Two deep lineages that diverged in the Pliocene (4.5 Myr) occur over wide areas of Brazil. Historical changes during the Plio-Pleistocene led to significant genetic differences between E. heros populations, which differentiated further in several biomes. The northern lineage is older, more diverse, and prevalent in the Amazon and Caatinga, while the southern lineage is younger, less diverse, and prevalent in the Atlantic Forest and Chaco biomes. Euschistus heros populations are expanding in size and range but at different rates, strongly affected by environmental variables. Secondary contact between the main lineages is now occurring, mainly in areas of intensive farming and particularly in the Cerrado, an important agricultural frontier. Individuals adapted to different environmental conditions and to large monocultures might currently be combining into a panmictic and hard-to-control pest population.The rapid pace of conversion of natural areas to agricultural systems is highly concerning, and the consequences for conservation and pest management are not yet fully understood. We examined mitochondrial (COI and Cyt The four major Brazilian biomes, the Atlantic Forest (AF), Cerrado , Amazon and Caatinga  have undergone profound changes during glaciation and interglacial cycles4. Due to this complexity and landscape composition, the processes of diversification by vicariance and habitat refugia are frequently invoked to explain the high levels of species endemism and diversity in this part of the planet8. Recent findings suggest that forest expansion and contraction and ‘historically stable areas’ may also have played a major role in the differentiation of lineages9.The drastic climate changes during the Plio-Pleistocene have been considered the main cause of high levels of diversification in many areas in Brazil11 and forms a natural barrier preventing the movement of organisms between the northern Brazilian biomes and the Atlantic Forest14. Recent human impacts on these biomes, particularly the expansion of agricultural areas, have drastically changed the landscape and likely the connection between ecosystems, rearranging patterns that began to be formed millions of years ago.Today, the Amazon and Atlantic forests are separated by a unique mosaic composed of savannas and woodlands that covers a large area between the two forest biomes, from Argentina and Paraguay (the Argentinean and Paraguayan Chaco) and continues through the central Brazilian Cerrado to the Caatinga in northeastern Brazil. This belt of mostly sparse dry vegetation is known as the ‘major South American disjunction’15. Fragments of native vegetation become embedded in a matrix of cropland and pasture that will eventually affect the species and ecosystem dynamics16. In some instances, habitat loss can limit the geographic range of endemic species, although certain species may thrive in their new surroundings. Cropland areas can provide exploitable resources, e.g. the expansion of soybean cultivation in Brazil in recent decades17. Soybean crops were formerly limited to southern Brazil (Atlantic Forest); in the early 1970s, advances in farming methods and new varieties allowed soybean farmers to expand into a new and important frontier, the Cerrado20.The expansion of agricultural frontiers necessitates conversion of the native habitat to agriculture22. On the other hand, Brazil is the second largest soybean producer after the USA, and soybeans account for an important share of its GDP23. For these reasons, it is important to understand both the impacts of soybean expansion on connecting natural populations, and the influx of insect pests from natural areas into soybean croplands.Farming in the Cerrado has had both negative and positive impacts over the last 40 years. The expansion of soybean crops caused extensive environmental impacts such as fragmentation and the loss of natural areas, a matter of great concern for ecologists and conservationistsEuschistus heros (Fabr. 1798) (Hemiptera: Pentatomidae), is one of the most important pests of soybeans24. This Neotropical native is widespread in South America, living in markedly different environments including the Amazon Forest, Cerrado, Caatinga and Atlantic Forest25. The dispersal ability of E. heros is not well known, but is considered to be poor, perhaps because of its limited flight activity and diapause behavior27. This polyphagous pest feeds on members of Fabaceae, Solanaceae, Brassicaceae, Compositae, and Malvaceae, and often reaches high population densities on soybeans31. Rarely reported before the 1970s33, E. heros has since increased in abundance and is now found in all major soybean-producing regions24. Recently, this pest was recorded in Argentina34 and Paraguay24, raising concerns regarding a possible range expansion to other locations in South, Central and North America24.The Neotropical brown stink bug, E. heros. We addressed several questions regarding the genetic diversity, population structure, and demographic history of E. heros populations in Brazil and Paraguay, examining the potential role of past events in the differentiation of lineages, and the recent events (i.e. soybean expansion) promoting the admixture of ancient lineages. Our first objective was to determine the genetic distribution, studying population divergence and population structure. Second, we investigated the demographic history of E. heros in different biomes in South America. Finally, we used a modelling approach to explore how environmental variables and soybean expansion can explain the genetic pattern of current populations of E. heros in Brazil.Here we present a genealogy of mitochondrial and nuclear DNA sequences from E. heros lineages separated by 52 mutational steps, and an estimated genetic distance of D = 0.042 , both from lineage S  of these haplotypes were private and only 18% were shared among individuals. The overall haplotype diversity, nucleotide diversity and mean number of nucleotide differences were h = 0.991, π = 0.03312 and K = 32.892, respectively (Table h = 0.984) and lineage S (h = 0.982); however, lineage N (π = 0.0090) had higher nucleotide diversity than lineage S (π = 0.0062). For the different biomes, the haplotype diversity among biomes (groups) was low, ranging from 0.967 in the Amazon to 1.000 in the Chaco. The nucleotide diversity varied more widely among biomes, from 0.00627 in the Atlantic Forest to 0.03147 in the Amazon Forest  than lineage S , previously defined by the mitochondrial network. Among biomes, the highest diversity was observed in the Chaco  and the lowest diversity in the Atlantic Forest   clade was 4.5 Myr (95% C.I 2.801–6.453 Myr), during the Pliocene, with intense diversification in the Pleistocene and Holocene Fig. .Figure 2Lineage N haplotypes were associated mainly with the Amazon Forest and Caatinga, with one, more recent clade (CW) associated with central Brazil, a transitional region among the Cerrado, Caatinga and Amazon Forest Fig. . LineageE. heros populations showed high genetic structure, as assessed by the Analysis of Molecular Variance (AMOVA). Differences among populations accounted for most of the genetic variances in mtDNA  and a high and significant value in ITS1 regions  . Furthermore, the larger portion of genetic variation within populations  indicates overall genetic differentiation in these populations , in which most of the genetic variation was within populations  Table . A test Considering the two lineages, significant negative values were found in both the Tajima’s D and Fu’s Fs neutrality tests, indicating population expansion or purifying selection. Considering the biomes, the neutrality test statistics did not fully agree with one another. Fu’s Fs statistic was significantly negative for all biomes, but only the Atlantic Forest biome had a significant negative Tajima’s D value Table .Table 4NP > 0.05), indicating a recent demographic expansion of lineage S but not lineage N (P = 0.04). For the biomes, a nonsignificant SSD (P > 0.05) was also found for the E. heros populations in all biomes but the Caatinga (P = 0.03) (Table P > 0.17) supports the spatial-expansion model of populations of lineages, biomes, and the entire group   and wi (0.09 and 0.14) (Table c = 35.35) was 22.29 units away from the null model (AICc = 57.64). The variable ‘latitude’ (0.50) was more important than ‘max temperature of the warmest month’ (0.43) in the best model according to the z-scored beta   and in the third-best model (0.66) compared to ‘mean temperature of wettest quarter’ (0.35) and ‘max temperature of the warmest month’ (0.56) . None of the three best models included soybean variables. The two soybean variables, ‘time since first soybean harvest’ and ‘soybean expansion rate’, ranked 7th and 16th in overall importance. The ‘time since first soybean harvest’ was strongly correlated with ‘latitude’ , ‘max temperature of the warmest month’ , and ‘annual mean temperature’ .Three models passed the cutoff (i.e. models that were less than 2 units away from the “best” model) to explain the presence (%) of the southern lineage at a given location . The best predictors were the ‘max temperature of the warmest month’, ‘latitude’, and ‘annual mean temperature’ Fig. . The mosE. heros in South America. The two COI-Cytb haplotype groups are separated by 52 mutational steps and have an estimated genetic distance of 4.2% (K2P). The number of mutation steps separating the two E. heros lineages is exceptionally high, raising the question of the possible presence of cryptic species. Rolston35 described a species in Middle America that is morphologically similar to E. heros, Euschistus atrox (Westwood) 1837, distributed in Colombia, Guiana, and Panama. However, examination of the external morphology, the sharing of ITS1 alleles by both lineages, and the admixture of lineages in the laboratory support the hypothesis of a single species that encompasses the two divergent lineages. Our results reveal the need for a thorough systematic review