Graellsia isabellae, and its two recognised host-plant species (Pinus sylvestris and P. nigra) in order to seek for any concordance useful to disentangle the evolutionary history of this iconic lepidopteran.Geographic and demographic factors as well as specialisation to a new host-plant may lead to host-associated differentiation in plant-feeding insects. We explored the phylogeography of a protected moth, G. isabellae studied in Spain and France comprising six groups mostly distributed along different mountain ranges. Reanalysis of a previously published chloroplast microsatellite dataset revealed a three and two-group structure for Spanish P. sylvestris and P. nigra, respectively. Overall, the population groupings of this protected moth did not match the ones of P. sylvestris and P. nigra.DNA variation in one mitochondrial marker and nine nuclear microsatellite loci revealed a strong phylogeographic pattern across 28 populations of P. sylvestris and the ones inhabiting P. nigra. The two major mitochondrial clades of G. isabellae likely diverged before the Last Glacial Maximum and geographically separated the species into a “southern”  and a “northern” lineage . The Eastern Iberian System, where this insect uses both host-plants, harboured the highest level of genetic diversity. Such a group independently colonised the West and East parts of the Pyrenees. Our results point to a native origin for the French populations occurring in the Alps, genetically related to the Eastern Iberian and Pyrenean sites. The Central Iberian group derived from Southern Iberian ancestors. Secondary contacts were inferred between the Southern/Central Iberian populations and Eastern Iberian cluster as well as between the two Pyrenean ones. The mito-nuclear discordance observed with regard to the Eastern Iberian cluster is congruent with a secondary contact after the evolution of mito-nuclear incompatibilities in geographically isolated areas.There was no evidence of host-associated differentiation between populations using The online version of this article (doi:10.1186/s12862-016-0708-y) contains supplementary material, which is available to authorized users. The diversity of plant-feeding insects is remarkable and host-associated differentiation has been advocated as a relevant trigger of such macroevolutionary diversification , 2. HoweGraellsia isabellae (Graells 1849) , a protected species in France and Spain after its inclusion in the Habitats Directive [Pinus sylvestris) and Black (P. nigra) pines. P. sylvestris is the host species of G. isabellae in the Central Iberian System, Pyrenees and Alps, where the presence of P. nigra is scarcer. This spectacular silkmoth inhabits forests of both P. sylvestris and P. nigra in the Iberian System [G. isabellae feeds exclusively on P. nigra in Southern Spain [L28, Fig. P. halepensis/P. pinaster, remains to be confirmed [Graellsia isabellae across its entire distribution range.Assess the levels of genetic variability and population structure of Evaluate whether host-associated differentiation and divergence of geographically isolated populations played a relevant role in the phylogeography of this iconic insect.Here we set out to disentangle the microevolutionary history of the Spanish Moon Moth irective , 8. Thisn System . Althougrn Spain , the hos Graells 849 (Lepionfirmed . Our aimPinus species are known for their variation in deterrent compounds (e.g. terpenoids) [G. isabellae to have experienced host-associated differentiation. If so, congruence between the population structure of G. isabellae and its host plant use  should be found.penoids) . Thus, iG. isabellae is expected to depend to some extent on the range shifts suffered by P. sylvestris and P. nigra during the Upper Quaternary [P. sylvestris and P. nigra in the Iberian Peninsula [P. sylvestris [G. isabellae in Spain is about three times larger than the one it presently occupies [G. isabellae was already present at the LGM, corresponding to the separate glacial refugia of P. sylvestris/P. nigra.The evolutionary history of aternary . Severaleninsula , 17. Botlvestris , 19. Howoccupies . TherefoCOI), a mitochondrial marker widely used in insect population genetics, and genotyped nine microsatellite markers specifically developed for Graellsia isabellae [Pinus sylvestris and P. nigra as a background. For this, we revised the available organelle and nuclear evidence for both pines at European level. We also surveyed the structure of their Iberian populations by reanalysing the chloroplast (cpDNA) microsatellite dataset published by Soto et al. [To achieve aim 1, we Sanger sequenced the second half of the cytochrome c oxidase subunit I gene  gene for 493 of the 796 sampled individuals. Amplifications were carried out in 30 μL volumes containing 1X PCR Buffer (5 PRIME), 1.5 mM MgCl2, 1U TaqDNA Polymerase (5 PRIME), 0.2 mM of each dNTP, 0.2 μM of each primer C1-J-1751 and C2-N-3661 [Bombyx mandarina mitochondrion genome), LepLEUr [We sequenced the 3′ end (832 bp) of the mitochondrial cytochrome c oxidase subunit I . Allelic peaks were independently called by two researchers, using GENEMAPPER 4.0 (ABI). Results from locus GI03 were not included in the present study due to its complex allelic pattern.The 796 samples were genotyped at ten microsatellite loci previously developed . PCR proCOI dataset with DNASP 5.1 [FIS values for each sampling location were calculated with FSTAT 2.9.3.2 [ΦST and FST as implemented in ARLEQUIN 3.5.1.2 [Nucleotide and haplotype diversities were analysed for the mitochondrial NASP 5.1 . Nuclear 2.9.3.2 . Allelic 2.9.3.2 . Analyse 2.9.3.2 . Genetic 3.5.1.2 .K values (groups of populations) from two to 10. Each run was repeated three times to check for consistency. In addition, we carried out population mixture analyses using the spatial clustering modules implemented for DNA sequence data in BAPS 6.0 [K = 10, K = 15 and K = 20 as upper bounds to the number of populations. In each repetition the program explores the space of partitions and informs of the optimal one .We explored global genetic structure using both the mitochondrial and nuclear datasets. We applied the Spatial Analysis of Molecular Variance (SAMOVA 1.0)  to the fBAPS 6.0 , 33, botK ranging from one to 29. We chose the best partition of the data by examining both the log probability of the data (ln Pr(X|K)) and the ΔK statistic, following Evanno et al. [K-value. Finally, DISTRUCT 1.1 [K ranging between 1 and 28. The best partition  was identified as explained above.Two Bayesian clustering algorithms were used to infer population structure from the microsatellite dataset: STRUCTURE 2.3.4  and BAPSo et al. . CLUMPP o et al.  was usedRUCT 1.1  was emplDC) were obtained with the aid of MICROSAT 1.5 [In addition, Cavalli-Sforza and Edward’s chord distances , revealed Actias luna (Linnaeus 1758) as an appropriate outgroup. In order to identify the earliest diverging haplotype/s, maximum connection limit was set to 70. Mitochondrial phylogenetic trees were reconstructed using Bayesian inference, with the aid of MRBAYES 3.2 [st and 3rd codon positions into different partitions, and eliminating the 2nd position of the COI alignment due to its lack of phylogenetic information (it only shows a single private change). We used a gamma model of rate variation across sites and sampled across the GTR model space in the Bayesian Markov chain Monte Carlo (MCMC) analysis itself [6 generations, with trees sampled every 103 generations. The analysis was carried out with default priors until the standard deviation of split frequencies dropped below 0.01, and the potential scale reduction factor for all parameters lay close to 1.0. Two completely independent analyses starting from different random trees were run. For the MCMC sampling of the target distribution, three heated chains and one cold chain were used. The first 25 % samples of the cold chain were discarded as burn-in. We used Bayes factor comparisons to test several topological hypotheses. Marginal model likelihoods were estimated by the stepping-stone method; strength of the evidence in favour of the better model was then assessed by the magnitude of the log-difference, following Kass & Raftery [−1 estimated for the COI gene in tenebrionid beetles [st or 3rd coding positions. The output cladogram summarizing the trees was visualized with FIGTREE 1.4.2 [A 95 % Statistical Parsimony (SP) network was calculated for the  TCS 1.2 . PrelimiAYES 3.2 , specifys itself , running Raftery . The str beetles , essentiEE 1.4.2 , retainiCOI dataset to plot the observed frequency distribution of pairwise nucleotide differences with their expected distribution by performing a mismatch analysis. We estimated the demographic parameters θ0, θ1 and τ from the mismatch distribution using MISMATCHHETMUTRATE [t = τ/2u; u = μLg, where μ is the estimated mutation rate (0.0177 substitutions/site/my), L is the length of the sequence (832 bp), and g is the generation time (1 year). In addition, we used Fu’s Fs and R2 tests, the more powerful ones for detecting departures from the null hypothesis of constant population size [P-values of these tests were obtained by Monte Carlo simulations  carried out with DNASP, based on the neutral coalescent process  and assuming no recombination. Coalescent simulations for the R2 statistic were conditioned on the number of segregating sites.We investigated the effects of fluctuations in population size on the genetic variation of the population clusters defined in this work. On the one hand, we used the TMUTRATE , based oTMUTRATE , so thation size , 50. ConhMD), obtained from the observed number of alleles at each locus through simulating the coalescent process for n loci, are compared to heterozygosities at HWE (hHW). During a bottleneck the number of alleles decreases faster than gene diversity, and therefore hHW should be higher than hMD. The analyses were performed across 10,000 iterations under both the infinite allele model (IAM) and the stepwise mutation model (SMM), which respectively correspond to the least and most stringent conditions to detect a bottleneck. We assessed the significance of the results using the Wilcoxon’s signed ranks test, implemented in the program. We also investigated the distribution of allele frequencies, checking for the expected distortions (mode shifts) produced by recent bottlenecks [As a separate approach, BOTTLENECK 1.2.02  was usedtlenecks .Pinus sylvestris, we inspected the mitochondrial results published by Sinclair et al. ([COI), Soranzo et al. [nad1, exon B/C). Then we surveyed the dataset from Naydenov et al. ([nad7 intron 1 and the nad1 intron B/C) and Pyhäjärvi et al. [With regard to  et al.  and 2.12 (PT1936). The available cpDNA SSR dataset for P. nigra consisted of 326 individuals, sampled in 14 natural forests and genotyped for the same six markers. In this case, all loci were used, as missing data ranged from 0.92 % (PT30204) and 3.37 % (PT1254). Preliminary runs revealed that the use of missing data would produce slight differences in the clustering pattern of P. nigra. Therefore, 30 individuals of Black pine  had to be discarded from the final analysis.We reanalysed the cpDNA SSR dataset published by Soto et al.  for IberK ranging between 1 and the number of sampled sites . The best partition  was identified as explained above.The overall genetic structure of both conifers was assessed using the Bayesian clustering