that, when combined and used with GH46F, will allow unbiased amplification of all six MhcCygu‐DRB1 alleles  assays. Briefly, the allele typing process requires three steps  are described in Appendix DRB1 locus within these C. gunnisoni populations, we calculated allele frequencies, observed heterozygosity (HO), expected heterozygosity (HE), and tested for Hardy–Weinberg equilibrium using a Markov Chain implemented in FSTAT 2.9  were excluded in all calculations that produced a mean value .To assess genetic diversity at the MHC‐9 Goudet , 2001. TDRB1 allele sequences found in C. gunnisoni were analyzed for evidence of selection acting on the MHC. To do so, our six MhcCygu‐DRB1 allele sequences were aligned with a human sequence, translated to amino acids  and nonsynonymous (dN) substitutions per site at 89 amino acid positions and the 21 putative ABS using the Nei‐Gojobori method and applying a Jukes–Cantor correction for multiple substitutions at a site . These MhcCylu‐DRB1 alleles were discovered by PCR amplicon sequencing, but their expression has not been validated at this time. All statistical analyses for the C. ludovicianus genetic data were performed as described above for C. gunnisoni. A maximum likelihood phylogeny was constructed for the DRB1 locus of both species of prairie dog using MEGA 6.0.6. The best nucleotide substitution model (AICc = 4081) was found to be Jukes–Cantor with a gamma distribution (JC + G). Confidence estimates for nodes on the tree were generated from 1000 bootstrap replicates.To further explore the evolutionary history of these alleles, we included five MHC‐r2, D', and G4) in the program PERMUTE  from microsatellite markers.To assess neutral genetic structure of the study populations, we calculated DRB1 locus using the software fdist2 , we tested for evidence of genetic bottlenecks in all four population samples. We used the 12 microsatellite loci in the program bottleneck  among the 94 C. gunnisoni analyzed in this study  that encodes a class II beta subunit molecule with no insertions, deletions, or stop codons  at the extreme 3′ end of exon 2 will robustly amplify all six DRB1 alleles in C. gunnisoni , marked with a superscripted “DRB1 locus when compared to ES or SE (the two plague sites). In fact, the greatest number of alleles (n = 6) was found at ES. There were only modest differences in allele frequency among these colonies  were fairly rare, with frequencies < 0.1. The level of HO in this MHC locus ranged from 0.27 to 0.57 (mean HO = 0.38 ± 0.1) and was virtually the same as HE (0.25–0.52) .Contrary to our prediction, the AV population did not exhibit greater variation at the MHC Appendix . Allele eles n =  was founDRB1 locus, particularly at four highly similar alleles . The uncorrected nucleotide distance of these four alleles ranged from 0.015 to 0.055, and together, they reach a very high combined frequency (80–92%) across all population samples. Most of the nucleotide diversity in the C. gunnisoni DRB1 locus is accounted for in the two remaining alleles, 02 and 04 (uncorrected distance 0.13–0.24). These two alleles appear to be trans‐species polymorphisms  and other ground squirrels  showing a close relationship (uncorrected distance = 0.011–0.027) and two additional alleles (MhcCylu‐DRB*02 and 04) contributing the majority of the observed nucleotide diversity (uncorrected distance = 0.10–0.22).The colonies that we sampled generally exhibited a low level of nucleotide diversity at the ms Klein  due to tDRB1 locus during the evolutionary history of both prairie dog species. A slightly higher rate of nonsynonymous substitutions to synonymous substitutions (dN/dS = 1.17) was observed for antigen‐binding sites (ABS) in C. ludovicianus only. Z‐tests of positive selection were nonsignificant for both species, whether considering all positions or only ABS sites . In codon‐based tests (PAML), models of neutrality (the null hypothesis) were not rejected in favor of positive selection models for C. gunnisoni. The model incorporating positive selection fit our C. ludovicianus data better than models that did not incorporate this factor based on AIC values; however, the likelihood ratio test was not significant at P = 0.05  and model 8 . However, model 2a has fewer parameters and is therefore the most parsimonious model that explains our data.We found no evidence of positive selection acting on the r2, D', and G4), only the r2 estimate was consistent with recombination in C. gunnisoni (P = 0.017). All three