of the genus Euschistus.Our results revealed two divergent deep lineages of E. heros lineages are geographically separated from one another, with one clade clustering the northernmost populations (i.e. northern and northeastern Brazil), and a second clade clustering the southernmost populations (i.e. southern and southeastern regions). Both mitochondrial lineages expanded to form a mixed zone upon secondary contact in the central and southwestern regions. It is not clear when the reunion occurred, but the formerly isolated populations seem to have come into contact before reproductive isolation was complete37. A related point to consider is that all but one of the northern haplotypes found in the Cerrado (CW) were phylogenetically grouped together in one clade, indicating a subgroup differentiation. The central-western (CW) subgroup likely occupied the region much earlier than the southern lineage arrived and before the first soybean fields were established. This is strong evidence against the hypothesis that the E. heros expansion was purely associated with the expansion of soybean cultivation during the 1970s.The two 38. Temperature cycles were also associated with more recent diversification events during the Pleistocene (i.e. differentiation of the CW group). Deep sequence divergence dating to the Pliocene is also reported for other organisms in the Neotropics40, and phylogeographic structure has been found in amphibians in the Atlantic Forest3, reptiles in the Cerrado41, and plants42.The divergence time of the two main clades is estimated as occurring during the Pliocene (i.e. 4.5 Myr). This divergence seems to be associated with a cooling and drying of the global environment, which caused the separation of the Amazon Forest from the southern part of the Atlantic Forest and the consequent expansion of grasslands and savannasE. heros. Separation into the Amazon Forest, Caatinga, Cerrado, Atlantic Forest and Chaco biomes seems to be the best way to explain the genetic variance hierarchically. Thus, separating insects by biomes can help us to understand the pattern of lineage mixing, diversity and demographic history. The haplotype diversity of E. heros was high and similar among biomes and lineages. This pattern is the result of the high number of private haplotypes found in E. heros populations in all biomes. The higher nucleotide diversity of lineage N compared to lineage S can be explained under the ‘historical climate’ stability models, where a stable environment such as the Amazon Forest can offer conditions for a population to persist, resulting in elevated intraspecific genetic diversity44. Unstable regions, on the other hand, would be associated with recent or multiple-event colonization, resulting in lower intraspecific genetic diversity and signatures of expansion4. Therefore, the northern biomes (Amazon Forest and Caatinga) were the most stable environment, while the Atlantic Forest was the least stable environment. Another consideration is that lineage S is associated with areas that have undergone intense transformation due to agricultural practices, and has experienced population dynamics linked with farming cycles and control tactics.Spatial genetic structuring by biomes was also found among subpopulations of E. heros’ limited dispersal capacity likely helped to preserve the pattern formed during the late Tertiary and Quaternary as an outcome of the climate changes, the last 100 years were an important turning point for E. heros populations (i.e. soybean introduction and expansion of farming starting at the end of the 19th century). It is plausible that farming and trade routes have increased the admixture process in certain areas, especially the Cerrado and connecting areas, even though there are still large areas where the two lineages have not yet encountered each other, showing that the pattern is still well preserved.Although E. heros lineages and in all biomes sampled. The inferences regarding population growth were supported by the neutrality tests, the unimodal mismatch distribution and the demographic expansion parameters (τ). Spatial expansion is also occurring, given that no significant Raggedness values were found. Apart from differences in test sensitivity, the lack of full agreement between tests for E. heros in each biome might indicate a more complex scenario. Multiple processes affecting local diversity and the noise from human intervention causing population reduction, population subdivision, bottlenecks, and facilitation of dispersal resulting in the secondary contact might affect the precise demographic estimates for a species47.Recent signals of expansion were detected for E. heros population growth in all areas overlaps with the period of intense changes caused by the increase of urban occupation and agricultural area in South America48. It may be that the resulting habitat loss not only did not affect E. heros populations negatively, but has even been advantageous. One possible hypothesis to explain the success of E. heros is shifting hosts from natural areas to agricultural fields, especially soybeans but also cotton and bean fields31. A second hypothesis is that one or more traits occur in a latitudinal cline51. The species’ association with environmental gradients should also be considered, given the possibility of differences in traits and adaptations such as reproductive diapause53.We also conducted a Bayesian Skyline analysis to test the hypothesis of recent expansion in all biomes and to determine how the effective sizes of the populations behaved over time. The period of E. heros’ evolutionary history. The time since the first soybean harvest correlates with latitude and other bioclimate variables, which likely decreased the importance of this variable in the model.We used environmental and soybean variables to make phylogeographic inferences to predict the predominance of lineage S over lineage N at a given location. Selected models had similar AICc scores and considerably reduced the number of variables, down to 4 for the percentage of lineage S models. The two most important variables were the maximum temperature of the warmest month and the latitude. Temperature and photoperiod both affect this species, and might induce quiescence behavior and other possible differences in physiological responses. Latitude, on the other hand, can be correlated with geographic distance, environmental gradients, and agricultural gradients, as in the case of the soybean expansion. Our data support the predictions of the latitudinal-gradient hypothesis, even though distinct demographic scenarios can be expected at different times of 54. It is possible that the northern and southern populations of E. heros are exchanging adaptations in admixture zones. However, knowledge of the differences between the two lineages is limited, because their presence was unknown until this point56. The changing status of E. heros from a secondary to a primary pest in soybean crops and the reasons for this are poorly understood. In recent years, the increase of population densities in soybean fields, the shorter quiescence period, larger host range (i.e. damage in cotton crops) and pesticide tolerance/resistance have been frequently reported in E. heros populations57. These concerns increase in a scenario of GM soybean introduction, no-till management, and expansion to diversity-hotspot areas.The reunion of the two long-separated lineages might have unforeseen consequences for one of the largest soybean-producing regions in the world. The two lineages are united again in central Brazil, where an agricultural revolution started in the 1970s and continues today, pushing soybean fields northwardE. heros were collected between 12/2015 and 07/2016 from 21 different localities across five South American biomes. Twenty sampling sites were in Brazil and one site in Paraguay. Specimens were collected as adults, from the canopy of soybeans, using a beating cloth under the plants. Individuals were preserved in ethanol (>95%) at –20 °C until laboratory manipulation, after which the remaining tissue from all specimens was stored at –80 °C. DNA was extracted from the head tissue of an adult specimen, using the modified CTAB protocol58.One hundred fifty-nine specimens of 59. The Cytochrome c Oxidase Subunit 1 (COI) fragment was amplified using the forward primer (5′-ACCGCACATGCATTTGTAATAA-3′) and the reverse primer (5′-GTGGCTGATGTGAAGTATGCTC-3′), and the Cytochrome b (Cytb) fragment was amplified using the forward primer (5′-GGATATGTTTTACCTTGAGGACA-3′) and the reverse primer (5′-GGAATTGATCGTAAGATTGCGTA-3′). To amplify the ITS1 rnDNA region  we used the forward primer CAS18SF1 (5′- TACACACCGCCCGTCGTACTA-3′) and the reverse primer CAS5p8sB1d (5′- ATGTGCGTTCRAAATGTCGATGTTCA-3′). The PCR reactions were performed in a total volume of 25 μL containing 80 ng total DNA, 1.5 mM/μL MgCl2, 0.1 mM/μL dNTPs, 0.4 pmol/μL of each primer, 1 U of Taq DNA Polymerase (Synapse Inc.) and Buffer (10 × Taq DNA Buffer). PCR cycles consisted of denaturation at 95 °C for 3 min, followed by 35 cycles with denaturation at 95 °C for 30 s, annealing at 54 °C for 40 s, polymerization at 72 °C for 1.5 min and final extension at 72 °C for 10 min. Subsequently, the PCR products were separated on agarose gel (1.5% w/v) and observed under ultraviolet light. The amplicons were purified using 0.33 μL EXO I, 0.33 μL FastAp and 0.34 μL of ultra-pure water together with 10 μL of each PCR product, held at 37 °C for 30 min, then at 80 °C for 15 min. The PCR product Sanger sequencing was performed by the Animal Biotechnology Laboratory at ESALQ, University of São Paulo.Fragments of two mitochondrial and one nuclear region were amplified by polymerase chain reaction (PCR), using specific mitochondrial primers developed for this project and previously developed ITS1 primershttps://www.ncbi.nlm.nih.gov/genbank/) with the accession numbers MG651970 - MG652128 (COI), MG652129 - MG652287 (Cytb), and MG654513 - MG654636 (ITS1).All sequences