implemented in BAPS 6.0. We applied a non-spatial genetic mixture analysis to groups  of haplotypes and the “linked loci” option. Again, we performed ten repetitions of the algorithm for each Pinus nigra in the South of Spain and Morocco inferred by Jaramillo-Correa et al. [P. sylvestris in the Northern Meseta (NW spain) surveyed by Robledo-Arnuncio et al. [P. sylvestris plus a sixth one, not included in Soto et al. [P. sylvestris, the only difference in terms of molecular markers is the exclusion of locus PT1254 in our dataset and the information provided by locus PT26081 in Robledo-Arnuncio et al. [The population structure of a et al.  was basea et al. . Howevero et al.  relied oo et al. . So, cono et al. . Lastly,o et al.  and all o et al.  are inclo et al.  we reanaL9 and L10 (see Results) might have been caused by reproductive parasites such as Wolbachia. We tested for presence of this bacterium in a set of 53 specimens of Graellsia following Kodandaramaiah et al. [Incompatibility between males from infected populations and females from uninfected ones may lead to asymmetrical gene flow, a phenomenon reported in moths . The mith et al.   and Montesquiu (L7), whereas EP.1 was present at Els-Port (L4) and all the Eastern Pyrenean sites (L5-L9)  showed the highest haplotype and nucleotide diversity  and EP.1 (mainly occurring at the Eastern Pyrenees). The first one was shared by three localities of the Iberian System L1, L and L4 aG. isabellae , Eastern Pyrenees , Western Pyrenees , French Alps , Central Iberian System , and Betic Mountains   to cluster EP and those specimens from Bronchales and Huerta (L2 and L3) bearing haplotype EI.5 to cluster FA.Mitochondrial data revealed six groups of populations of ed) Fig. . The spaGI26) and 37 (GI11). Loci GI18 and GI23 showed significant departures from HWE in more than two localities. This was likely due to segregating null alleles, as supported by the geographic distribution of these departures, i.e. locus GI18 deviated from HWE at three sites (L1-L3) from the Iberian System and two  from the Betic Mountains, whereas HWE was rejected for GI23 at three localities from the Eastern Pyrenees (L5-L8)  showed the highest levels of gene diversity (He = 0.731–0.760). When it comes to private allelic richness, the southern site of Sierra-María (L28) showed a similarly high level to the Iberian System localities (0.35–0.39) , one in the Central Iberian System (L21) and the last one in the Betic Mountains (L28). The Western Pyrenean sites (L10-L15) revealed a particular substructure, as all pairwise comparisons but one (L12-L13) were significant  was supported only by Evanno’s method: the first cluster joined the French Alps, Eastern Pyrenees and Western Pyrenees (FA-EP-WP), whereas the second one grouped Eastern Iberia, Central Iberia, and Southern Iberia (EI-CI-SI) . Cluster and sampling locality only matched at Rascafría L21 and Sierra-María L28.The most likely partition of the microsatellite dataset was six groups , as from STRUCTURE Fig. . A seconion Fig. . Three oCOI alignment produced a rather shallow haplotype phylogeny, with many polytomies. Both in the phylogenetic trees  differ by three mutational steps. Although the first group was not as well supported in the Bayesian trees (posterior probability = 0.83) as the second one (posterior probability = 1), the monophyletic relationship between them was strongly preferred by the Bayes factor test (log difference = 21 log units). The clock-wise evolution of these sequences was also very well supported by the corresponding test (log difference of 35 log units in favour of the strict clock against the non-clock model). The split between these groups would have taken place ca. 400 kya , with the most recent common ancestor (MRCA) of EI-FA-EP-WP dated at 300 kya (95 % HPD: 195–405) and that of CI-SI at 190 kya (95 % HPD: 95–310).The 33 polymorphic sites observed along the ees Fig.  and the ees Fig.  of the CL8), phylogenetically closer to the haplotypes of the Iberian System  but differed by a single mutational step from the dominant haplotype at the Eastern Pyrenees (EP.1), whereas EI.5 was sampled at L2 and L3 and also differed from the French haplotype (FA.1) by one substitution. Two more features of the mitochondrial network should be pointed out. Firstly, the only haplotype found at the Central Iberian System (CI.1) derives directly from the dominant haplotype at the Betic Mountains (SI.1), in agreement with their joining in a monophyletic group by the Bayesian tree reconstruction. Secondly, the also unique haplotype of the French Alps (FA.1) derives from haplotypes of the Iberian System (cluster EI), not from those of the geographically intervening Pyrenees (clusters EP and WP). Turning again to the phylogenetic tree for corroborative evidence, we used a Bayes factor test to contrast the hypothesis that FA and EI form a group with the hypothesis that EI forms a group with the populations from the Pyrenees (EP and WP) instead, obtaining a strong preference (5 log units) for the former topology.Clusters CI, SI, FA, EP and WP generally showed either a single haplotype or a highly predominant one with others arising from it by a single mutational step Fig. . The onlDC) between clusters showed the highest bootstrap support (93 %) for the branch separating EI-CI-SI from FA-EP-WP. The same partition was first unveiled by STRUCTURE (if K = 2) and recovered by BAPS  with its expected value in mutation-drift equilibrium   Table . We conss) Table  scenarioGraellsia isabellae somehow resembles the one of Pinus nigra salzmannii [The distribution area of lzmannii , 63. Thelzmannii . Indeed,lzmannii . Lastly,lzmannii . In addilzmannii . More dePinus sylvestris when compared to other European populations. Within the Iberian Peninsula, the mitochondrial evidence revealed a particular differentiation of Sierra Nevada, one of the two extant populations of the Scots pine in Southern Spain. However, the other southern locality of Baza (only 80 km apart) showed similar haplotype frequencies to the Central Iberian System [Both chloroplast  and nucln System –56. The n System –56. The n System ).Pinus sylvestris (five markers) yielded K = 2 as the best partition. The southernmost locality  revealed as the first cluster, whereas the remaining 29 forests grouped together. Removal of this divergent population, led to a two-group partition with a clear West–east orientation  BAPS indicated again K = 2 as the most likely partition. The two southernmost Betic localities  grouped with the Central Spanish locality  and the Eastern Iberian forest coded as #XIV (Vistabella). All other localities clustered together. Such a two-group structure does not match the taxonomic division within P. nigra salzmannii, i.e. site #XIV (pyrenaica) grouped with the Central Iberian System and the southernmost Betic Mountains, whereas sites #IV and #X (hispanica) clustered with the Pyrenees and the northern Betic Mountains . The EIs nigra, ). Convervised by ). The SIG. isabellae. This hypothesis is congruent with the basal position of haplotype EI.4 and the rich diversity and highly reticulated pattern displayed by the EI haplotypes  as well as to loops caused by ambiguous phylogenetic relationships. Firstly, we argue for incomplete lineage sorting to be the reason why variants EP.2 and EI.5 seem to be geographically “misplaced” if compared to their closest haplotypes Fig. . Secondlsabellae . MoreoveG. isabellae in the Western Pyrenees revealed by the nuclear markers , whose natural origin has never been in dispute. A similar result was obtained for the Northern Pine Processionary Moth (Thaumetopoea pinivora) [P. nigra salzmannii) for G. isabellae existed in Southeastern France even before the LGM [P. sylvestris was traced (based on macrofossils) from the easternmost part of the Pyrenees northwards [We propose a native origin of the French populations (cluster FA) of the Spanish Moon Moth. Some authors considered them to be the result of a deliberate human introduction from Iberian individuals in the early 20ry, e.g. , 78. Howinivora) . Secondl the LGM , (ii) thrthwards  and (iiirthwards .G. isabellae did not support host-associated differentiation as a relevant factor preventing gene flow between populations using Pinus sylvestris or P. nigra. The indiscriminate use of the Scots and Black pine has been reported in other oligophagous plant-feeding insects [P. sylvestris and P. nigra have been reported for the pine processionary moth Thaumetopoea pityocampa [P. nigra vs P. sylvestris) differentiation has also been reported in T. pinivora [P. nigra instead of its usual host P. sylvestris [G. isabellae at Sierra-María, likely using P. pinaster/P. halepensis [P. nigra  is finally confirmed as the host-plant of G. isabellae at Sierra-María, future research will have to evaluate if its genetic differentiation of this southern locality is just the result of genetic drift  makes tift Fig.  or whethGraellsia isabellae reside in geographical areas identified as glacial refugia for Pinus sylvestris by Cheddadi et al. [G. isabellae, the “southern” (CI and SI clusters) and “northern” lineages , fits a two glacial refugia scenario. These two groups would have initiated their divergence approximately 400 kya  indicatvised by , 87). Thpinaster  and supppinaster , 19, 57.G. isabellae, we have just argued that genetic drift may account for a pre-Holocene origin of the WP cluster  was somig. 7 by . Or mayba myconi , and thetructure .Pinus nigra during the LGM and Mid-Holocene [P. nigra was the host species that allowed G. isabellae to reach the Eastern Pyrenees at some point during the Holocene. It is worth noting that the expansion of P. sylvestris from both the Western and Eastern Pyrenean refugia had already covered most of the Spanish slopes of that mountain range at the Mid-Holocene  an EP locality (Montesquiu) having one specimen carrying the most frequent haplotype (EI.1) of cluster EI, (ii) an EI site (Els-Ports) having four individuals showing the most frequent haplotype (EP.1) of cluster EP, and (iii) the seemingly “misplacement” of haplotype EI.20 being present in only one male from Els-Ports and just one substitution apart from EP.1 . Testing alternative hypotheses  for the colonisation of EP will require a more thorough sampling of the Catalonian areas where the distribution of both pine species overlap.We advocate that cluster EP was founded by individuals belonging to EI, independently and after the establishment of cluster WP. The EI-EP corridor was noticeable in the reconstruction of potential habitat for Holocene . TherefoP. sylvestris from its southernmost glacial refuge, as depicted at Fig. 7 by [P. sylvestris, either because of a temporal mismatch or because it actually never existed and G. isabellae reached Central Spain by means of its other host-species, P. nigra. Both P. sylvestris and P. nigra were likely to be used by G. isabellae when the split between its northern and southern mitochondrial lineages took place before the LGM. Then, the Central Iberian system (CI) was colonised by ancestors of the SI cluster. P. nigra was and currently still is most abundant than P. sylvestris in South Spain. By contrast, the occurrence of the Black pine in the Central Iberian System is currently minimal. Regardless of the pine species used to arrive to each of these mountain ranges, it is clear that the only possibility for the moth to survive was to