measures were nonsignificant in C. ludovicianus.Of the three commonly used measures of linkage disequilibrium employed by the program PERMUTE (HO (range 0.51–0.57) and HE (range 0.50–0.59) reached similar levels at SE, ES, and both samples of AV. All markers were in Hardy–Weinberg equilibrium in the four population samples, with the exception of marker IGS‐1 in the SE population (Appendix P = 0.02). Direct comparisons of Ho (excluding the AV2006 subset) were not significantly different . The total number of alleles per locus was low (range 2–6), and no difference was observed in the mean number of alleles per population sample (3.4–4.1)  estimated by the microsatellite markers (Table FST (excluding the AV2006 subset) was 0.098 (95% CI: 0.035–0.149). No evidence of differentiation over time (FST = 0) was observed between the two AV population samples from 2006 and 2009 (AV2006 vs. AVC in Table C (FST = 0.09–0.12), which is expected given its greater geographic distance (~66 km) from the SE and AV colonies. The SE colony is <6 km distant from AV at its closest border, but was significantly differentiated from both the AV2006 and AVC samples (FST = 0.05 and 0.08).The level of population structure (rs Table  was low‐FST = 0.033) compared to neutral microsatellite markers (Table C) displayed about half the level of differentiation at the DRB1 locus  compared to microsatellites . Interestingly, colonies AVC and ESC displayed very low differentiation at DRB1 , which was significantly less than the 95% CI of the microsatellite loci for these two colonies . The relatively narrow range of FST variation at DRB1 compared to microsatellite loci could be an indication of past selective pressure acting on the MHC. However, these results do not suggest a simple correlation with plague presence/absence in these prairie dog colonies.The MHC locus generally displayed lower levels of genetic differentiation , we would expect to see FST values that are closer to zero than the neutral microsatellite loci. Although the observed FST estimates for the MhcCygu‐DRB1 locus were low, they all fell within the lower 95% CI of the simulated fdist2 distributions estimated from microsatellite markers  that was significantly greater than the DRB1 locus (0.033). It is important to note that the fdist2 result shown in Figure We also used a simulation technique in cus Fig. . As the ers Fig. . The relHE) excess in 11 of 12 markers at ES (P = 0.002). The SMM model yielded nine markers with HE excess at ES (P = 0.09). The only other colony with a bottleneck signature was SE, with 11 of 12 markers showing HE excess under the IAM (P = 0.034) but not under the SMM (P = 0.733). It is worth noting that the IAM is a less conservative model and has been known to identify heterozygosity excess in nonbottlenecked populations  . An important caveat to this result is that the sample sizes were very unbalanced: 55 prairie dogs from ESC and AVC had at least one copy of allele 01, but only five individuals did not carry it. MHC heterozygosity at the DRB1 locus was not a significant factor in determining survival during plague challenge , and of the 34 prairie dogs that were homozygous for allele 01, 19 survived and 15 died (Appendix n = 3) died when challenged with plague, including a 0104 heterozygote.Comparison of Kaplan–Meier survival curves Fig.  among C.Yersinia pestis is endemic in western North America and a significant pathogen of native rodents and associated wildlife, such as black‐footed ferrets. The four species of prairie dogs in the United States have experienced recurring selection pressure from plague since the 1930s, with mortality rates approaching 100% at affected colonies  while others survived the highest dose . This type of variation is necessary for developing an evolutionary response to a highly virulent pathogen such as Y. pestis. Here, we discuss the association of MHC alleles in prairie dogs experimentally challenged with plague and present the first description of MHC diversity in C. gunnisoni.DRB1 locus in C. gunnisoni was lower than in most other rodent species, but similar to that observed in ground squirrels. With just six alleles, C. gunnisoni allelic richness is extremely low compared to mice and rats , although not at AV. Fortunately, the nucleotide diversity among existing alleles in C. gunnisoni (0.015–0.24) and C. ludovicianus (0.011–0.22) remains high and is similar to that reported for other ground squirrel species  compared to spotted suslik (0.63–0.89), eastern