were aligned and edited manually using the software Sequencher 4.0.1 . To eliminate missing data, sequences were interrupted at 607 bp for the COI gene, 386 bp for the Cytb gene and 638 bp  for the ITS1 region. There were no insertions or deletions in the sequences obtained. All sequences (datasets) obtained in this study were deposited in NCBI-GenBank  indels that introduce frameshifts, (ii) out-of-place inframe stop codons that lead to premature termination of protein translation, and (iii) lack of codon position substitution bias toward the 3rd position, that lead to a higher rate of non-synonymous mutations. The presence of signatures (i) and (ii) is enough to consider a given sequence a numt. No numts were detected in the COI or CtyB sequences; therefore, we included all mitochondrial sequences in our analysis. The posterior analyses were performed using concatenated mitochondrial genes (COI-Cytb).The presence of nuclear paralogs of mitochondrial origin (termed numts)62. Preliminary analysis revealed two putative mitochondrial lineages associated with E. heros populations in Brazil and Paraguay. The genetic distance (D) between the two mitochondrial lineages was inferred by dividing the haplotypes in two groups and calculating the 2-parameter Kimura method (K2P) in MEGA v.5.2 software61.The genealogical relationships between haplotypes of the mitochondrial and ITS1 regions were reconstructed by a network of median-joining haplotypes, using the PopArt softwareh), nucleotide diversity (π) and mean number of nucleotide differences (S) were estimated using the DNAsp v.563.The diversity analysis was performed by dividing individuals into two groups according to the mitochondrial lineages, or into five groups according to the biome to which the individuals belonged: Amazon Forest, Cerrado, Caatinga, Atlantic Forest or Chaco. Number of haplotypes, haplotype diversity 66 that corresponds to 3.54% pairwise divergence per Myr. Three independent runs were performed for 150 million generations, sampling every 1000 steps and discarding 20% as burn-in. TRACER v.1.6 was used to determine convergence, measure the effective sample size (ESS), and calculate the mean and 95% highest posterior density interval (HPD) for divergence times. Effective sample size (ESS) for all parameters exceeded 200, and the three runs converged to similar distributions. Runs were then combined with LogCombiner v.1.4.764.We estimated the relative age of divergence between the two mitochondrial lineages using the Bayesian relaxed phylogenetic approach implemented in BEAST v.1.8.467. The analyses were conducted to examine the presence of genetic structure among individuals, considering all sites sampled , among populations according to the sampling location (populations) and among biomes in three hierarchical levels.Variance Analysis (AMOVA) was performed in Arlequin with parametric bootstrap (1000 replicates) using a 5% significance level67. Both tests used 1,000 permutations using coalescing simulations. Fu’s Fs statistic was considered significant at the 95% confidence level when the P-value was less than 0.02. For each biome, we also estimated tau (τ) with its 95% confidence intervals, using a generalized least-squares approach and 1,000 coalescent simulations in Arlequin v.3.5. The parameter τ denotes the age of the expansion (t), so that t = τ/2 u; u = μLg68. The parameter μ represents the estimated mutation rate, L is the length of the sequence, and g is the generation time. For E. heros, we did not estimate t directly, because the number of generations per year cannot be estimated straightforwardly. Thus, if we assume that the estimated mutation rate has not changed in E. heros (substitution rate = 1.345%), then u will be constant and we can consider that a smaller τ value indicates a newly established population, and a larger τ value an older one.Tajima’s D and Fu’s Fs neutrality tests were calculated using Arlequin v.3.5r) statistics, and their associated P-value were calculated using Arlequin v.3.5. A nonsignificant SSD value means that the hypothesis of population expansion cannot be rejected, and a nonsignificant raggedness index indicates a good fit of the data to the spatial expansion model. We also used a Bayesian Skyline Plot (BSP) in Beast to reconstruct the demographic history, using TRACER v.1.6, based on the COI-Cytb data using 10 groups. We used the same substitution model and molecular-clock model that were used to estimate the divergence time.We conducted a mismatch distribution analysis using a spatial expansion model. The sum of square of deviations (SSD), raggedness index 75. We selected models with AICc less than two units away from the best model. We also evaluated the Akaike weight of the best models, to assess the probability that a model is the best76. All variables were standardized by z-score, and the significance of each predictor was assessed by a GLM. We also assessed the importance of each variable by summing the Akaike weight for the models in which the variable appeared. Variables that appear many times in the top models, tend to be more important. We used the cutoff of 0.8 to separate the most important variables under the weight criterion70.We evaluated the fit and plausibility of possible candidate models using glmultiSupplementary Information


DNA) and nuclear genetic (microsatellite) diversity among three populations of a migratory bird, the greater white‐fronted goose , which breeds discontinuously in western and southwestern Alaska and winters in the Pacific Flyway of North America. Significant genetic structure was evident at both marker types. All three populations were differentiated for mtDNA, whereas microsatellite analysis only differentiated geese from the Cook Inlet Basin. In sexual reproducing species, nonrandom mate selection, when occurring in concert with fine‐scale resource partitioning, can lead to phenotypic and genetic divergence as we observed in our study. If mate selection does not occur at the time of reproduction, which is not uncommon in long‐lived organisms, then mechanisms influencing the true availability of potential mates may be obscured, and the degree of genetic and phenotypic diversity may appear incongruous with presumed patterns of gene flow. Previous investigations revealed population‐specific behavioral, temporal, and spatial mechanisms that likely influence the amount of gene flow measured among greater white‐fronted goose populations. The degree of observed genetic structuring aligns well with our current understanding of population differences pertaining to seasonal movements, social structure, pairing behavior, and resource partitioning.An understanding of the genetic structure of populations in the wild is essential for long‐term conservation and stewardship in the face of environmental change. Knowledge of the present‐day distribution of genetic lineages (phylogeography) of a species is especially important for organisms that are exploited or utilize habitats that may be jeopardized by human intervention, including climate change. Here, we describe mitochondrial (mt This isAnser albifrons) in the Pacific Flyway of North America  populations nesting in high latitudes in Alaska and western North America, but also found deep cleavages in mtDNA haplotypes between what were previously called the large‐ and small‐bodied Canada goose lineages, findings that contributed to taxonomic revision of the group into two species, the Canada goose and the cackling goose (B. hutchnisii), respectively , and Quinn . Geese were captured and banded under the auspices of the U.S. Fish and Wildlife Service Region 7, and the USGS, Alaska Science Centre, under Federal Permit # MB789758.Blood, feather, or eggshell membranes were collected from greater white‐fronted geese at three different locales in Alaska within the Pacific Flyway .Genomic DNA was extracted from blood, muscle, feather, or eggshell membranes using a “salting out” procedure  in the mtDNA control region are within control region domain I. Using the methods in Lanctot et al.  control region using the primer pair WFGL1M (5′–ACTAACCGCGAACTCCCAAA–3′) and H542 , observed and expected heterozygosities, Hardy–Weinberg equilibrium (HWE), and linkage disequilibrium (LD) for each microsatellite locus and population in FSTAT ver. 2.9.3 , and nucleotide diversity (π—the average number of nucleotide differences per site between two randomly chosen DNA sequences) for the mtDNA control region sequence data. An unrooted phylogenetic tree for mtDNA control region was constructed in NETWORK 4.6.1.3 using the median joining method  for microsatellite data. For sequence data, pairwise ΦST was calculated using the best‐fit nucleotide substitution model, as identified in MODELTEST 3.06  iterations. The analyses were repeated ten times for each K to ensure consistency across runs. We used the ∆K method of Evanno, Regnaut, and Goudet , to determine the most likely number of groups at the uppermost level of population structure.We also used a Bayesian‐clustering program, STRUCTURE 2.2.3  for mtDNA from each population independently. We used a Bayesian analysis with 1,000,000 recorded genealogies sampled every 50 steps, with a burn‐in of 100,000 (10%) genealogies. Priors were flat with the upper limit for growth set to 15,000.Demographic histories based on mtDNA sequence data were evaluated using two approaches: standard qualitative test statistics and coalescent‐based estimations. To test for genetic signatures of recent effective population size changes, we calculated Fu's Fs Fu,  and Taji Tajima,  on the b Kuhner,  to calcu2.6m) over the last several generations using allelic frequency data and does not assume that populations are in migration–drift or HWE. MIGRATE, in contrast, estimates recent  and historical (mtDNA) migration rates (Nem and Nfm), respectively , recent (long term), and historical gene flow among sampled sites were calculated using two different models: assignment methodology in BayesAss v3.0 , migration