use P. nigra in the South and P. sylvestris in Central Spain  melanogaster and the seed beetle Callosobruchus maculatus .G. isabellae between Renanué and Baiasca  may reflect the onset of their hybridisation mediated by male gene flow. The increase of reforested pine woodland  and unvPyrenees  as well Pyrenees , 94–96).Pyrenees . (2) OurGraellsia isabellae showed a strong phylogeographic pattern. The six differentiated groups revealed by the microsatellites and mtDNA showed a one to one correspondence with each of the main mountain ranges inhabited by the species. The mitochondrial data further clustered those groups into two major lineages, “southern” and “northern”, which likely diverged before the LGM. There was no evidence of host-associated differentiation between populations using P. sylvestris and the ones utilising P. nigra. Eastern and Western Pyrenees were most likely colonised by individuals closely related to modern Eastern Iberian populations through independent asynchronous events. The past and present wider distribution of P. nigra when compared to P. sylvestris suggests that G. isabellae used the former to reach several mountain ranges where it currently lives on the latter. This seems to be the case for the populations of the Spanish Moon Moth from the Eastern Pyrenees and French Alps, which likely derived from the Holocene fragmentation of the continuous forest of P. nigra connecting different populations of P. sylvestris in Northeastern Spain and Southeastern France. The Central Iberian system descends from Betic ancestors, whose populations showed genetic footprints of both a population expansion and posterior bottlenecks. Subsequent gene flow between both the Central/Betic mountains and the Eastern Iberian System was revealed by the nuclear dataset and was compatible with the overlapping potential distribution area of the Scots and Black pines during the Holocene. The mito-nuclear discordance involving the Eastern Iberian cluster is congruent with male-biased gene flow and/or a secondary contact after the evolution of mito-nuclear incompatibilities in geographically isolated areas.3) years ago; MRCA, most recent common ancestor; mtDNA, mitocondrial DNA; mya, million (106) years ago; na, not applicable; SI, Southern Iberia; SSD, sum of squared deviations; SSR, single sequence repeats; vs, versus; WP, Western Pyrenees.Ca, circa; CI, Central Iberia; cpDNA, chloroplast DNA; EI, Eastern Iberia; EP, Eastern Pyrenees; FA, French Alps; HPD, High Posterior Density; ISSR, Inter Simple Sequence Repeat; Kya, kilo (10


Corbicula fluminea, is an invasive alien species (IAS) originally from Asia that has spread worldwide causing major ecological and economic impacts in aquatic ecosystems. Here, we evaluated C. fluminea genetic , morphometric and sperm morphology variation in Portuguese freshwater ecosystems. The COI marker revealed a single haplotype, which belongs to the Asian FW5 invasive lineage, suggesting a common origin for all the 13 Portuguese C. fluminea populations analysed. Morphometric analyses showed differences between the populations colonizing the North (with the exception of the Lima River) and the Centre/South ecosystems. The sperm morphology examination revealed the presence of biflagellate sperm, a distinctive character of the invasive androgenetic lineages. The low genetic variability of the Portuguese C. fluminea populations and the pattern of sperm morphology have been illuminating for understanding the demographic history of this invasive species. We hypothesize that these populations were derived from a unique introductory event of a Corbicula fluminea FW5 invasive androgenic lineage in the Tejo River, which subsequently dispersed to other Portuguese freshwater ecosystems. The C. fluminea asexual reproductive mode may have assisted these populations to become highly invasive despite the low genetic diversity.The Asian clam,  Corbicula fluminea  is nowadays globally distributed and its invasive success is responsible for major ecological and economic impacts [C. fluminea may be related to their biological traits, namely: (i) rapid growth, (ii) early sexual maturation, (iii) short life span, (iv) high fecundity, (v) high filtration rates, (vi) broad dispersal capacities , (vii) ability to inhabit different substrate types, (viii) competitive success over native species and (ix) interactions with human activities [C. fluminea populations may reach great densities and biomass being also consumed by higher trophic levels [Biological invasions by bivalve mollusc species have become a worldwide problem due to their dispersal capacity and effects on biological diversity, and ecosystems functions and services –3. The A impacts . The sucMüller, 174 is nowtivities . Major etivities ,9. As a tivities ,9. C. flc levels ,9. Regarc levels ,10–12.C. fluminea are substantially documented, their taxonomic status is still unclear. Initially, morphology-based taxonomy led to an excessive number of species within the genus Corbicula [Even though the ecological and economic impacts caused by orbicula . This waorbicula ,15, whicorbicula . Howeverorbicula –19 and morbicula ,21–propoorbicula ,22,23.Corbicula presents different reproductive strategies, being able to reproduce sexually [Corbicula. This phenomenon occurs after the self-fertilization process—by an oocyte and a biflagellate sperm, which is a distinctive character of androgenetic lineages of the genus Corbicula found in both native and invasive populations [Corbicula, with the sperm from one lineage being able to fertilize the egg of another lineage [Corbicula species fitness and may contribute to the invasive success of the four genus Corbicula invasive lineages. Three of these, namely FW1 (form B), FW4 (form Rlc) and FW5 (forms A/R) have been reported in the native (Eastern Asia) and in the non-native range (Europe and North America). The fourth, FW17 (form C/S) has been detected outside the native range but not yet in Eastern Asia [Interestingly, the genus sexually ,25 and asexually –30. Prevulations ,31–35–whulations ,27,31,32 lineage ,36. The  lineage ,37–42. H lineage ,40. SuchC. fluminea presents a widespread geographic distribution and has invaded ecosystems throughout Europe, North and South America, and more recently North Africa [C. fluminea was first detected outside its native range (Eastern Asia) in 1924 in Vancouver Island, British Columbia [C. fluminea was firstly introduced in the North American continent as a food source for humans. However, the introductions into Europe and South America are believed to have occurred via ballast water [C. fluminea dispersed within the continents by different dispersal vectors—commercial transportation and other human activities but also by natural vectors such as birds and mammals—which promoted their rapid spread [C. fluminea was first detected in the Tejo River in 1980 [Currently, h Africa –47. RecoColumbia ,45. By tColumbia  and reacColumbia . BrittonColumbia  suggested spread ,50. In P in 1980  and a fe in 1980 , Minho [ in 1980 , Lima [5 in 1980 , Mondego in 1980  and Guad in 1980  Rivers. C. fluminea Portuguese populations employing molecular, morphometric and morphological sperm analysis. The obtained results were compared with other available worldwide data of Corbicula spp. from native and invaded regions applying population genetics and phylogeographical inference methodologies.The main objectives of this study were to assess the genetic variability and the phylogeography of The study did not involve any kind of endangered or protected species. No specific scientific research permits were required for the sample collection of these highly invasive invertebrates.C. fluminea were randomly collected in 16 different sites belonging to 13 distinct ecosystems: Minho , Lima , Tâmega , Tua , Sabor , Douro , Paiva , Mondego , Tejo , Sado , Mira , Guadiana  Rivers and Pateira de Fermentelos Lake , using a scoop net or by handpicking , 10 pmol of a specific set of primers– LCO1490 and HC02198 for the COI [Total genomic DNA was extracted from 328 samples of t method . The mit the COI , the HOL the COI  and CBF6 the COI . The fol the COI . The 18S the COI . The CYT the COI . All PCR the COI ,62.Corbicula spp. sequences of the mtDNA COI gene were retrieved from the NCBI-GenBank [Neocorbicula limosa was used as an outgroup for phylogenetic and phylogeographic analysis. The mtDNA, 18S rDNA and CYTb sequence alignments were performed employing the default parameters of ClustalW in MEGA 6 software [th generation and a total of 25% of the generated trees were discarded. The tree convergence was evaluated in MrBayes by analysing the parameters set values of the Potential Scale Reduction factor (PSRF) and the Estimated sample size (ESS). In addition, further visual and numeric convergence was assessed using Tracer v1.6 software [A total of 93 -GenBank ,63 and Nsoftware ,65. DnaSsoftware . Phylogesoftware  and Maxisoftware . Both BIsoftware ,70. The software . The BI software .C. fluminea specimens—from the Minho, Lima, Mondego, Sado, Mira, Tejo and Guadiana Rivers—were measured for length, height and width using a digital calliper (±0.2 mm). A Principal Component Analysis (PCA) was carried out using the three morphological measurements and the determination of the PCA components was performed using the correlation matrix in the “princomp” function of the R statistical software [The shells of 275 software .Corbicula fluminea (N = 10 from the Douro River) was collected to perform sperm morphology analyses. The sperm was obtained by collecting one drop of the specimens fresh gonadal tissue/fluid [A sample of ue/fluid  in a glaCorbicula spp. populations retrieved from GenBank [The obtained mtDNA COI sequences from all the 328 analysed individuals presented a unique haplotype which was phylogenetically compared with 93 other sequences from worldwide  GenBank –63. BothCorbicula specimens from different geographical ranges within Asia, Europe, North and South America, Africa and Oceania (0.97/96 node support) and C. madagascariensis, which is an outgroup of the freshwater Corbicula spp. lineages. For phylogenetic analysis purposes the classification “Group I-V” was employed in this study and does not imply the existence of haplotype similarity.The phylogenetic analysis demonstrated the existence of two well supported clades: the estuarine and the freshwater . The latter clade splits into five groups—I, II, III, IV and V—which includes The evaluation of Portuguese freshwater populations—Minho, Lima, Tâmega, Tua, Sabor, Douro, Paiva, Tejo, Sado, Mira, Guadiana Rivers and Pateira de Fermentelos Lake—revealed only one mtDNA COI haplotype (belonging to group IV with 0.90/85 node support), which is identical to previously reported haplotypes from Europe (form R), North America (form A), South America and the FW5 invasive lineage from Asia .Corbicula haplotypes from the non-Asian range, namely from: Europe, South America, Africa and Oceania (with the exception of Israel which is a Western Asian country), as well as the FW17 (C/S form), detected exclusively in non-native regions, namely Europe, Africa and South America.Group I (0.97/96 node support) encompasses two invasive lineages, the FW1 (form B) from Asia and North America and the FW4 (form Rlc) present in Asia and Europe, whereas group IV (0.90/85 node support) represents the invasive lineage FW5 (form A/R) from Asia, Europe and North