woodchuck (0.77–0.83), and alpine marmot (0.51–0.63) . These microsatellite HE estimates fell in the middle range of C. ludovicianus (0.53–0.63)     at the MHC locus compared to the level at neutral markers (Table DRB1 FST estimates (0.018–0.051) is also much narrower than that for 12 microsatellites  . It is possible that MHC differentiation across these same 34 rangewide locations will be much lower.Selection appears to have played an important role in shaping patterns of genetic structure at the MHC tes Fig. . Howeverers Fig. . The samace Hill . In thisC. gunnisoni that were used in the previous experimental plague challenge , an Asian ground squirrel that naturally occurs in areas with native plague. Ancient polymorphisms shared by ground squirrels species would probably have arisen before the divergence of Y. pestis from its ancestor (Y. pseudotuberculosis) in the past 6000 years  that shared high sequence similarity between nus Fig. . These tnus Fig. . AllelesAppendix . Therefoci Klein  and may ci Klein  or conveci Klein . Some ofY. pestis attacks the immune system with a complex array of virulence mechanisms. Our study is generally consistent with this idea, because the class II MHC locus is clearly not the only factor determining, which prairie dogs will survive plague. In the prairie dog challenge experiments, very few individuals of either species produced a strong antibody response, indicating that other immune mechanisms  may be more critical for surviving plague infection  contribute to high resistance levels in Mus spretus  to suppress MHC class II antigen presentation . We found weak evidence for positive selection acting on the DRB1 locus in the black‐tailed prairie dog, but not in Gunnison's prairie dog, which may be a result of recent population reductions or the short time frame during which plague has been acting as a selective force. Plague is probably not the only source of selective pressure in this system, and other diseases  or parasites may play an important role in shaping variation at immune system loci in prairie dogs (Zeidner et al. C. gunnisoni at both neutral loci and genes under selection. We are currently investigating whether greater microsatellite and MHC diversity existed prior to the introduction of plague to the western United States by genotyping C. gunnisoni specimens collected prior to 1930. These studies will contribute to an understanding of the development of plague resistance in prairie dogs and other rodents, and help elucidate the influence of diseases in shaping natural genetic variation in wild populations.Characterizing genes integral to the immune system is an important first step in identifying possible mechanisms of resistance to diseases in nonmodel species like prairie dogs. This is the first published report of an expressed MHC class II The authors declare no conflict of interest.KU695893–KU695896 and KR338362–KR338368.Sequence data have been submitted to GenBank under accession numbers GenBank accessions


Urinary tract infections (UTI) are a common condition in children. Vesicoureteral reflux (VUR) represents a common associated condition with childhood UTI. UTI susceptibility appears to have a genetic component based on family and UTI cohort studies. Targeted analysis of innate immune system genetic variations indicate that these variations are important in UTI susceptibility. In this overview, we discuss how current cohorts and genetic strategies can be implemented to discover new susceptibility loci in patients with UTI. Under normal circumstances, the urinary tract is a sterile system. In children, 3%–7% of girls and 1%–2% of boys will experience a urinary tract infection (UTI) by age 6 [Vesicoureteral reflux (VUR) is one of the most common anomalies associated with APN in children. VUR is the abnormal backflow of urine from the bladder to the kidneys and affects 1%–2% of all children . Up to 6Clinicians have had difficulty determining a consensus for treatment of children with VUR. Current management strategies are focused around preventing UTIs while limiting a child’s exposure to potential side effects from medicines or invasive procedures. Because only a small fraction of children will have recurrent UTI and most will “out-grow” their VUR, the debate regarding how best to approach a child with VUR continues. A child with VUR can be treated with daily antibiotic prophylaxis, have corrective procedures performed, or be carefully watched and intervention initiated when recurrent