rate (m), and inbreeding (F). Subsequent runs incorporated different delta values to ensure that proposed changes between chains at the end of the run were between 20% and 40% of the total chain length to maximize log likelihood values and ensure the most accurate estimates , Δm = 0.15 (37%), and ΔF = 0.80 (33%). We performed five independent runs  with different random seeds to ensure convergence across runs. Convergence was also assessed by examining the trace file program Tracer v1.6 to ensure proper mixing of parameters , and all pairwise migration parameters were estimated individually from the data. Gene flow was estimated using a maximum‐likelihood search parameters; 10 short chains , ten long chains , and five static heated chains . Full models were run ten times to ensure the convergence of parameter estimates.MIGRATE was run with a full migration model, θ (433.1n = 30) and a late time period . CIB samples were also broken down into early  and late  collections. We found no differences across time for either YKD geese , or CIB geese . This result was not unexpected, as geese have a long generation time  characterized by 33 variable sites  and nucleotide diversity (π = 0.0095–0.0215) were observed across populations, with the CIB and BBL populations having the lowest observed levels of genetic diversity  ranging from 4.8 to 5.4 (Table FIS) ranged from 0.050 to 0.082 across sampled sites with an overall mean of 0.063; no FIS value differed significantly from zero. All populations were in HWE, and no signature of linkage disequilibrium was detected between any pair of loci.In 115 greater white‐fronted geese examined, the number of alleles per autosomal microsatellite locus ranged from 3 to 13, with an average of 7.0 alleles per locus. Molecular diversity indices were similar across populations with allelic richness  with all pairwise comparisons showing significant differentiation . We estimated the upper limit of FST as 0.359 and 0.938 for RST; thus, the standardized FST and RST is 0.056 and 0.139, respectively, which accounts for the maximum possible level of genetic structure based on our data set. In contrast to mtDNA sequence data, however, only the CIB (Tule) population was differentiated from the other populations based on autosomal loci (Table r > 1), suggested that the most likely number of populations is two, with the CIB population comprising one genetic cluster and other populations assigned to the second cluster  overlapped or approached zero (LAMARC estimates are biased upward), consistent with a stable population size  with lower but significant differentiation in allelic frequencies involving CIB (RST = 0.130) which is concordant with morphological differences within the Pacific Flyway  populations nesting on Wrangel Island, Russia (north and south wintering populations), and Banks Island, Canada , show a signature suggestive of winter pairing  where two morphs feed in different habitats and also prefer different substrates for spawning, which further restricts gene flow  is in contrast to most species of ducks (Oxyurini, Tadorinini, Aythinim, Anatini, Mergini; Anderson, Rhymer, & Rohwer, 5The moderate‐to‐high degree of genetic structuring and relatively low estimates of gene flow indicate the three populations of greater white‐fronted geese in the Pacific Flyway are at varying degrees of reproductive isolation and may be considered to be an example of early stage or incomplete radiation (whereby a lineage rapidly diversifies with emerging branches evolving different traits that may be adaptive; Givnish & Sytsma, None declared. Click here for additional data file. Click here for additional data file.


Melocanna baccifera (Roxb.) Kurz is an economically important bamboo of North-East India experiencing population depletion in its natural habitats. Genetic variation studies were conducted in 7 populations sampled from 5 districts of Manipur using ISSR molecular markers. The investigation was carried out as a primary step towards developing effective conservation strategies for the protection of bamboo germplasm. ISSR marker analysis showed significant level of genetic variation within the populations as revealed by moderately high average values of Nei's genetic diversity (H 0.1639), Shannon's diversity index (I 0.2563), percentage of polymorphic bands (PPB 59.18), total genetic variation (Ht 0.1961), and genetic diversity within population (Hs 0.1639). The study also divulged a high genetic variation at species level with Shannon's diversity index (I), Nei's genetic diversity (H), and percentage of polymorphic band (PPB%) recorded at 0.3218, 0.1939, and 88.37, respectively. Genetic differentiation among the populations (Gst) was merely 19.42% leaving 80.58% of genetic variation exhibited within the populations. The low genetic diversity between populations was consistent with AMOVA. The low genetic differentiation among populations coupled with existence of significantly high genetic diversity at species level indicated the urgent necessity of preserving and protecting all the existing natural bamboo populations in the region. Melocanna baccifera (Roxb.) Kurz, an evergreen arborescent, non-clump forming bamboo, is a dominant species of Manipur with its multiple uses in making of houses, furniture, handicraft, and other household items [ M. baccifera using ISSR markers and no reports are available on the population genetic studies of the bamboo in Manipur. The present study aimed to investigate the genetic diversity and population genetic structure of M. baccifera in 5 districts of Manipur using ISSR markers. This study could contribute to the development of strategies for effective conservation and sustainable utilization of this bamboo for ecological and economic gains by better understanding the genetic diversity profile at the species and population level.Bamboos are multipurpose forest tree grasses having more than 1000 different uses , 3. Melold items , 5. Thisld items . Overexpld items –10. Thisld items , 12. DNAld items –18. The ld items –21. Howeld items . ISSR mold items , 31. Theld items –34. Ther M. baccifera representing 7 populations were collected from different locations spreading across 5 districts of Manipur in North-East India, namely, Bishnupur, Thoubal, Imphal West, Imphal East, and Chandel . The selected primers showed good, reliable, repetitive, and distinct bands which enabled effective scoring for genetic diversity study within and among the populations.The genomic DNA was extracted from the collected leaf samples using the CTAB method with slight modifications . The leaμL contained 25 ng template DNA, 1x PCR buffer, 1.5 mM MgCl2, 200 mM dNTPs, 1 μM primers , and 0.6 U Tag DNA polymerase and double distilled sterile water. The PCR components were prepared as master mix for each primer to minimize the pipetting error. The amplification reaction was performed in a thermal cycler (Eppendorf Mastercycler nexus X2) with amplification cycle condition of initial 4 minutes' strands separation at 94°C followed by 40 cycles of 94°C for 45 secs, 53°C for 1 min, and 72°C for 2 minutes and final extension at 72°C for 10 minutes. The products obtained after PCR amplification were electrophoresed in 2% agarose gel in 0.5x TBE buffer at 100 V for around 3 hours and gel was stained with ethidium bromide (0.5 μg/mL). The fragments after staining were visualized in gel documentation system . A 1000 bp DNA ladder (Hi-media) was used as a size marker for every gel run.The DNA amplification mixture of 25 Na), the effective number of alleles (Ne), Nei's genetic diversity (H), Shannon's information index (I), total genetic diversity (Ht), genetic diversity within population (Hs), Nei's genetic differentiation index among populations (Gst), and gene flow estimates between populations (Nm) were determined with POPGENE version 1.31 [ M. baccifera using Mega 5.10 [K was set from 1 to 8 with 10 independent runs for every K value. The results of the STRUCTURE outfile were utilized to determine the optimum K value following the simulation method of Evanno et al. [Distinct, reproducible, well resolved fragments were scored as present (1) or absent (0) for each ISSR reaction and were displayed as part of a binary matrix. The data matrices obtained were analyzed using POPGENE version 1.31 . Geneticion 1.31 . Geneticion 1.31 . A studyion 1.31 . An analion 1.31 . The unbega 5.10 . Principega 5.10  to deterega 5.10 . Ancestro et al.  employino et al. . M. baccifera and generating high level of genetic polymorphism. High genetic variation at species level was observed in the present investigation with the recording of percentage of polymorphic bands (PPB) at 88.37%. The values for Na, Ne, H, and I at species level were also recorded at 1.8837, 1.2751, 0.1939, and 0.3218, respectively, showing a relatively high level of genetic diversity (Ne) varied from 1.1626 to 1.3923 with an average value of 1.2625, while the observed number of alleles (Na) ranged from 1.5116 to 1.6977 averaging at 1.5940. Nei's genetic diversity (H) also extended from 0.1149 to 0.2282 with an average of 0.1639, and Shannon's information index (I) spanned from 0.1927 to 0.3400 with an average value of 0.2563. THBL-A and CHDL exhibited highest genetic diversity at population level while the lowest variation was found in IMW-B.The five different ISSR markers amplified a total of 93 individuals representing 7 different populations ofiversity . HoweverHs) and the total genetic diversity (Ht) of the species were recorded at 0.1639 and 0.1961, respectively. The observed genetic differentiation among populations (Gst) was 0.1942 demonstrating the presence of 19.42% of genetic variation among the populations. The finding was consistent with the result of analysis of molecular variance (AMOVA) which revealed 22% of the genetic variation existing among populations and 78% within the populations (P = 0.001) .The genetic diversity within population (ulations . The dif= 0.001) . The resr = 0.311; P = 0.240)  . The genectively . The denectively . But CHDectively . IndividThe principal coordinate analysis (PCoA) was conducted to determine the spatial representation of genetic distances observed among individuals of different populations and also to check the consistency of population genetic differentiation as defined by cluster analysis. The two-dimensional PCoA plot showed the first principal coordinate accounting for 16.32% while the second coordinate produced 10.90% of the total genetic variation. The individuals of seven bamboo populations were distributed in the plot in accordance with the cluster analysis. The individuals belonging to CHDL and IMW-B were scattered separately from the rest of the populations . The remK which determined the best value of K was obtained from STRUCTURE HARVESTER and was found to be shown at K = 3  was 0.1942 which was higher than the mean value (Gst 0.073) observed for 121 woody species examined using allozyme markers [Gst (0.252) in ISSR analysis of Dendrocalamus membranaceus populations in Yunnan province of China. The low Gst value recorded for M. baccifera indicated low level of genetic differentiation among the populations. Low genetic differentiation between populations had also been confirmed by AMOVA . The finding was also consistent with high Nm (2.5455) recorded which was much higher than Nm (0.0101) recorded for Dendrocalamus giganteus by Tian et al. [Nm 2.545) was observed in spite of poor seed dispersal mechanism of M. baccifera. The flower cycle of this bamboo is relatively long and large seeds which fall near the foot of plants are mostly eaten by rats and rodents. However, M. baccifera being one of the economically important bamboos of the region might have undergone human mediated movement of genotypes through local people, farmers, and entrepreneurs. This may lead to enhancement of gene flow among populations and produce overlapping and intermixing of bamboo genotypes from different populations. The easy genetic movement might also be possible as the regions of population sampling in the present study were confined mostly to plain valley area. In fact majority of the populations under study were located in valley districts except for the one population sampled from hill district of Chandel. The STRUCTURE result also revealed high degree of admixture among the 7 populations in three genetic clusters. The existence of great extent of admixture may be attributed to cultivation and utilization pattern of the bamboo species. Geographic isolation is another major factor influencing the genetic differentiation among populations by limiting the amount of gene flow via both pollen and seeds and also through human activities [Nm 2.5455) ruling out the possibility of inducing the genetic differentiation among populations of M. baccifera due to geographical isolations. The results from the Mantel test also supported the above observation as it showed no correlation between the genetic and geographic distances between different populations. The ultimate goal of conservation is to ensure the continuous survival of population and to maintain their evolutionary potential by preserving natural levels of genetic diversity within and between populations [ M. baccifera resources in the selected sites have been exploited by locals for many years leading to loss of plant resources, habitat destruction, and fragmentation. The dramatic fall of bamboo natural population due to increasing demand for its multiutility characters ensures that there is urgent requirement of effective conservation. The population size of M. baccifera sampled from different locations was small requiring population enhancement to maintain standard effective size for high genetic diversity preservation. Genetic drift in very small population might also cause rapid genetic erosion and increased the risk of extinction of the bamboo species. Considering the M. baccifera maintaining significant diversity at species level but low genetic variation among populations and that many native habitats and ecosystems are being destroyed by human interferences, great effort should be made to preserve individuals of the existing populations. Exploitation of bamboos and habitat destruction should be prohibited in the region till effective in situ and ex situ conservation strategies are established. This will prevent further reduction of population size and conserve the overall genetic base and structure of the bamboo species. The in situ efforts to conserve the remaining habitats should be combined with ex situ approach through seed and vegetative propagation of culm cutting, division, and tissue culture technology. These activities should be performed with a view to establishing a new generation of plants both cultivable and wild. A more effective protection strategy may also be framed especially for BISH, IMW-A, IMW-B, and THBL-B which displayed low genetic diversity at population level. Sensitization of locals about the prevailing genetic scenario of alarming bamboo populations and involvement of local communities in framing protection policies will be a highly effective approach to bamboo conservation. The presence of high genetic variation within populations of M. baccifera emphasizes the necessity of preserving and conserving all the existing 7 natural populations and their habitats in Manipur.The coefficient of genetic differentiation among populations ( markers . Yang et markers  also recn et al. . Gene fligh Nm 2.55 recordulations , 57. The


Alouatta caraya, are arboreal primates, inhabitants of Neotropical forests, highly susceptible to the yellow fever virus, considered early 'sentinels' of outbreaks, and thus, of major epidemiological importance. Currently, anthropogenic habitat loss and modifications threatens their survival. Habitat modification can prevent, reduce or change dispersal behavior, which, in turn, may influence patterns of gene flow. We explored past and contemporary levels of genetic diversity, elucidated genetic structure and identified its possible drivers, in ten populations (n = 138) located in the southernmost distribution range of the species in South America, in Argentina and Paraguay. Overall, genetic variability was moderate . Significant evidence of inbreeding was found in a population that was, later, decimated by yellow fever. Population-based gene flow measures , hierarchical analysis of molecular variance and Bayesian clustering methods revealed significant genetic structure, grouping individuals into four clusters. Shared haplotypes and lack of mitochondrial differentiation (non-significant θST) among some populations seem to support the hypothesis of historical dispersal via riparian forests. Current resistance analyses revealed a significant role of landscape features in modeling contemporary gene flow: continuous forest and riparian forests could promote genetic exchange, whereas disturbed forests or crop/grassland fields may restrict it. Estimates of effective population size allow anticipating that the studied populations will lose 75% of heterozygosity in less than 50 generations. Our findings suggest that anthropogenic modifications on native forests, increasingly ongoing in Northeastern Argentina, Southern Paraguay and Southeastern Brazil, might prevent the dispersal of howlers, leading to population isolation. To ensure long-term viability and maintain genetic connectivity of A. caraya remnant populations, we recommend preserving and restoring habitat continuity. To conserve the species genetic pool, as well, the four genetic clusters identified here should be considered separate Management Units and given high conservation priority. In light of our findings and considering complementary non-genetic information, we suggest upgrading the international conservation status of A. caraya to “Vulnerable”.Black-and-gold howler monkeys  Howler monkeys (Primates: Atelidae) are amongst the largest New World monkeys, inhabitants of several Neotropical ecoregions, from central Mexico to northeastern Argentina . NowadayAlouatta caraya; hereafter denoted as BGHM), arboreal primates which inhabit several ecoregions in South America . We received specific approval to conduct this study by the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) from Argentina (no. 11420110100322CO). Additional specific sampling permits were obtained from Chaco and Corrientes Provinces, Argentina (Permit Number: 01071), and from Ministry of Ecology, Misiones Province, Argentina (Permit Number: permit no. 04/15); tissue samples were collected as part of an investigation conducted jointly by the Global Health Program, Wildlife Conservation Society and the Ministry of Ecology, Misiones Province, Argentina (Permit Number: 304/09). All methods used for tissue sampling complied with the guidelines recommended by the Protocol for Primate Sample Methods . Fecal collection was conducted without capturing the animals and therefore does not cause any harm to the studied species. The specific coordinates for each sampling location are: Pop1: 27,275°S 57,684°W; Pop2: 27,314°S, 58,646°W; Pop3: 27,550°S, 58,679°W; Pop4: 26,791°S, 59,631°W; Pop5: 25,970°S, 58,177°W; Pop6: 28,307°S, 57,457°W; Pop7: 27,467°S, 55,827°W; Pop8: 25,574°S, 54,075°W; Pop9: 26,500°S, 53,833°W; Pop10: 29,445°S, 56,800°W.This study was carried out in strict accordance with Argentinean laws for research on non-human primates, and following the recommendations of ‘Principles for the Ethical Treatment of Primates’ of the American Society of Primatologists ,,Fig 1), 2, 0.2 mM of each dNTP, 1 U GoTaq DNA polymerase , 4 pmol of each forward primer with an M13 tail, 4 pmol of each reverse primer, and 0.4 mg of Bovine Serum Albumin . The second step consisted of a 12.5-μL reaction with 5 μL of a 5:100 dilutions of the first multiplex PCR product as template, 0.875 mM MgCl2, 4 pmol of a forward primer with an M13 tail, 4 pmol reverse primer, and 4 pmol 5´FAM or HEX-labeled M13 primer [Genotypes from populations 1, 2 and 3 were obtained in previous studies , 32. The3 primer . Cycling3 primer . Product5’-CTACCATCAACACCCAAAGC-3’) [5’-CCAGGTTAAGAGGGTGATAGC-3’, this paper). Amplifications were performed at a final volume of 25 μL, containing 25 ng of single DNA extractions,1x GoTaq buffer , 1.75 mm MgCl2, 0.2 mM of each dNTP, 1 U GoTaq DNA polymerase , 4 pmol of each primer, and 0.4 mg BSA. Cycling parameters were: initial denaturation (5 min at 94°C), followed by 35–40 cycles of denaturation (1 min at 94°C), annealing (30 s at 50°C), extension (1 min at 72°C), and a final extension(3 min at 72°C). All products were sequenced bi-directionally in an Applied Biosystems 3500 Genetic Analyzer using BigDye® Terminator v3.1 Cycle Sequencing Kit , and with the samereverse and forward primers used foramplifications. Quality of the sequences was eyed-checked inspecting the electropherograms in Sequencher 5.3 software .Sequences were edited and aligned using the Musclealgorithm [® search in the National Center for Biotechnology Information (NCBI) website (https://blast.ncbi.nlm.nih.gov/Blast.cgi). For this search, the filters and mask options were clicked off, word size was set to a value of 28, match/mismatch scores were set to 1/-2 and gap creation/extension penalties were set to ´linear´.A491-bp fragment of the left domain of the mitochondrial DNA Control Region  was amplified using primers How RA-1 (AAGC-3’)  and RC-Blgorithm  in MEGAvlgorithm . As in [lgorithm , the posPID-SIBS)[n = 5, the smallest sample with complete genotypes at all loci  [Ne) of each group was estimated using a single-sample linkage disequilibrium method with jackknifing, as implemented in LD Ne v3.1 [n), the correlation in allelic frequencies arises from sampling a limited number of individuals from the population for estimating gene frequencies and disequilibrium [Ne is based on the expectation that small populations accumulate more disequilibrium over time [Ne [Genotypes were screened for null alleles, stuttering, or scoring errors using Micro-Checker v2.2.3 . ConformPID-SIBS) were comPID-SIBS). To accoPID-SIBS), based oee R, of  was comp≥ 0.375)  that cou Ne v3.1 , for a m Ne v3.1 . The preilibrium . The LD ver time . The mettime [Ne .a priori way to predict the genetic structure. Under restricted dispersal, we would expect a pattern of positive autocorrelation, where individual-by-individual genetic distances should be more similar at shorter geographical distances [r, were calculated with 10,000 bootstraps. The distance class at which r is no longer significant can be interpreted as an approximation of the extent of detectable positive spatial genetic structure [K (ranging from 1 to 10) was conducted using the admixture model with correlated allele frequencies, sampling locations as prior (LOCPRIOR), and 1,000,000 MCMC iterations after a burn-in of 50,000 replicates. Given our uneven sampling sizes we applied the correction method proposed by [n = 138 individuals, 10 sites), and three trimmed datasets prepared to achieve more even sampling schemes; 2) a dataset obtained by randomly removing individual genotypes, without replacement, from the two best sampled populations to reach a sample size of 15 individuals in each, plus all other samples ; 3) the original dataset, removing the populations with n ≤ 6 ; and 4) the sub-sampled even-sized reduced dataset, plus removing the populations with n ≤ 6 . For each one of these sampling strategies, runs were performed with the above-mentioned settings. We then collected the outputs and computed the corrected estimators MedMeaK, MaxMeaK, MedMedK, and MaxMedK[K identified by MaxMedK and MedMedK for a 0.80 threshold, as these should be more conservative, less influenced by the presence of migrants, and less affected by an incorrect a priori grouping of some individuals into populations [K, identified as detailed above.We first investigated genetic structure using microsatellites genotypic data by running both individual- and population-based analyses, at fine and regional spatial scales. For example, we evaluated spatial genetic autocorrelation at a fine scale, using GenAlExv6.5. This method is appropriate when there is no istances . Geograptructure . In additructure . A serieposed by  to accoud MaxMedK which red MaxMedK.For eachulations . We usedulations  to visuahttp://due.esrin.esa.int/page_globcover.php). The elements of the landscape were classified into three categories: 1) “tree cover”  and resistance distances, as well as partial Mantel tests between Dps and resistance, controlling for Euclidean distances , and between Dps and geographic distance, partialling out the effects of resistance distances [R. If landscape features influence gene flow, we would expect the Mantel statistic between Dps and resistance and the partial Mantel statistic (controlling for the effect of geographic distance) to be significant, whereas the partial Mantel statistic between Dps and geographic distance (controlling for resistance distances), to be non-significant [FST values) were also computed between all pairs of populations in Fstat v2.9.3.2.We analyzed the possible influence of landscape features on the genetic structure of BGHM by conducting analyses based on current theory in Circuitscape v 4.0 . For thidisperse , 2) rivedisperse  in R, weistances . The proistances  in R. Ifnificant . Populath) and nucleotidic diversity (ð) were calculated in DnaSP v5.0 [R2[FS [Fs and R2 have more power to reject the null hypothesis [R2 and negative Fu's FS values were significant. A Median-Joining haplotype network [Rg) and the Sums of Squared Deviations (SSD) indexes [Tau [ut, where u is the mutation rate of the assayed fragment. We used 0.15 mutations per site per million years, as in previous studies in howler monkeys [rlequinv3.522by conducting an analysis of molecular variance (AMOVA) [θST between populations and population groups.Haplotype frequencies, nucleotide composition, the number of transitions and transversions, and the number of polymorphic sites were calculated in Arlequin v3.522 . HaplotyaSP v5.0 . Standar v5.0 [R2, Tajima’ v5.0 [R2, and Fu'0 [R2[FS , and the0 [R2[FS . Under tpothesis . Selecti network  was buil network . To insp network  in Arleq network . The smo indexes . The sigxes [Tau , using t monkeys . To expr monkeys . Finally (AMOVA)  comparinn = 98) all ten loci were amplified, whereas in 40 samples a minimum of seven loci were amplified. We found no evidence of linkage between any pair of loci (P< 0.05), nor evidence of significant deviations from Hardy-Weinberg Equilibrium. We found evidence of first-order relatives (R ≥ 0.375) within populations (Pop) 2 and 3, and removed one individual from each of those dyads for further diversity and structuring analyses . BGHM populations exhibited moderate levels of microsatellite diversity  was 0.443±0.025 overall populations; uHe of Pop 2 differed significantly from uHe of Pop 6, 7, and 10, and uHe of Pop 9 differed significantly from that of Pop 7 and 8 . A significant signal of inbreeding was found only in Pop 9 (Ne estimates were 40 (95%CI: 21–112) for Pop 2, and Ne = 26 (95%CI: 15–52) for Pop 3. The remainder populations yielded Ne with infinite 95% CIs.For most samples (iversity . The meain Pop 9 . Ne estin = 72), 15 of which were new to this study , an overall haplotypic diversity of 0.930 ± 0.11, and an overall nucleotidic diversity of 0.01± 0.007. Pop 2 was the most diverse in terms of number of haplotypes, but these were moderately divergent from each other  and Cebidae (three species) family. Identity of fragment sequences ranged from 77% to 99% with E-values ranging from 0.00 to 0.0057, respectively. Therefore, the presence of NUMTs in our fragment sequences can be considered negligible.Thirty-six transitions defined 22 mtDNA CR haplotypes , Fs (-1.889), and R2(0.099) were non-significant, supporting the neutrality of the assayed fragment. The unimodal pattern of the mismatch distribution (data not shown) was consistent with a demographic expansion scenario estimated to have occurred 9,183 years ago . At the population level, significant SSD and Rg were observed in Pop 5 and Pop 9, which could be interpreted as a demographic expansion  only at the first distance class (0–27 km), indicating that BGHM were more closely related to other members of the same population than to BGHM from other populations  resulted in the same number of clusters , while that between Dps and geographic distances was not . The partial Mantel test between Dps and resistance  was significant , while that between Dps and geographic distance  was not .The average inter-individual genetic distance was 7.0 ± 3.0. Results of spatial autocorrelation analyses revealed a significant positive spatial structure (ulations . The rese others . The ManFST for microsatellites was 0.13 ± 0.34 and significantly different from zero (p = 0.001). Pairwise FST values between populations indicated substantial genetic differentiation, with 32 significant out of 45 comparisons (Table C in θST for mtDNA CR fragments was 0.18 ± 0.22 and significantly different from zero (p = 0.001). Pairwise θST values showed that Pop 3 and Pop 6 differed significantly from the others, except for Pop 1 and 2; in turn, Pop 5 differed significantly from 6 and 10, and Pop 10 differed significantly from Pop 9 , and that 2.62% variation was significantly partitioned among ecoregions ; likewise, pairwise FST values between ecoregions were significant, except for APF-AMAF and AMAF-SCMS comparisons. In agreement, results of the hierarchical AMOVA for mtDNA showed that 46.14% of variation was significantly distributed among populations within ecoregions  and that 11.56% of variation was significantly partitioned among ecoregions . Pairwise θST values showed significant genetic differentiation (p< 0.0001) between AMAF and the other ecoregions, and between SCMS and all ecoregions except for PFS. The Mantel statistic for mtDNA genetic distance and geographic distance between populations was non-significant .The global Loss or alteration of native forest