America. Both of these groups include COI haplotypes from both the native and non-native range. Groups II and V  are strictly confined to Eastern Asia. Group III (0.69/55 node support) is the only group that includes most of the genus C. fluminea populations. This finding, suggests that Portuguese C. fluminea populations present a low genetic variability for these two markers. Further analyses employing these two markers were not carried out due to insufficient data available in the Genbank database.A subsample comprising a total of 110 specimens was used to evaluate the genetic variability of the mitochondrial CYTb and the nuclear 18S rDNA genes. The analysis revealed that only two haplotypes—one for the 18S rDNA (accession no. KT878642) and one for CTYb (accession no. KT878643)–were detected in Portuguese C. fluminea specimens from the Minho River; and centre/south cluster (C/S) comprising specimens from Mondego, Tejo, Sado, Mira, Guadiana Rivers. However, the majority (31) of the specimens from the Lima River (N = 40), located in the North of Portugal, grouped within the C/S cluster. Therefore, morphologically they present more similarities with the C. fluminea populations from the centre and south.The two PCA components explaining most variation  were jointly plotted to seek clusters related to the river/region location of each specimen . The PC1C. fluminea inhabiting the Douro River clearly revealed the presence of biflagellate spermatozoa  and three other rare haplotypes (each represented by only one specimen) in the Minho River [C. fluminea massive mortality events that occur recurrently in the Minho River [C. fluminea population recovered rapidly from these die-offs and attained its previous biomass and density [A previous study also reported the existence of this mtDNA COI haplotype in ho River . Howeverho River ,76 potenho River . Even th density , our datC. fluminea populations. The mtDNA CYTb haplotype has also been reported in Japanese populations [C. fluminea populations from the main Portuguese basins. Thus, this seems to be a general pattern in C. fluminea Portuguese populations. In addition, the sperm morphology revealed that at least one sample of the Portuguese C. fluminea lineage is biflagellate, a distinctive character of the asexual androgenetic lineages [C. fluminea has derived from an androgenetic invasive asexual lineage with low mitochondrial genetic variability. In fact, some case-studies have reported successful invasions with low or no genetic variation in animals and plants regardless of their reproduction mode—in Africa diverse genotypes of the water flea Daphnia pulex have been replaced by a single non-native clone from the American continent [Linepithema humile in California present a loss of genetic diversity which is associated with reduced intraspecific aggression and form interspecific dominant supercolonies [Fistularia commersonii exhibits low genetic variability in comparison to the Indo-pacific native range [Eichhornia crassipes from the Amazon basin presents one main clonal genotype in China [C. fluminea asexual reproductive mode may assist these populations to become highly invasive despite their observed low genetic diversity [C. fluminea populations found in the present study, we hypothesize that an asexual reproduction strategy might seem to increase their reproductive potential, thus contributing to their high invasive success [C. fluminea in Portugal, we hypothesize that the low genetic variability found in populations from main rivers all over the country, is a result of the introduction of an asexual lineage with a reduced genetic pool in Tagus River—where this species was first reported –that rapidly spread to other Portuguese freshwater ecosystems. This spread may have occurred through different dispersal mechanisms that may include human activities or natural dispersion by birds, mammals and fish as previously reported [C. fluminea populations exhibited low genetic variability, morphological differences have been detected in the present study  still remains to be explained [Corbicula spp. habitats that are less subjected to human mediated activities may in fact be inhibiting the spread of these haplotypes that derive from well-established populations [Most of the groups resolved in the phylogenetic inference present polytomies , indicatal scale . From a  regions . Howeverxplained . PerhapsCorbicula spp. invasion in Europe was exclusively by water ballast transport from America [Corbicula spp. may have also been introduced into the European continent directly from the Asian populations  within Portugal nor the main population source (North America and/or South America or directly from Asia).


Most species exist as subdivided ex situ daughter population(s) derived from a single original group of individuals. Such subdivision occurs for many reasons both natural and manmade. Traditional British and Irish pony breeds were introduced to North America (U.S.A. and Canada) within the last 150 years, and subsequently equivalent breed societies were established. We have analyzed selected U.K. and North American equivalent pony populations as a case study for understanding the relationship between putative source and derived subpopulations. Diversity was measured using mitochondrial DNA and a panel of microsatellite markers. Genetic signatures differed between the North American subpopulations according to historical management processes. Founder effect and stochastic drift was apparent, particularly pronounced in some breeds, with evidence of admixture of imported mares of different North American breeds. This demonstrates the importance of analysis of subpopulations to facilitate understanding the genetic effects of past management practices and to lead to informed future conservation strategies. The maintenance of populations to preserve maximal genetic variability and future adaptation potential is a common management challenge of conservation efforts in both domestic and wild species. Every finite population risks losing variability through stochastic sampling particularly when the number of reproducing individuals is restricted, creating founder effects and bottlenecks, for example, during: reintroduction of wild animals to previous habitats, formation of ex situ captive breeding groups, and formation of domestic species . Mitochondrial sequence data for the corresponding breeds of North American origin analyzed by Prystupa et al.  were pra et al.  were dowSSR genotyping of the samples were performed for 17 SSR loci using commercially available kits and protocols as described previously for the U.K., Winton et al. , and theP < 0.05) from Hardy–Weinberg expectations using the exact test in Arlequin 3.5  was calculated using STRUCTURE Harvester v0.6.7 (http://taylor0.biology.ucla.edu/struct_harvest/) as per Evanno et al.  were calculated using Arlequin 3.5 (Excoffier and Lischer An unbiased Bayesian approach using Markov chain Monte Carlo (MCMC) clustering of samples was conducted via the STRUCTURE v2.2.3 software . In line with other studies, hypervariable nucleotide positions 15,585, 15,597, and 15,650 were removed (downweighted to 0) and nucleotide positions 15,604, 15,659, and 15,737 were downweighted (from 10 to 5). Nucleotide positions refer to that of the complete horse reference sequenced by Xu and Arnason  (Drummond and Rambaut Summary statistics are presented in Table HO), and inbreeding coefficients (FIS), with the exception of the U.S. Welsh Pony and the U.S. Connemara , with 6.91% of total variation existing between populations within U.S. and U.K. groups. There was no significant variance found between U.S. and U.K. breeds. Nonetheless, Bayesian analysis using STRUCTURE of the log likelihood (Ln Pr(X/K)) of the posterior probability for a given K suggested the optimum value was K = 2. Delta K plots also showed a secondary peak for K = 4 clusters , with the exception of the Connemara. Where K = 4 clusters of sample origin were also evident, but the Fell and Shetland populations cluster together irrespective of geographical origin. Pairwise FST comparisons were performed based on the sum of squared allele size differences (RST) and substantiated the relationship between populations of U.K. and U.S. origin (Table H (4Neμ) values for autosomal data . Excepting the Fells and Dartmoors, transcontinental populations of the same breed did not show significant divergence in maternal genotypes as indicated by pairwise comparisons and estimated Nm  demonstrating the associated reduction in ancestral diversity as a result of a recent bottleneck in U.S. populations  raggedness index was found for the Welsh Section A, U.S. Connemara, Shetland, and U.S. Shetland population, which indicated that they existed in a multimodal distribution consistent with a stationary population for some time.Skyline plots generated using mitochondrial sequence data illustrated that the effective population size of the U.S. breeds was lower than that of U.K. counterparts at a given time-point , excludes this being the result of from laboratory-based genotyping discrepancies. Divergence between captive or wild subpopulations and source populations has been demonstrated for other species, where increasing gene flow or multiple restoration/reintroduction sources can limit the effects . Autosomal divergence between the U.K. and U.S. populations suggests that stochastic drift has occurred between source and daughter populations. The clustering according to breed rather than country of sample origin, evidenced by STRUCTURE analysis and pairwise The Welsh breeds were an exception to the typical shared characteristics of an origin–daughter relationship observed within this study. It was not possible to identify an individual Welsh Section as the founder of the U.S. Welsh pony. The U.S. Welsh consisted of mitochondrial signatures that were shared by both U.K. sections As and Bs, yet the association with the Section Bs was most pronounced. This female amalgamation was also reflected in the Skyline plots whereby, unlike other U.S. populations, the U.S. Welsh pony showed an overall increase and a higher effective population size at the same relative time-point compared to the Section A. This is consistent with the U.S. Welsh pony sample used here having originated from a “mixed” population of Welsh sections A and B ancestors. As such, the U.S. Welsh population captures ancestral mitochondrial diversity from a larger population of origin than the U.K. Section A pony does alone. In effect, the bottleneck caused by breed formation was more recent in the Welsh Section As compared to the “mixed” origin of the U.S. Welsh. While cross-breeding and amalgamation seems to have occurred, particularly in the formation of the U.S. Welsh Pony, this contrasts with estimates of Nm for maternal data indicating American populations are more divergent from each other. This is therefore likely evidence of two processes. In one, founder and/or drift effects resulting in allele frequency differences  have driven populations in the U.S. apart and given lower estimates for previous population sizes due to loss of haplotypes passing through the bottleneck. In the other, cross-breeding or use of a common external breed  as a crossing sire/dam in the U.S. has brought some of them together, but to a lesser degree than the stochastic changes that have caused the evident divergence.As an example of this, SSR