UTIs occur. Our research group studies why only certain children with VUR get UTIs and why only some develop sequelae. This review of the presentation will serve as an overview for why studies in the genetics of UTI are critical for directing medical decision making in patients at risk of UTI complications.versus APN) can also be seen to be over-represented in different families [versus recessive but rather appears to be a complex genetic trait. Therefore, UTI susceptibility is likely a polygenic phenomenon with environmental and host factors also playing a role.A growing body of literature exists implicating the genetic contribution to UTI susceptibility. Several studies have identified inheritance patterns that confer higher UTI risk in family members of affected individuals ,12,13,14families ,11. GeneThe host innate immune system is the first-line defense of the kidney and urinary tract to prevent microbial invasion. The innate immune system, unlike the adaptive immune system, has constitutively active components and components that are rapidly up-regulated upon microbial presence. The host’s innate immune system is complex but simplistically consists of antimicrobial peptides and molecules that rapidly kill bacteria, chemokines and cytokines that promote an inflammatory response, pattern recognition receptors that recognize pathogens, and phagocytes that consume bacteria ,16. ThesDEFB4, with four copies occurring most frequently. CNVs in the DEFB4 gene and other key components of innate immunity have been reported in susceptibility to Crohn’s disease and HIV [Genetic variations in humans range from single nucleotide polymorphisms (SNPs) to large microscopically visible chromosome defects. SNPs occur once every 300 base pairs across the genome and represent a common source of genetic variation . On the  and HIV ,20,21,22The first challenge in an unbiased approach for determining genetic risk factors that contribute to UTI risk is recruiting a well-phenotyped, longitudinal cohort of UTI-susceptible individuals. The Randomized Intervention for Children with Vesicoureteral Reflux (RIVUR) Study  recently completed in North America . All chiThe second challenge to determine what genetic determinants may contribute to UTI risk in humans is selecting the appropriate genome-wide approach to assess genetic variations. Classic approaches to determine genetic causality rely on either rare variants to be over-represented for rare diseases or large pedigrees with genetic similar individuals with affected and unaffected individuals. Applying these techniques to a complex genetic trait such as VUR and UTI risk represented by the RIVUR study cohort would likely cause key disease modifying genes to be overlooked . FurtherHigh-resolution aCGH utilizes a whole-genome tiling array with oligonucleotide probes (25–85 base pairs) spaced every 1–2 kilobases and covers the entire genome. Patient DNA is labeled with a fluorescent dye and a reference sample is labeled with a different colored fluorophore . Equal aVariations in innate immunity genes lead to UTI susceptibility. In this review, we provide a novel approach and rationale for using aCGH to interrogate genomes for multi-allelic genes such as the alpha and beta-defensin loci that may be critical in UTI susceptibility.


Chorocaris sp. 2 and the squat lobster Munidopsis lauensis—that are common at deep-sea hydrothermal vents in the western Pacific. While Chorocaris sp. 2 has only been observed at hydrothermal vent sites, M. lauensis can be found throughout the deep sea but occurs in higher abundance around the periphery of active vents We sequenced mitochondrial COI genes and deployed nuclear microsatellite markers for both species at three sites in Manus Basin and either North Fiji Basin (Chorocaris sp. 2) or Lau Basin (Munidopsis lauensis). We assessed genetic differentiation across a range of spatial scales, from approximately 2.5 km to more than 3000 km. Population structure for Chorocaris sp. 2 was comparable to that of the vent-associated snail Ifremeria nautilei, with a single seemingly well-mixed population within Manus Basin that is genetically differentiated from conspecifics in North Fiji Basin. Population structure for Munidopsis lauensis was more complex, with two genetically differentiated populations in Manus Basin and a third well-differentiated population in Lau Basin. The unexpectedly high level of genetic differentiation between M. lauensis populations in Manus Basin deserves further study