and yellow fever outbreaks represent severe threats to black-and-gold howler monkeys in the southernmost limit of the species range. In light of this scenario, this study assessed their regional genetic diversity, gene flow and connectivity patterns, revealing that they conform four genetically differentiated clusters. Our results may contribute to the upgrade of BGHM conservation status, and provide guidelines for the future management of remnant populations.Alouatta pigra: 0.430 [Alouatta belzebul:0.640 [Alouatta palliata: 0.584 [In natural populations, genetic diversity provides the basis for the maintenance of evolutionary potential and adaptive capacity of individuals to face threats such as environmental change and disease . The popa: 0.430  and 0.58a: 0.430 , Alouattul:0.640 , Alouatta: 0.584 , suggesta: 0.584 , 23. BGHa: 0.584 , 25 coulSSD and Rg indexes, which could be interpreted as a demographic expansion  were concordant in detecting significant present-day genetic structuring among the examined BGHM populations (θST) seem to support the hypothesis of dispersal via riparian forests, which act as biological corridors enabling the movement of BGHM [th century [Black-and-gold howler monkeys are arboreal primates still present in some patchy and impoverished forests , 30. Theulations . Four diulations .This res of BGHM . Past im of BGHM , interru century , 78. Pop century .Circuistscape analyses , applying the equation for heterozygosity loss (Eqn. 4 in [Alouatta caraya seems to rely heavily on the species’ wide geographic distribution range (Alouatta caraya to “Vulnerable”.Based on the estimates of the effective population size obtained for the two biggest populations (qn. 4 in ), we canqn. 4 in ), the stqn. 4 in  and Brazqn. 4 in , that coon range , which iIn sum, the present study contributes novel evidence supporting contemporary restricted gene flow between BGHM inhabiting the southernmost portion of the species’ geographic distribution range, and identifies four distinct Management Units for conservation. We also anticipate that most of the studied populations would loss heterozygosity in the mid-term and recommend that the IUCN global conservation status of BGHM is upgraded to “Vulnerable”. Lost habitat connectivity can play a significant role in preventing gene flow between isolated populations and, if not reverted, such a pattern may severely affect the survival capacity of BGHM. Our results have direct implications for the conservation of howlers and should be taken into account by policy makers when taking decisions, drafting management plans or designing reintroduction projects of these vulnerable Neotropical primates.S1 FileTable A. Summary estimates of mitochondrial diversity. Genetic diversity estimates, results of neutrality tests and demographic parameters for black-and-gold howler monkeys sampled in ten populations from Northeastern Argentina and Southern Paraguay, based on 491-bp mtDNA Control Region fragment sequences. H: number of haplotypes (n: number of sequences). PS: number of polymorphic sites; h: haplotypic diversity± standard deviation (SD); π: nucleotidic diversity ± standard deviation (SD); Tajima’s (1989) D; Fu’s (1997) Fs; Ramos-Onsins and Rozas’s (2002) R2; SSD: Sum of Squared Deviations, Rg: Raggedness index; Tau: mode of the unimodal mismatch distribution; TSE: time since population expansion (in years before present). Table B. Results of modifiedtructureSprocedure. The most likely number of differentiated genetic groups  found to better explain the variation observed in the genotypic dataset (ten microsatellite loci) of black-and-gold howler monkeys from Northeastern Argentina and Southern Paraguay. The Max-ofMedian (MaxMedK) and Median-of-Median (MedMedK) indexes , taken at the 0.80 threshold of membership proportion, were used as conservative estimators of K. The different sampling schemes tested were: 1) the full dataset , 2) subsampling Pop 2 and 3 to obtain more even sample sizes , 3) original dataset but removing the least sampled populations , and 4) the dataset obtained by a combination of the two latter strategies  (see main text for further details). For each sampling scheme, 20 replicate STRUCTURE runs were performed from K = 2 to K = 10. Table C. Genetic structure observed in populations of black-and-gold howler monkeys from Northeastern Argentina and Southern Paraguay. Pairwise FST values for ten microsatellite loci  and pairwise ΦST values for 512-bp mtDNA Control Region fragment sequences . Significant values after Bonferroni correction are shown in bold (α = 0.0005).(PDF)Click here for additional data file.


Bdelloura candida  is an ectocommensal symbiont on the American horseshoe crab Limulus polyphemus, living on the book gills and appendages, where it spends its entire life. Given its limited dispersal capabilities and its inability to live outside of the host, we hypothesized a genetic structure that parallels that of its host. We obtained 84 planarian individuals from 19 horseshoe crabs collected from 10 sites from Massachusetts to Florida. We amplified the mitochondrial 16S rRNA and the nuclear internal transcribed spacer 2 and conducted phylogeographic and population genetic analyses, which show a clear and strong genetic break between the populations in the Atlantic and the Gulf coasts. Among the Atlantic populations, two additional, weaker barriers located along Cape Hatteras and Cape Cod restrict gene flow. Even though previous studies have suggested that the populations of the host may be in decline, those of B. candida remain stable, and some even shows signatures of expansion. Our results indicate that the phylogeography of these marine ectocommensal triclads closely mirrors that of its Limulus host, and highlight the challenges to both host and symbiont to genetically connect populations across their distribution.The online version of this article (doi:10.1007/s00227-017-3132-y) contains supplementary material, which is available to authorized users. Symbion, and these have never been found in non-nephropid hosts . In general, symbiont and host share gene flow and their genetic structure is linked, the symbiont mirroring the genetic structure of the host . The members of the genus Bdelloura are characterized by the absence of eye lenses, a posterior adhesive caudal disk set off from the rest of the body, and numerous testes distributed throughout the body  and its host, the American horseshoe crab, dy Leidy . The gen Girard, , B. proped Sluys .Bdelloura candida was described from Chelsea Beach, Massachusetts  following manufacturer’s instructions, with a slight modification in the incubation of the tissue in lysis buffer (overnight). We selected two markers, the mitochondrial 16S rRNA and the nuclear ribosomal internal transcribed spacer-2 region (ITS2). The 16S rRNA marker was amplified in 83 individuals with the primer pair 16Sa–16Sb , 0.5 μL 10 mM dNTPs , 0.13 μL GoTaq® Taq Polymerase , and 0.25 μL of each forward and reverse primers (10 mM). The PCR program for both markers included an initial denaturing step at 95 °C for 2 min, 35 cycles of denaturing at 95 °C for 30 s, annealing at 45 (for 16S rRNA) to 49 °C (for ITS2) for 30 s, and extension at 72 °C for 1 min, and then a final extension step at 72 °C for 1 min. The PCR products were vacuum cleaned and sequencing reactions were done using BigDye Terminator v3.1  and a sequencing program with an initial denaturing step at 94 °C for 3 min, then 25 cycles of denaturing at 94 °C for 10 s, annealing at 50 °C for 5 s, and extension at 60 °C for 4 min. The purified PCR products were sequenced using the same primer pairs in a 3730xl DNA Analyzer  at the Harvard Center for Systems Biology.PCR reactions were conducted using 1 μL of template DNA, 18 μL molecular grade distilled water, 5 μL GoTaq http://ncbi.nlm.nih.gov). Accession numbers for 16S sequences in GenBank are KY499968-KY500051 (84 sequences) and those of ITS2 are KY500082-KY500133 (52 sequences).Chromatograms were edited and assembled using Sequencher 5.0.1 . Sequences were checked for contamination using BLAST . For 16S rRNA, the L-INS-i strategy was used  was 1.53. ITS-2 had similar settings, with the only change being the E-INS-i strategy  .Number of haplotypes and segregating sites, haplotype diversity, nucleotide diversity and mean number of pairwise differences were obtained in DNAsp 5.10.1 , and subsequently applies Monmonier’s distance algorithm to identify areas that hinder gene flow among sites, i.e., the zones where ФST differences between pairs of sites are the largest.Genetic differentiation between populations was measured by computing pairwise te Narum . To testte Narum  with 16,Differentiation between groups of populations was assessed by conducting hierarchical analyses of molecular variance (AMOVA) using genetic distances, and testing their significance by running 16,000 permutations in ARLEQUIN. We grouped the populations in the following sets, based on results from BARRIER see “” sectionD test of neutrality to test for bottlenecks and population expansions , Group 2 , Group 3 , and Group 4 ] (Ramos-Onsins and Rozas We performed the Tajima’s s Tajima . The hishttp://beast.bio.ed.ac.uk/Tracer). Migration rates (Mt) were expressed as the number of migrants per generation Mt = n/µ, where n is the immigration rate per generation, and µ the substitution rate.Given the high genetic divergence detected between the populations of Florida (“Gulf”) and those from the rest of the Eastern coast of the US (“Atlantic”) shown in “All values of genetic diversity are detailed in Table B. candida collected from the same host were genetically identical .Using the matrix of Cod Fig. a, b, groCod Fig. , 5. HoweCod Fig.  and whenD statistics were not significant in any of the populations, indicating no evidence of selection  for ITS2  for the 16S rRNA marker  of B. candida were similar to those of other triclads Supplementary material 2 (PDF 297 kb)Below is the link to the electronic supplementary material.