analyses demonstrated closer relationships between the U.S. Welsh and the Section A, with nearly four times the number of migrants exchanged than with the Section B, in contrast to the mtDNA patterns. It thus seems likely that U.S. Welsh ponies have a different maternal and paternal history; seemingly a product of mares from at least both sections of Welsh ponies from the U.K., while stallion introductions to the U.S. have been largely Section A. The diversity seen in the U.S. Welsh ponies today is therefore a consequence of original breed formation and subsequent repeated “migration” from one subpopulation of the source group. This highlights the importance for conservation strategies more generally to appreciate the degree of genetic variability and the level of gene flow among each subpopulation (Ciofi and Bruford Skyline plots in the U.S. populations tend to be dominated by evidence of recent bottleneck effects, as only a limited signal of previous population changes can survive through the bottleneck to the current population. This is illustrated by mitochondrial haplogroup I, predominantly found in Section A and Fell ponies, but in only a single sample from each respective U.S. population. The branching star-shaped pattern of haplogroup I indicates these lineages may have existed within the earliest ancestral ponies of Britain and Ireland and is strongly characteristic of some of the more isolated populations (Winton et al. The recently characterized Kerry Bog is a prime example of the importance of consistency between conservation management organizations or breed societies. Our results indicate more restricted matrilines and lower diversity values in the U.S., but with no major divergence from the maternal source population. However, the recently established American Kerry Bog Pony Society accepts pied coloration not found in Ireland Anon, ; this diWhile statistically significant comparisons cannot be made for individual Skyline plots between the U.K. and U.S. populations, together they provide a comparative story of reduced population size and recent bottlenecks in the U.S. breeds. As expected, the results in FigureBy studying ex situ subpopulations and putative source populations through the analysis of genetic diversity of ponies, we have established it is possible to identify different genetic characteristics that are compatible with known histories of those populations. We detected clear indications of founder effect and stochastic drift in most daughter populations, more pronounced in some breeds than others, and evidence of admixture of imported mares of different breeds within the U.S.A. To conserve a valuable resource and maintain sufficient adaptive genetic variability, it is important that derived populations remain representative of the original source once ex situ and that enough founder lines are present within the original or subsequently exported animals to maintain diversity levels and avoid extreme stochastic drift. It is imperative that the objectives of daughter breed societies/management groups are clear and consistent from the outset. We demonstrate that genetic analysis of subpopulations can assist in understanding the genetic effects of past management practices and inform future conservation strategies even in the absence of recorded histories.


Saxifraga granulata along two rivers in central Belgium. We hypothesized that rivers might be essential for gene flow among increasingly isolated populations of this species. Genetic diversity was high (SH = 0.68), which to a certain extent can be explained by the octoploid nature of S. granulata in the study area. Populations along the Dijle and Demer rivers were also highly differentiated  and showed significant isolation-by-distance, indicating moderate levels of gene flow primarily between populations that are geographically close to each other. Along the river Demer population genetic diversity was higher upstream than downstream, suggesting that seed dispersal via the water was not the primary mode of dispersal. Overall, these results indicate that despite increasing fragmentation populations along both rivers were highly genetically diverse. The high ploidy level and longevity of S. granulata have most likely buffered negative effects of fragmentation on genetic diversity and the spatial genetic structure of populations in riparian grasslands.Due to changes in land use, the natural habitats of an increasing number of plant species have become more and more fragmented. In landscapes that consist of patches of suitable habitat, the frequency and extent of long-distance seed dispersal can be expected to be an important factor determining local genetic diversity and regional population structure of the remaining populations. In plant species that are restricted to riparian habitats, rivers can be expected to have a strong impact on the dynamics and spatial genetic structure of populations as they may enable long-distance seed dispersal and thus maintain gene flow between fragmented populations. In this study, we used polymorphic microsatellite markers to investigate the genetic diversity and the spatial genetic structure of 28 populations of  Habitat loss and fragmentation decrease the size and increase the spatial isolation of many plant populations in Europe and elsewhere in the world ,2. PlantIn riparian habitats, rivers can be expected to be important to maintain gene flow between populations. Natural river landscapes consist of a mosaic of habitats that are tightly connected to each other by water movement ,8. RiverSparganium emersum and Populus nigra disperse upstream via birds and wind, while downstream migration might have occurred via river systems [Given that river systems can be characterized by unidirectional water movement seed dispersal via the water (i.e. hydrochory) will most likely occur downstream. When downstream migration is slightly higher than upstream migration, genetic diversity could accumulate downstream, while populations upstream may gradually lose genetic diversity and in the most extreme case may disappear due to stochastic events . This ph systems ,23.The occurrence and frequency of long-distance seed dispersal via rivers also depends on the distance to the streamline. Several studies of riparian plant species have shown that there is a clear relationship between distance to the river and population genetic differentiation ,19. SomeSaxifraga granulata along two rivers in Belgium. Over the last decades, populations of S. granulata have become smaller and more isolated due to habitat loss and fragmentation [S. granulata along two dynamic river systems. More specifically, we asked the following questions:The aim of this study was to assess whether rivers shape the genetic diversity and spatial genetic structure of fragmented populations of the grassland perennial entation ,27. The entation . Hence, S. granulata genetically differentiated within both river systems?Are populations of Does gene flow mainly occur between populations that are geographically close to each other?Do we find accumulation of genetic diversity downstream, indicating that the river is the main dispersal vector?S. granulata, meadow saxifrage, is a self-compatible, insect-pollinated, protandrous, perennial rosette herb [tte herb . The spette herb . Individtte herb . Populattte herb , mainly tte herb ,27.S. granulata used to be a widespread species in the first half of 20th century, but nowadays is confined to riparian grasslands. The study was conducted along the rivers Dijle and Demer  and two small ceramic beads. DNA was extracted from a total of 560 plants following the NucleoSpin Plant II protocol for genomic DNA . For cell lysis we used buffer PL1 and incubated the cell lysis suspension for 60 min. DNA concentration and quality were measured using a NanoDrop ND-2000 spectrophotometer .S. granulata is octoploid in our study area [S. granulata is presumably an auto- rather than an allopolyploid [Nine recently developed microsatellite markers were used for genetic analysis. In a previous study these markers indicated that udy area . All locolyploid .The markers were amplified in two separate PCR multiplexes in a 2720 Thermal Cycler . The total PCR multiplex reaction volume was 10 μl; containing 5 μl Qiagen Multiplex PCR Master Mix , 3 μl RNAse-free water, 1 μl of one of the two multiplexed primer combinations (25 ng/μl) and 1 μl of template DNA (50 ng/μl). Both PCR multiplexes followed the same thermocycler program with initial denaturation of 15 min at 95°C; 27 cycles of 30 sec at 95°C, 1.5 min at 58°C and 1 min at 72°C; and a final elongation of 30 min at 60°C. Then, 1 μl of the PCR reaction was added to a solution of 8.8 μl formamide and 0.2 μl of GeneScan 500 LIZ  size standard (Applied Biosystems). Fragments were sized on an ABI Prism and analyzed by capillary electrophoresis using the 3130 Genetic Analyzer (Applied Biosystems). Samples from all populations were randomly distributed across the ABI plates to prevent influence of gel-artifacts in the data. The raw genetic data were scored using GeneMapper Software v4.0 (Applied Biosystems) using the default settings for microsatellites. Panels and bins for GeneMapper were manually constructed and all data were visually checked to make sure that the loci were identified correctly.S. granulata in the study area, population genetic data were analyzed using the program GenoDive 2.0 [GenoDive: the number of alleles, the effective number of alleles , and the so-called ‘gametic heterozygosity’ (HS) [HE) in diploid species [Because of the octoploid nature of Dive 2.0  and the Dive 2.0  in R 2.1Dive 2.0 . Three mDive 2.0  were caly’ (HS) , which i species . Analyse species , since iGST, G”ST [DEST [GenoDive to assess the genetic structure among populations along both river systems. These population differentiation statistics have their specific advantages and disadvantages. GST , for instance, relates the amount of genetic variation among populations to the total genetic variation over all populations and is determined by the amount of within-population diversity (HS). Hence, GST = (HT−HS)/HT [GST may be underestimated. For this reason, we also calculated G”ST and DEST, which have the advantage that they are not negatively dependent on the amount of within-population diversity [G”ST) values were calculated as well.Several measures of population differentiation; T, G”ST  and DEST [DEST , were caHS)/HT . Howeveriversity . To asseGenoDive and a spatial Principal Component Analysis (sPCA) [G”ST) and the other log-transformed distances. sPCA was used as an alternative spatial analysis method and includes the genetic variation as well as the spatial structure of populations along both rivers. Based on the spatial coordinates a Delaunay triangulation connection network was built between populations and the largest eigenvalues, based on genetic variance and spatial structure, were retained for the analysis [GenoDive [For both river systems, the spatial genetic structure of populations was analyzed using a Mantel test in s (sPCA)  using thanalysis . To idenGenoDive . The simGenoDive .S. granulata along the two rivers, we first related genetic diversity to population size and distance to the river. We expected genetic diversity to increase with increasing population size and that populations closer to the river would be more genetically diverse since the chances of receiving