since it has implications for conservation and management of diversity in deep-sea hydrothermal vent ecosystems.Studies of genetic connectivity and population structure in deep-sea chemosynthetic ecosystems often focus on endosymbiont-hosting species that are directly dependent on chemical energy extracted from vent effluent for survival. Relatively little attention has been paid to vent-associated species that are not exclusively dependent on chemosynthetic ecosystems. Here we assess connectivity and population structure of two vent-associated invertebrates—the shrimp  Riftia pachyptila: Bathymodiolus thermophilus: Rimicaris exoculataIfremeria nautilei:Alviniconcha spp.: In the deep sea, studies of gene flow and population structure have disproportionately focused on numerically dominant taxa from chemosynthetic ecosystems, where it is relatively easy to collect sufficient individuals for population genetic analyses Bathymodiolus spp. Alviniconcha spp. Ifremeria nautileiAlviniconcha sp. 2 Ifremeria nautileiAmong the first-order questions that arise regarding gene flow and population structure in deep-sea chemosynthetic ecosystems is whether there is greater community and population differentiation among back-arc basins than along mid-ocean ridges, due to the isolated nature of the basins Bathymodiolus thermophilus) on the East Pacific Rise are divided into northern and southern populations across the Easter Microplate  is commensal in Bathymodiolus thermophilus, are undifferentiated across the same boundary  and often correspond to geomorphological features Chorocaris sp. 2 and the galatheid squat lobster Munidopsis lauensis—to determine the extent of population differentiation within and among back-arc basins for vent-associated species. Shrimp in the genus Chorocaris are so far only reported at or near hydrothermal vents Chorocaris sp. 2 has, to date, only been observed in Manus and North Fiji Basin . Squat lobsters in the genus Munidopsis occupy numerous deep-sea habitats, including cold seeps M. lauensis is frequently observed in large aggregations near active hydrothermal venting M. lauensis is an opportunistic species that is broadly distributed in the deep-sea but is attracted to the rich food source at hydrothermal vents, whereas Chorocaris sp. 2 is more patchily distributed and restricted to regions around active hydrothermal venting. We expected that opportunistic vent taxa, like Munidopsis lauensis, would show the least population structure, while the vent-associated shrimp, Chorocaris sp. 2, would exhibit greater population structure in comparison.In this study, we explore population structure in two western Pacific back-arc basin vent species—the alvinocarid shrimp Samples were collected with permission of the governments of Papua New Guinea, Fiji, and Tonga. These field studies do not involve any endangered or protected species.Chorocaris spp. and Munidopsis lauensis were collected from three hydrothermal-vent sites in Manus Basin—Solwara 8, Solwara 1, and South Su (M/V Nor Sky research campaign in June-July 2008 (Chief Scientist: S. Smith). Samples were collected using an ST200 work class ROV modified for biological sampling. To allow comparisons of population structure at multiple spatial sales, two to four discrete aggregations of Chorocaris sp. 2 or Munidopsis lauensis were sampled from each site within Manus Basin .South Su —during tus Basin . Additious Basin  were proTissues were preserved in 95% ethanol prior to DNA extraction. Genomic DNA was isolated using a standard Chelex-Proteinase-K extraction  in 600 µl 10% Chelex-100 resin  overnight at 60°C, heated to 100°C for 15 min, and centrifuged at 10,000 rpm for 5 minutes; Chorocaris sp 2. mitochondrial COI fragments were amplified using the following reaction conditions: 10 to 100 ng of DNA template was combined with 2 µL 10× PCR buffer , 2 mM MgCl2, 0.6 mM dNTPs, 0.5 µM LCOI1490 and 0.5 µM HCOI2198 primers Munidopsis lauensis mitochondrial COI fragments were amplified using the following reaction conditions: 10 to 100 ng of DNA template was combined with 2.5 µL PCR buffer, 2 mM MgCl+, 0.6 mM dNTPs, 0.05 µM GALA COIR primer (5′-GAA YAG GRT CTC CTC CTC CTA C -3′) and 0.05 µM GALA COIF primer (5′- CAT CAC TWA GWT TRA TYA TTC CAG CAG AA-3′), and 1 unit of Taq polymerase in a 25 µL reaction with the following PCR protocol: initial melting temperature of 94°C for 240 seconds; 35 cycles of 94°C for 60 seconds, 55°C for 120 seconds, 72°C for 210 seconds; and a final extension of 72°C for 600 seconds. Reactions were stored at 4°C until purification.COI sequences for each individual are provided as FASTA files , 0.05 µl Antarctic Phosphatase , 0.05 µl Exonuclease I  at 37°C for 60 min followed by 85°C for 15 min to inactivate the enzymes. Sequencing reactions (both directions) were performed using Big Dye Terminator v3 reactions . Dye Terminator removal was performed using AMPure magnetic beads , sequencing products were analyzed on an ABI 3730xl DNA Analyzer , and sequence chromatograms were edited with CodonCode Aligner . Consensus sequences were compared against the NCBI GenBank database to confirm species identity when available TA files  and S2.Maximum-parsimony phylograms of aligned mitochondrial sequences were assembled in MEGA version 5 , haplotype diversity (Hd), nucleotide diversity (π), and Fu's SF were calculated using DnaSP version 5.10.01 STφ and permutation tests were used to identify significant departure from genetic homogeneity and Sequential Bonferroni was used to correct for multiple tests Statistical-parsimony networks were assembled in TCS version 1.21  were amplified from Chorocaris sp. 2 in Manus Basin following methods reported in Zelnio et al.Chorocaris sp. 2 in North Fiji Basin due to low sample size (n = 9). Nine microsatellite markers  were amplified from Munidopsis lauensis in Manus and Lau Basin following methods reported in Boyle et al.Six microsatellite markers  between aggregations, sites, and basins was analyzed using Microsatellite Analyzer. Alpha levels were adjusted via Sequential Bonferroni to correct for multiple tests Chorocaris sp. 2 and Munidopsis lauensis. Equilibrium heterozygosity (Heq) was estimated under the TPM model allowing for 4% multi-step mutations (1000 iterations).Analysis of molecular variance (AMOVA) was used to analyze hierarchal population structure in Arlequin. Pairwise genetic differentiation , and Chorocaris sp. 2  were identified from 179 individuals from Manus Basin. Haplotype diversity among aggregations in Manus Basin was high, ranging from 0.70 to 1.00  did not vary significantly among patches, mounds, or sites  for population differentiation in Manus Basin indicated no significant differentiation at any spatial scale . AMOVA performed on microsatellites across basins indicated that almost 35% of the observed genetic variability was accounted for by differentiation at the basin level, i.e., between Manus and North Fiji Basin (p<0.05) and significant pairwise population differentiation was detected between Manus Basin and North Fiji Basin , but not South Su. (two-tailed p>0.05) suggesting a recent bottleneck.Analysis of Molecular Variance (AMOVA) for al scale . AssignmCOI haplotypes (454 bp) were amplified from Munidopsis lauensis , all of which adhered to Hardy-Weinberg expectations  in Manus Basin . Chorocaris sp. 1 is not known from other sites in the western Pacific . Three individuals of Munidopsis antonii, a squat lobster closely related to M. lauensisM. antonii is broadly distributed Cryptic or miss-identified species were discovered in samples of shrimp and squat lobsters from western Pacific back-arc basins. Chorocaris sp. 2 haplotypes and microsatellite markers within Manus Basin is consistent with high gene flow among the study sites. Although larvae are likely the primary dispersal vector, the mobility of juvenile and adult shrimp may also allow individuals to travel among neighboring vent sites, reducing the potential for local population structure to emerge.The homogeneous distribution of Chorocaris sp. 2. The excess of rare COI haplotypes (significantly negative Fu's SF) and departure from mutation-drift equilibrium for microsatellite markers at Solwara 1 and Solwara 8 suggest that these two populations encountered a recent bottleneck followed by a population expansion. South Su also departed from mutation-drift equilibrium based on COI  but not for microsatellite markers. This could indicate that the bottleneck encountered by the South Su population was not as strong as bottlenecks affecting other populations, or that the recovery from the bottleneck was faster in South Su. Bottlenecks followed by population expansions have been found in other species Rimicaris exoculata shrimp on the Mid-Atlantic Ridge Chorocaris sp. 2 sampled from multiple sites in Manus Basin, the null alleles did not influence the overall outcome.There is a high frequency of rare, private haplotypes (78.3% are singletons) from Manus and North Fiji Basin samples of COI