seeds via the water is higher. These relationships were assessed using linear models in R. Finally, we tested the hypothesis that the rivers are the main seed dispersal vectors  by relating the position of each population along the river to the population genetic diversity measure ‘number of alleles’, using a linear model in R. Position along the river was quantified as the log transformed distance between populations starting from the first upstream population.To investigate patterns of genetic diversity of t26 = 4.0, P < 0.001 and HS: t26 = 4.8, P < 0.001; Populations along the river Demer were significantly more diverse than populations along the Dijle . All population differentiation statistics were highly significant (P < 0.001) for both rivers. Pairwise genetic differentiation values (G”ST) for populations along the river Dijle ranged from 0.033 to 0.715 with an average of 0.264 . However, this was caused mainly by the highly differentiated population ‘Heverlee’, which was situated in the middle of the sampling range. When we excluded this population from the analyses we did find a significant isolation-by-distance effect along the Dijle  and populations along the river Demer were actually significantly more diverse upstream than downstream .Based on the distribution of genetic diversity along the two rivers, we did not find any indication for an increase in genetic diversity downstream as expected when the river is the main dispersal vector . PopulatF1,13 = 0.36, P = 0.56; Demer: F1,11 = 0.0002, P = 0.99), indicating that populations closer to the stream did not receive more alleles via the water than populations that were situated further from the river. Finally, genetic diversity was not significantly related to population size .High population differentiation in combination with a significant isolation-by-distance effect indicates moderate levels of gene flow, primarily between populations that are geographically close to each other. Mantel’s Succisa pratensis has shown that current genetic similarity between populations was affected by past connectivity as a result of the long life-span and a persistent seed bank of S. pratensis [S. granulata used to be more abundant in the study area and was not entirely restricted to riparian grasslands, but also occurred in meadows and grasslands of the traditional small-scale agricultural landscape. Current population genetic diversity patterns might reflect historical connectivity, since clonal propagation and polyploidy can buffer loss of genetic diversity.On the other hand, the observed population differentiation patterns might also reflect historic connectivity. A study of the grassland perennial ratensis . In the Helmholtzia glaberrima persistent founder effects also increased population differentiation [The observed differences in population differentiation between the two studied rivers were unexpected, since the distances between the populations along the Dijle were smaller, while populations showed higher genetic differentiation. These differences might be explained by a higher population turnover rate along the Dijle. For instance, population ‘Heverlee’ shows signs of a founder event, it has the lowest effective number of alleles but it does not have the lowest number of alleles, suggesting that this population probably had one high frequency allele at most of its loci that were uncommon in other populations. In ntiation .S. granulata. Polyploid populations contain more copies of each gene than similar sized diploid populations. Hence, they could be more genetically diverse due to a higher number of mutations and a lower impact of genetic drift [Populus nigra showed similar high levels of genetic diversity [The studied populations also showed high levels of genetic diversity within populations, which could be explained by the polyploid nature of ic drift . Most otic drift ,58,60,61iversity . High geS. granulata has ways of dispersing seeds, pollen or vegetative structures upstream, like other riparian plant species [S. granulata could occur via pollen and seed flow mediated, for instance, by animals, while downstream seed dispersal across large distances could occur during flooding events that inundate the river valleys and riparian grasslands. In Origanum vulgare, another inhabitant of riparian grasslands, occasional long distance seed dispersal events could be traced back to extreme-floods of the river Meuse and local colonization was mostly observed after regular flooding events [S. granulata occurred at a distance from the river, flooding events in the past could have been important for gene flow, shaping the spatial genetic structure of the studied populations.We did not find evidence to support the hypothesis that the river is the main seed dispersal vector, which would lead to accumulation of genetic diversity downstream. In contrast, genetic diversity along the Demer River was higher in populations upstream instead of downstream. Hence,  species ,58,62–64g events . Thus, eS. granulata in Western Europe, populations along the Dijle and Demer are still genetically diverse. The high ploidy level and longevity of this species may have buffered the negative effects of increasing isolation due to anthropogenic habitat modification. The levels of population differentiation found in this study imply a history of moderate levels of gene flow, mostly between populations that are geographically close to each other. However, these patterns might also reflect historical connectivity when populations were more widespread. In the riparian plant Myricaria germanica the historic pattern of gene flow in one of the studied catchments was mainly directed downstream, while contemporary gene flow was bidirectional. This indicates that the importance of individual dispersal vectors  can change over time [S. granulata relies on other dispersal vectors that maintain connectivity between increasingly isolated populations.Despite the recent decline of ver time , for ins


The basicranium has been described as phylogenetically informative, developmentally stable, and minimally affected by external factors and consequently plays an important role in cranial size and shape in subadult humans. Here basicranial variation of subadults from several modern human populations was investigated and the impact of genetic relatedness on basicranial morphological similarities was investigated. Three-dimensional landmark data were digitized from subadult basicrania from seven populations. Published molecular data on short tandem repeats were statistically compared to morphological data from three ontogenetic stages. Basicranial and temporal bone morphology both reflect genetic distances in childhood and adolescence (5–18 years), but not in infancy (<5 years). The occipital bone reflects genetic distances only in adolescence (13–18 years). The sphenoid bone does not reflect genetic distances at any ontogenetic stage but was the most diagnostic region evaluated, resulting in high rates of correct classification among populations. These results suggest that the ontogenetic processes driving basicranial development are complex and cannot be succinctly summarized across populations or basicranial regions. However, the fact that certain regions reflect genetic distances suggests that the morphology of these regions may be useful in reconstructing population history in specimens for which direct DNA evidence is unavailable, such as archaeological sites. Homo sapiens based on geographic provenance, population affinities, and dietary proclivities )livities . The vasPrevious studies have demonstrated that the basicranium is a phylogenetically informative region among human and nonhuman primate adults –15. HumaNonetheless, since the cranium starts to develop before birth –18 and cPrevious studies have compared the shape of the developing facial skeleton among groups from different geographic locations and genetic backgrounds to determine whether similar ontogenetic processes characterize divergent groups –10. A wiTo date, there have been extensive studies on the morphology of the temporal bone and its applications to phylogeny –34, evol Homo sapiens and found that significant differences among populations originate early in ontogeny. Specifically, individuals from different populations can be differentiated based on temporal bone shape prior to the eruption of the first molar [Modern human populations can be readily distinguished from one another based on one particular component of the basicranium and temporal bone shape –4, 34. Mst molar . Subsequst molar . These fSeveral researchers have attempted to determine whether the basicranium is a more reliable region for reconstructing genetic distances than other regions of the cranium , 3, 32. (H1)Human populations differ significantly in the shape of the basicranium and its various components irrespective of ontogenetic stage.(H2)Differences among human populations in basicranial morphology are significantly correlated with their genetic distances throughout ontogeny.In light of the previous work and unresolved research questions outlined above, the aims of this study are to determine at what point during the ontogenetic process population-specific basicranial morphologies emerge and to compare basicranial ontogeny among human populations. Specifically, we test the following hypotheses:Three-dimensional data on basicranial morphology were collected by one of us (DHD). The data were collected from skulls of seven modern human populations at various ontogenetic stages  from the t-test was performed, comparing the Procrustes residuals from the different trials on each skull. The paired t-test did not yield significant differences , indicating that the individual landmarks were consistently measured without significant error.Prior to traveling to the museums, an intraobserver error study was conducted to ensure the accuracy of the data collection. Two adult crania were digitized ten times each. To minimize the effect of investigator fatigue, the data were collected on two separate days. A paired samplesForty-four landmarks were collected from the basicranium and subcategorized into temporal, occipital, and sphenoidal regions Tables . These cData on individual genotypes for 783 short tandem repeats (STRs) were compiled from Ramachandran et al. , 41 and STRs are composed of back-to-back repeating segments of two to six nucleotides and are found at many locations within the genome , 39. TheThe morphological data were analyzed using a Generalized Procrustes Analysis (GPA), in which the digitized points were rotated and translated and specimens were scaled to the same size, such that the only remaining differences among them were directly attributable to shape. The GPA was followed by a Principal Components Analysis (PCA) using MorphoJ . Adult sIn order to assess the degree to which basicranial morphology can be utilized to correctly classify individuals of various ages into the population from which they derived, a discriminant function analysis (DFA) was conducted using the Principal Component (PC) scores from the PCA. The DFA was used to determine whether groups could be classified reliably or if there was excessive morphological overlap. This analysis was conducted for the morphology of the basicranium and then for each of its major components, the temporal, occipital, and sphenoidal regions. These tests were conducted with