sequences between populations of Chorocaris sp. 2 from Manus and North Fiji Basins, the presence of two shared COI haplotypes between basins indicates that some migration must occur or have occurred in the recent past or that the populations are experiencing incomplete lineage sorting. The absence of COI haplotypes descended from the North Fiji clade in Manus Basin suggests that migration, if it occurs, may be directional, from Manus into North Fiji Basin, though this could be the result of sample bias, given the small number of individuals (n = 9) sampled from North Fiji. A Manus to North Fiji route is consistent with the regional circulation patterns and recent models of larval transport in the southwest Pacific Despite strong signals of genetic differentiation in Chorocaris sp. 2, COI and microsatellite-based population studies of the related Rimicaris exoculata from hydrothermal vents along the Mid-Atlantic Ridge found no population structure across more than 5,000 kilometers R. exoculata populations (Hd = 0.69 to 1.00) is similar to populations of Chorocaris sp. 2 (Hd = 0.70 to 1.00). Teixeira et al.R. exoculata population is the product of a recent founder event followed by demographic expansion along the Mid-Atlantic Ridge, while population structure of Chorocaris sp. 2 appears to arise from a barrier to gene flow from basin to basin in the southwestern Pacific.In contrast to COI haplotype diversity in Munidopsis lauensis is low (Hd = 0.07–0.09) and is an order of magnitude lower than that observed in other vent-associated species in Manus Basin , causing inconsistencies and failures in microsatellite amplification and amplification of multiple fragments Munidopsis polymorphaMunidopsis lauensis for this study.Past microsatellite-based studies of galatheid squat lobster population structure have been confounded by the presence of mobile, cryptic, microsatellite-flanking transposable elements in squat lobster genomes Munidopsis lauensis yielded evidence for fine-scale population structure among sites in Manus Basin, but not within sites. M. lauensis from Solwara 1 are genetically differentiated from those of South Su (only ∼2.5 kilometers apart) and Solwara 8 (∼40 km apart), but M. lauensis from South Su and Solwara 8 (∼40 km apart) are genetically undifferentiated. While family effects—the appearance of genetic differentiation due to a region being colonized by closely related propagules—could explain the observed genetic differentiation between Solwara 1 and the other Manus Basin sites, no indication of significant relatedness among individuals from Solwara 1 was detected. Isolation of M. lauensis at Solwara 1 could be the result of physical or hydrographic barriers to gene flow that limit colonization or migration from other sites in Manus Basin. Solwara 1 lies along the northwest flank of a large, submerged, and active volcano (North Su), which physically separates Solwara 1 from South Su M. lauensis possesses larva that remain near the sea floor, the path of the St. George's Undercurrent could prevent M. lauensis larvae from effectively dispersing up-current, from Solwara 1 southwestward to South Su.Microsatellite-based estimates of genetic differentiation in Munidopsis lauensis is genetically differentiated between Manus and Lau Basins based on microsatellite data, but the less variable COI sequences do not detect this differentiation. These divergent results from two types of genetic markers highlight one of the challenges in conservation genetics. Because multiple phenomena likely shape the genetic diversity and distribution of M. lauensis throughout the western Pacific, no single gene, or suite of similar genetic loci , can provide a complete picture of population structure M. lauensis. On an evolutionary time-scale, COI data suggest that homogenizing processes  have reduced the genetic diversity of M. lauensis. The lack of a recent population bottleneck detected in the microsatellites supports a selective sweep on COI. On an ecologic time-scale, microsatellite data suggest locally differentiated populations and restricted gene flow over relatively short distances (2.5 km); the extent to which locally differentiated populations persist over multiple generations of M. lauensis is unclear and requires an assessment of temporal variability among these differentiated populations. A similar phenomenon of divergent estimates of population structure based on differing marker types was observed among species in the tubeworm genus Escarpia that inhabit cold seeps in the Gulf of Mexico, Gulf of California, and West Africa; no significant population differentiation was observed based on mitochondrial markers, but microsatellites revealed some significant differentiation among regions M. lauensis, this kind of ascertainment bias is apparent in two microsatellite loci, Mp12 and Mp21 (as these markers were developed on M. lauensis from Manus Basin), which failed to amplify in individuals from Lau Basin. It is possible that these loci are not present in Lau Basin samples. The frequency of alleles for the other seven markers fell within Hardy-Weinberg expectations, with no evidence of null alleles, and suggests that they are not influenced by ascertainment bias. The failure of two microsatellites to amplify, suggests that the observed genetic divergence between M. lauensis from Manus and Lau Basin may be even more pronounced.When a suite of microsatellite loci is developed for one particular population, it may not work as well in additional distantly related populations, resulting in null alleles Chorocaris sp. 2 and Munidopsis lauensis in Manus Basin are isolated from other back-arc basin vent systems. Three provannid snail species  also exhibit strong population isolation between Manus Basin and other regional back-arc basins  have a high degree of connectivity, providing evidence that South Su may serve as an effective reservoir of genetic diversity for some species. For Munidopsis lauensis, however, Solwara 1 and South Su populations are genetically distinct, and, in this case, South Su would not act as an effective reservoir of genetic diversity. Population differentiation between Munidopsis lauensis from Solwara 1 and other sites is perplexing. If the squat lobsters are genetically isolated through some as yet-to-be-determined mechanism, it is not clear that this genetic lineage could be sustained in the face of severe population reduction. Genetic differentiation at such a local scale, however, is a surprising outcome for this species and requires further investigation.Characterization of patterns of genetic diversity and connectivity within and among populations is a valuable tool for managing and mitigating the effects of anthropogenic disturbance at deep-sea hydrothermal vents Chorocaris sp. 2 and Munidopsis lauensis and conspecific populations in other western Pacific back-arc basins. This suggests that if maintenance of genetic diversity is an environmental management objective, then management tools must be applied on a regional basis  for these species. Ongoing monitoring of genetic diversity of key taxa before and after mineral extraction would allow managers to assess the impact of the activity on connectivity and population structure and inform best practices.There is limited connectivity between the Manus Basin populations Figure S1Maximum likelihood tree for a subset of Chorocaris spp. sampled from Manus and North Fiji Basin. Sequences are 600-base pairs in length. Substitution model is Tamura 3-parameter determined by Find Best Model application in Mega 5. Representatives of Chorocaris sp. 1 and sp. 2 were chosen at random. Chorocaris sp. 1 and Chorocaris sp. 2 indicated by horizontal bars. Chorocaris vandoverae  presented for comparison. Bootstrap values greater than 0.50 reported on branches. Scale bar is number of substitutions per base pair.(TIF)Click here for additional data file.Figure S2Observed and expected mismatch curves of pairwise mitochondrial COI nucleotide differences for Chorocaris sp. 2 sampled from Manus and North Fiji Basin and Munidopsis lauensis sampled from Manus and Lau Basin. Each graph represents a comparison between simulated curves for pairwise nucleotide differences and observed pairwise differences for (A) Chorocaris sp. 2 under a model of constant population size, (B) Chorocaris sp. 2 under a model of population growth and decline, (C) Munidopsis lauensis under a model of constant population size, and (D) M. lauensis under a model of population growth and decline.(TIF)Click here for additional data file.File S1FASTA format file for all Chorocaris spp. COI sequences.(NEX)Click here for additional data file.File S2FASTA format file for all Munidopsis lauensis COI sequences.(NEX)Click here for additional data file.File S3GENPOP format file of all Chorocaris spp. microsatellite markers.(XLS)Click here for additional data file.File S4GENPOP format file of all Munidopsis lauensis microsatellite markers.(XLSX)Click here for additional data file.