cross-validation using SPSS version 11.0.1 . This analysis indicated how well basicranial shape discriminates among populations at the three subadult stages of ontogeny .A matrix of Slatkin's molecular distances among the molecular representative of these populations was calculated from the published molecular data using Arlequin 3.11  Table 3Table 3. A series of descriptive analyses were conducted to evaluate the ontogenetic trajectory in the sample. Principal Component scores were regressed against centroid size and biological age  to deteProcrustes distances based on basicranial morphology of populations sampled in this study were found to be statistically significant for all combined-age subadult samples . When seDFAs were performed on all regions of the basicranium: temporal, occipital, and sphenoidal. The DFA for the entire basicranium resulted in cross-validated classification rates ranging from 13.3–34.8% . The indThe Egyptian population was one of the most correctly classified in the basicranial (33.3%), temporal bone (55%), and occipital bone (35.7%) data sets Tables –11. The FST distances based on STR data using Mantel tests. In the combined subadult sample, the morphology of the basicranium and occipital bone was significantly correlated with molecular distances , thus supporting (H2)  for these data sets. The temporal bone and basicranium also reflect genetic distances during AC2 (5–12 years of age) when the Egyptian population is excluded. The morphology of the sphenoid bone does not reflect genetic distances for subadults of any age category; however, its morphology can be used to discriminate among populations with slightly higher rates than the other two cranial regions. Therefore, (H2) is not supported for the sphenoid, AC1 , or the occipital bone for AC2.The regression of biological age with Principal Components scores Tables  and 15 fThe regression analysis of centroid size with PC scores for the entire basicranium revealed significant correlations between size and PC1 (14.83% of the variance), PC2 (9.31% of the variance), PC3 (7.57% of the variance), and PC4 (6.53% of the variance) Tables . The regIn order to further explore how populations and age categories differed with regard to basicranial morphology, we conducted a shape space exploration in which wireframes of each cranial data set were morphed along the major PC axes to visualize how the shape varied along each PC.In basicranial shape, subadults were commonly located on the +PC1 axis . A positAssociated with a positive PC2 score , the EAMTo date, extensive research has been conducted on the temporal bone and how it reflects genetic relationships in nonhuman primates e.g., –33, 48)., 48.33, (H1)Human populations differ in the shape of the basicranium and its various components irrespective of ontogenetic stage.The hypotheses that formed the basis for the study were found to be only partially supported. In actuality, the patterns of morphological variation turned out to be more complex than the relatively simply stated hypotheses. The patterns of the individual basicranial bones differed from each other and across the various age categories. Thus, the ontogenetic processes driving basicranial development cannot be succinctly summarized across all regions of the basicranium.(H2)Differences among human populations in basicranial morphology are significantly correlated with their genetic distances throughout ontogeny.This hypothesis was not supported by our results. All populations were found to differ significantly in basicranial morphology in the combined subadult samples. However, in the separate age cateories there was a trend toward increasing differentiation with age. In AC1, several population were not significantly different, by AC2 most populations differed, and by AC3 all populations were significantly different. Interestingly, of all the individual cranial bones, the sphenoid was revealed to be the most distinct among populations and therefore the most reliable region for population classification.This hypothesis was partially supported for several subsets of our data. Our results show that morphology of the basicranium, occipital, and temporal bones each significantly reflects genetic distances in the combined subadult sample. The sphenoid bone, however, is not a good indicator of genetic distances in subadults in the combined subadult sample or at any ontogenetic stage. In the separate age categories for the basicranium, temporal, and occipital regions, the older the individuals, the more congruent the patterns between the genetic and morphological datasets, such that AC3 was the most highly correlated, followed by AC2 and then AC1.The discriminant function analyses revealed that the Egyptian population was the most correctly classified population for the basicranium, occipital, and temporal bone in subadults. The Egyptian population was most likely classified correctly because it is the most geographically and genetically distant of the populations in this study. This finding is consistent with the previous description of Egyptian cranial morphology as unique and distinctive .Unsurprisingly, the closely related Native American groups of Utah and Mexico were often classified as each other in basicranial, temporal, and occipital bone for all subadults. This trend has been supported by previous studies , 7 and sBased on the findings of this research, the basicranium, temporal, and occipital bone reflect genetic distances in childhood and adolescence, but this study suggests that these differences are not seen in infancy. AC1 was not correlated with molecular distances among populations for any of the cranial regions. Two possible scenarios may explain these results in infancy. First, infant basicranial morphology may be similar among populations and the observable differences in later ontogenetic stages have not yet developed. However, an alternative explanation is that as a result of the fact that many basicranial bones are not fully fused in infants, our sample sizes in the AC1 age category may have been insufficient to reveal subtle differences among populations in infancy.These findings contrast with some of the results found previously for the temporal bone , 31, whiInterestingly, while morphology of the sphenoid was not found to reflect the molecular distance matrix at any ontogenetic stage evaluated , it did Overall patterns revealed by this study show that the shape of the basicranium, the temporal bone, and occipital bone, for all populations excluding the Egyptian population, reflects genetic distances in subadults. Removing the Egyptian population from the analyses yielded significant results in the temporal bone, occipital bone, and age categories AC2 and AC3. It cannot be definitively ascertained why the Egyptian population deviated from the patterns of the rest of the included samples, but one possibility is the relatively high degree of mismatch between the morphological Egyptian sample and its molecular population representative, the Mozabite. The Mozabite people live in Algeria and speak a Berber language. As one of the few northern Saharan populations to have been extensively studied for neutral molecular loci, they are the closest well-typed molecular representative for the Egyptians, but certainly not a perfect match.Patterns on the basicranial PC wireframe showed that the Egyptian crania appeared on the +PC2 scale, indicating that the basicranium in the Egyptian population is relatively shorter anteroposteriorly compared to the other populations. Most of the Egyptian population clustered towards an increasingly long occipital bone  and increasing large temporal bone. It appears that the Egyptian cranium might have a relatively smaller overall basicranium but that the temporal and occipital bone increased in length, with the sphenoid decreasing in length to compromise.A study by A. C. Berry and R. J. Berry  found thOne potentially limiting factor of this study is the degree of mismatch between the molecular and morphological population representatives. The morphological samples were chosen based on the availability of subadult cranial material in museum collections. Thus, we were not able to include a wide geographically distribution of populations. In North American natural history collections, there is a natural bias towards Native American specimens. Consequently, the present study included a few Native American samples. Similarly, the molecular representative for each population was not a perfect match with these morphological populations. However, there is precedent for this approach. As noted by Roseman (2004), such mismatch is not necessarily problematic, but the correlations obtained from this type of analysis should be interpreted as minimal approximations of their actual value. Thus, the significant correlations obtained here should be interpreted as minimum estimates of a real biological relationship between morphology of the cranial region and genetic relatedness.The landmark dataset employed in the present research expands the smaller landmark sets used by previous studies. Harvati and Weaver  and SmitOccipital bone morphology is valuable for assessing relatedness in adolescents starting around the age of 13 years old. The sphenoid bone, on the other hand, does not reflect genetic relatedness at any ontogenetic stage; however, despite this, the morphology is distinct enough among populations to allow unidentified specimens to be classified by population with rather high accuracy. This is likely because the sphenoid body of basicranium reaches adult size and shape more rapidly than other portions, presumably because vital cranial nerves (II–VI) run through the cranial base in the sphenoid region . This maPopulation relatedness can be inferred using the basicranium, temporal, and occipital bone of subadults, especially those of 13–18 years of age. The findings of this study have implications for future studies of archaeological specimens for which genetic material is not well-preserved. Given that the morphology of the temporal bone and basicranium reflect genetic distances in young subadults, the morphology of these cranial subsets can be used to sort human populations with a reasonable degree of precision . This could be useful for child or adolescent cranial specimens of unknown affinity found at archaeological sites.If future studies examine other hominoid (ape) species in a similar manner, the findings combined with hose of the present study would have implications for the development of hominoid brain size, posture, and evolution. First, the angle of the midline cranial base is hypothesized to correlate with the volume of the brain relative to its basicranial length –52. ThusIn this study, the basicranium, occipital, and temporal regions were found to reflect genetic distances among populations in childhood and adolescence. The sphenoid bone, however, is not a good indicator of genetic distances in subadults, but its morphology may still serve as a reasonably accurate means of classifying individuals by population affinity. Unsurprisingly, the Native American populations, and especially the Utah sample, were commonly classified as another Native American population, as would be predicted if basicranial shape reflects genetic relatedness. These findings reveal valuable information on the population differences in basicranial morphology at various ontogenetic stages.


In total, 23 chloroplast haplotypes and 24 nuclear haplotypes were recovered from the 21 natural populations and six hebarium specimens. Phylogenetic analysis based on the combined plastid and nuclear dataset revealed two distinct lineages of L. buchtormensis, which inhabit the disjunct areas on both sides of the desert zone. The molecular dating analysis indicated that the divergence between the southeastern and the northwestern populations occurred in the middle Pleistocene, concomitantly with the desert expansion. The geographical vicariance likely contributed to the present disjunct distribution of L. buchtormensis across the deserts in Northwest China. Populations in the southeastern region may have migrated from the northwestern region, and seem to be a peripheral distribution of L. buchtormensis.In Northwest China, aridification and desert expansion play significant roles in promoting desert plant diversification and speciation. However, to date, little is known about the effects of the desert barrier on the population structure of montane, non-desert species in the area. In this study, we sequenced chloroplast DNA regions ( Located in central Eurasia, the arid Northwest China  is one of the most arid zones in the world, covering the western parts of the Inner Mongolia Autonomous Region and Ningxia Provinces, across the Hexi Corridor in Gansu Province to the Xinjiang Uygur Autonomous Region  1,2]. I. I2]. InBased on the varied geography and climate and on the plant diversity, the flora of the arid Northwest China is generally classified into the Asian desert flora subkingdom and the Eurasian forest subkingdom ,6. With Libanotis buchtormensis (Fisch.) DC. (Umbelliferae) presents an interesting model for studying the intraspecific divergence and spatiotemporal population dynamics of montane species in the arid Northwest China. This perennial herb is widely distributed in the mountain regions of Central Asia, and western and eastern Siberia [Libanotis buchtormensis prefers sunny rocky slopes at 700–3000 m altitude [Libanotis buchtormensis is insect–pollinated and mainly gravity–dispersed [L. buchtormensis. Moreover, morphological differences between the two regions have been observed [ Siberia ,12. Libaaltitude . Libanotispersed . The speispersed . The desobserved . Individobserved .trnL–trnF and trnS–trnG) and a bi–parentally inherited nuclear gene (rpb2) to investigate the phylogeographical history of L. buchtormensis distributed alongside the desert zone in Northwest China. The main goals were to answer the following questions: (1) How does the desert zone in Northwest China contribute to the genetic variation among L. buchtormensis populations? (2) When did the NW and SE populations diverge and expand/contract? and (3) What are the main driving forces for the disjunction of L. buchtormensis?In this study, we used maternally inherited chloroplast DNA sequences  and the Western Sichuan Plateau (three) were also used in the study. We used Libanotis spodotrichom K. F. Fu and Peucedanum praeruptorum Dunn as outgroups [In total, 256 individuals from 21 populations were sampled, covering the possible geographical range of in China , Fig 1. utgroups ,16. VouctrnL–trnF and trnS–trnG, were amplified and sequenced in all L. buchtormensis samples (including herbarium specimens) following Taberlet et al. (1991) [rpb2, i.e., the 23rd intron of RNA polymerase beta subunit II, was amplified to provide nuclear DNA (nrDNA) polymorphisms at the population and possibly individual level. This region has been used successfully in phylogenetic and phylogeographic studies of various plant taxa [trnL–trnF), LC072695–LC072708 (trnS–trnG), LC072862–LC072866 (products of primers LB1F and LB1R), and LC072867–LC072890 (products of primers LB2F and LB2R).Genomic DNA was extracted from silica–gel dried leaf material based on the modified CTAB method . Two pla. 1991)  and Hami91  and Hant taxa –22. Initant taxa . To sequh) and nucleotide (π) diversity at different levels  were calculated using DnaSP (ver. 5) [NST) and unordered (GST) alleles/haplotypes, and the comparison result can be used to investigate whether phylogeographic structure exists at the population level. The genealogical relationships of the cpDNA and nuclear haplotypes were constructed using the median joining (MJ) algorithm in Network 4.6 [Haplotype ((ver. 5) . A U–sta(ver. 5)  for the work 4.6 . In thiswork 4.6 .L. buchtormensis were inferred using Bayesian inference (BI) and maximum likelihood (ML) analysis, based on cpDNA data and rpb2 data, respectively. The best–fit model of nucleotide substitution (cpDNA: TPM1uf + I and rpb2: HKY + I) was determined by jModelTest 2.1.5 based on the Akaike information criterion (AIC) [rpb2 datasets (see Phylogenetic relationships among the native populations of on (AIC) . The ML on (AIC) . Likelihon (AIC) . Markov sets see , we combLibanotis were available, a mean substitution rate reported for cpDNA sequences was used for calibration. Wolfe et al. [−9 substitutions per site per year (s/s/y) based on the comprehensive study of chloroplast genes. Considering the uncertainties of the rate values, we assumed a mean of 2 × 10−9 s/s/y and a deliberately standard deviation of 6.080 × 10−10 s/s/y with a normal distribution prior for the two cpDNA regions (trnL–trnF and trnS–trnG) in this study [7 chains were performed, sampling every 1,000 generations following a 10% burn–in in each chain. We used the HKY + I substitution model, a strict clock, a constant population size coalescent tree prior, and a UPGMA starting tree. Tracer 1.4 was used to confirm the sampling adequacy and convergence of the MCMC output parameters [Divergence times were estimated using the cpDNA dataset, with a Bayesian approach as implemented in BEAST 1.8.1 . As neite et al.  estimateis study ,33. Two rameters . Both lorameters  was usedL. buchtormensis. The sum of squared deviations (SSD) and Harpending’s raggedness index (HRag) were chosen to test the validity of the expansion model and quantify the smoothness of mismatch distributions [T = τ/2u was used to estimate the expansion time, where T is the expansion time in number of generations, τ is the expansion parameter detected by the mismatch distribution, and u is the neutral mutation rate of the entire cpDNA sequences per generation [u was calculated as u = μkg, where μ is the substitution rate per nucleotide site per year , k is the average sequence length of the analysed cpDNA region, and g is the generation time in years (approximated as 4 years).We conducted mismatch distribution analyses based on the demographic expansion model in Arlequin to detect independent demographic expansion events in the regional populations of ibutions ,36. For neration . The valL. buchtormensis since the Last Glacial Maximum , a species distribution model was run in MAXENT 3.3.1 using the maximum entropy method [www.cvh.org.cn) and the National Specimen Information Infrastructure of China (www.nsii.org.cn) were also included. To achieve higher accuracy, we excluded misidentified and uncertain herbarium information. Based on a total of 65 records, a current distribution model was developed using seven bioclimatic data layers  from the WorldClim dataset at 2.5 arc–min resolution (available at http://www.worldclim.org/download). The seven bioclimatic data with high gain values were selected by a Jackknife Test to avoid highly correlated variables, which may cause potential overfitting [http://www.worldclim.org/download) to infer the sphere of suitable habitat during the LGM. The accuracy of the predicted model was assessed by calculating the area below the receiver operating characteristic curve (AUC) [To infer the distributional dynamics of y method . In addirfitting ,40. Thenve (AUC) .L. buchtormensis using RASP 3.1 [L. buchtormensis can be categorized into five areas: (A) Altai Mountains (populations 1–8), (B) steppe mountains from Altai Mountains to Western Tienshan Mountains (populations 9 and 10), (C) Western Tienshan Mountains (populations 11–13), (D) Western Sichuan Plateau (populations 14 and 15), and (E) Qinling Mountains (populations 16–21). The MCMC output and condensed tree exported from BEAST were loaded as ‘trees file’ in this analysis.Statistical dispersal–vicariance analysis (S–DIVA) was performed to investigate the hypothetical historical biogeographic areas of RASP 3.1 . Due to trnL–trnF and trnS–trnG regions were both successfully amplified and sequenced in the 262 individuals from 21 natural populations and six herbarium specimens of Libanotis buchtormensis. The total alignment of the combined plastid regions was 1333 bp long. In total, 23 plastid haplotypes were identified based on 19 polymorphic sites  and nucleotide diversity (πcp = 0.0033 ± 0.00055 and πnr = 0.0037 ± 0.0009) were revealed at the species level in L. buchtormensis (hcp = 0.921 ± 0.009 and hnr = 0.883 ± 0.013) was higher than that within the SE populations (hcp = 0.606 ± 0.033 and hnr = 0.561 ± 0.048). Population level analysis of sequence data revealed that the BJ, YN, YL, and TK populations in the NW region had the highest genetic diversity  was significantly greater than GST (0.671), suggesting a distinct phylogeographical signal in L. buchtormensis.High levels of haplotype diversity . A large proportion of variation was attributable to differentiation among geographical regions  and between populations within geographical regions . Dispersal events from the Altai Mountains (A) to the Tienshan Mountains (C) and the steppe mountains from the Altai Mountains to the Western Tienshan Mountains (B) were detected  and 20.4 (95% CI = 0–460.5) ka BP, respectively.The unimodal distribution pattern obtained from the mismatch analysis suggested that both the NW and SE populations underwent recent expansions . Non–sigL. buchtormensis showed a good predictive model performance. The present–day predicted distribution of the species was similar to the actual distribution. Under the simulated LGM climate scenario, we found that most habitats suitable for L. buchtormensis were reduced relative to present day conditions  for the present potential distribution of nditions . In the nditions .Libanotis buchtormensis at the species level. The genetic differentiation among populations contributed to the total genetic divergence [L. buchtormensis of China comprised two geographically distinct lineages, the northwestern lineage distributed in the Xinjiang region (NW region) and the southeastern lineage occupying both the Qinling Mountains and the Sichuan region (SE region). In the nuclear haplotype network, this well–defined genetic structure was recovered as well  estimated among the L. buchtormensis populations evidenced this [L. buchtormensis is generally incapable of long–distance dispersal, except maybe on rare occasions. Thus, the desert zone in Northwest China has a pivotal effect on the genetic differentiation of L. buchtormensis, acting as a geographical barrier. We likely proposed a vicariance scenario to explain the current disjunct distribution of L. buchtormensis. The genetic structure, along with morphological variation such as leaf texture and fruit shape in populations of the two regions (NW and SE), may imply subspecies formation as a result of geographical isolation. Further work is needed to test this.Two distinct genetic lineages were identified in the geographically disjunct populations of ced this . ConsideL. buchtormensis was dated to the middle Pleistocene  [L. buchtormensis. These findings indicated that the SE populations might have originated from ones in the NW region by occasional dispersal events. However, the genetic imprint of the founding event may not be detectable after a few generations, especially when anthropogenic destruction exists [In the present study, the NW lineage had a higher amount of ancestral and unique haplotypes than did the SE lineage . Most posorting) ,48. Furtsorting) ,50. The n exists . AccordiL. buchtormensis would have been further influenced by the subsequent glacial retreat to refugia and interglacial recolonization. The NW region was identified as the main centre of genetic variation and haplotype endemism. Refugia are expected to possess high levels of genetic diversity and haplotype uniqueness [L. buchtormensis contracted dramatically during LGM, and it was restricted to the western Altai Mountains and western Tienshan Mountains  harbored higher levels of genetic diversity and the greatest number of unique haplotypes. The refugia identified have also been supported for other plant species in the area [Following the initial genetic divergence of the NW and SE lineages, the current distribution of iqueness . Compareountains . Influenthe area ,53,54. Tthe area . Multiplc. 136–76 ka BP) [L. buchtormensis in the NW region [The unimodal mismatch distribution  revealed6 ka BP) . We shouW region .c. 19–11 ka BP) [L. buchtormensis, the SE region may have a chance to inherit only a small portion of the genetic diversity present in the ancient parental distribution (NW region). The distribution pattern of L. buchtormensis in the SE region is island–like to some extent, and characterized by low genetic diversity. Studies on island populations have shown that long–term isolated and small habitat sized populations would be threatened by genetic drift and inbreeding [L. buchtormensis in the Qinling Mountains and Sichuan Plateau [Regarding the SE lineage, populations in the region expanded their range at 20.4 ka BP, approximately coinciding with the second Taibai glacial period in East Asia (1 ka BP) . The tim1 ka BP) . Althougbreeding ,56. A si Plateau . Thus, iS1 FigNumbers below the branches were BI posterior probabilities (PP) and ML bootstrap values (LB), respectively. The dash lines indicated that the branches were not supported in the phylogenetic analyses.(TIF)Click here for additional data file.S2 FigNumbers below the branches were BI posterior probabilities (PP) and ML bootstrap values (LB), respectively. The dash lines indicated that the branches were not supported in the phylogenetic analyses.(TIF)Click here for additional data file.S1 TableAll sequences are compared to the reference plastid haplotype C1.(DOC)Click here for additional data file.S2 TableAll sequences are compared to the reference haplotype H1.(DOC)Click here for additional data file.