Atherinomorus stipes, a small schooling fish found around islands throughout the Caribbean. Our field observations of A. stipes in the cays of Belize and the Florida Keys found that populations tend to be in close association with the shoreline in mangrove habitats. Due to this potential island‐based population structuring, A. stipes represents an ideal system to examine questions about gene flow and isolation by distance at different geographic scales. For this study, the mitochondrial gene nd2 was amplified from 394 individuals collected from seven different Belizean Cays (N = 175) and eight different Floridian Keys (N = 219). Results show surprisingly high haplotype diversity both within and between island‐groups, as well as a high prevalence of unique haplotypes within each island population. The results are consistent with models that require gene flow among populations as well as in situ evolution of rare haplotypes. There was no evidence for an isolation by distance model. The nd2 gene tree consists of two well‐supported monophyletic groups: a Belizean‐type clade and a Floridian‐type clade, indicating potential species‐level differentiation.Little is known about the natural history, biology, and population genetic structure of the Hardhead Silverside,  However, the identification of genetic structuring within marine species is notoriously difficult due to the lack of clearly identifiable barriers to gene flow and dispersal commonly found within terrestrial systems. Early genetic studies on marine systems worked under the assumption that populations existed in a state of panmixia in the absence of extrinsic barriers, such as ocean currents and continental barriers Avise, . HoweverDispersal capabilities, in particular, play a major role in shaping the genetic connectivity within marine species. Studies have shown that taxa with high dispersal capabilities maintain high levels of gene flow and express low levels of geographic structuring due to the homogenization of genetic diversity  are the Belize Cays and Florida Keys island‐groups genetically homogeneous and (ii) are populations within each island‐group homogeneous? We predict that populations of A. stipes exhibit genetic structuring both within and between the island‐groups. We also predict that the isolation by distance (IBD) model will explain genetic divergence in relation to geographic distance.We examine the population structure of 22.1A. stipes has been described as Caribbean‐wide, but an explicit habitat characterization is lacking  in July 2014, with the exception of B‐CB1_A01, and all Floridian samples in August 2015. All specimens were caught by 2‐m seine net with 3.2‐mm mesh. Individual B‐CB1_A01 was received as a voucher specimen from the University of Kansas Biodiversity Institute (Voucher Specimen #USNM 349215).A total of 394 individuals of Caudal fin clips were stored in individual tubes in either 95% ethanol (Belizean samples) or Chaos Buffer  and annotated using the R package “ggtree”  exhibited highly differentiated haplotypes from all other Floridian individuals. As outliers, they were removed from the Florida dataset used in all of the following analyses. DnaSP v5.10.01  between individual island populations within each island‐group were also generated in Arlequin.Analyses of molecular variance (AMOVA) was conducted in Arlequin 3.5.1.2 .When genetic structuring is observed, the role of the IBD model of evolution can be assessed Wright, . An anal33.1nd2 gene were successfully determined and aligned for 394 individuals of A. stipes. There were 178 variable sites among all individuals, and 94 of these were parsimony informative sites.On average there were 3,487 nucleotide differences among haplotypes from the Belizean Cays and 2,301 nucleotide differences among haplotypes from the Florida Keys. In Belize, the average Hd was 0.875 (range: 0.825 ≤ Hd ≤ 0.897), and the average nucleotide diversity (π) was 0.00294  three haplotypes  were “universal,” defined as being shared among all populations; (ii) haplotype 2 was shared among five populations; (iii) haplotype 31 was found in three populations; and (iv) and three haplotypes  were found in two populations  haplotype 62 was “universal”; (ii) haplotype 65 was shared among six island populations; (iii) haplotype 66 was observed among four island populations; and (iv) six haplotypes  were found in two populations. The remaining 95 haplotypes were “unique” , while none of the Belizean populations were found to be statistically significant  and all populations from the Florida Keys  between populations on the Gulf of Mexico side of the Florida Keys versus those populations on the Atlantic side  corroborated a tree topology identifying two distinct, well‐supported, monophyletic groups Figure : (i) a cWithin the Floridian‐type/Barbadian‐type clade, haplotypes from the Florida Keys and haplotypes from Barbados formed two separate, well‐supported monophyletic groups Figure . The genN = 14–52; Figure The statistical parsimony haplotype networks for Belizean‐type haplotypes and Floridian‐type haplotypes differed greatly in their structures. The Belizean‐type haplotype network exhibited a more complex structure with four major “universal” haplotypes found in varying frequencies . Mantel tests comparing the pairwise FST to the Euclidian geographic distance between populations within the Belizean and Floridian island‐groups revealed that there was only a slightly significant correlation within the Belizean island‐group (p = .0475).Linear regression analyses using log and standardized axes resulted in nonsignificant correlations within both the Belizean and Floridian island‐groups (adjusted 4nd2 from A. stipes were highly divergent (ca. 4.5%) between the Belize Cays and Florida Keys island‐groups. The two specimens from Barbados, the type locality of A. stipes, formed the sister group to the Floridian clade  high levels of haplotype diversity (Hd) and low levels of nucleotide diversity (π); (ii) high proportions of rare “unique” haplotypes associated with particular island populations; and (iii) significant molecular variation among populations. Combined, these genetic characteristics are consistent with island‐group populations that have experienced a recent rapid expansion with subsequent accumulation of novel mutations after one or more bottleneck events  with a genetic divergence of 4.5%  processes, respectively. The Floridian populations exhibited a classic starburst pattern haplotype network Figure  and had A. endrachtensis within isolated marine lakes in Palau  limited gene flow among populations, where rare, “unique” haplotypes result from in situ evolution over time diminishing the frequency of the common haplotypes; and (ii) moderate or higher amounts of gene flow among populations, where rare haplotypes are not necessarily “unique.” Both of these assume that the common haplotype is relatively older than the rarer haplotypes, but differ in their interpretation of the proportion of “unique” haplotypes within each population. The classification of a haplotype as “unique” is based upon sampling. In this case, our sample sizes  may not have been sufficiently large to detect haplotypes in very low frequencies, and therefore, overestimated the uniqueness of haplotypes and the degree of isolation among populations. Common haplotypes have a higher probability of being exchanged among populations compared to rarer haplotypes and, hence, explain the prevalence of common haplotypes in many populations. Our data are not sufficient to reject either of these hypotheses.A. stipes, mangroves and sea grass beds, around the shoreline of the key is not continuous. Similarly, in Belize, South Water Cay (N = 29) and Carrie Bow Cay (N = 31) differed by 21 “unique” haplotypes despite being separated by only 1.4 km of open water.Some of the results indicate more isolation of populations than would be expected from the second hypothesis. The genetic compositions of the samples from the two Marathon Key, Fla., sites suggest relative isolation of these geographically proximate localities. The sample taken on the Atlantic side (F‐MK) had only nine individuals that had eight different haplotypes for which six were “unique.” The Gulf of Mexico sample from Marathon Key (F‐MA) had 30 individuals with 18 haplotypes of which 16 were “unique.” Despite the fact that they were collected on opposite sides of the same key (5.8 km between the sample sites), none of the “unique” haplotypes from the small Atlantic side sample were found in the larger sample from the Gulf side of Marathon Key (no Atlantic vs. Gulf of Mexico effect was found among the Floridian samples). That said, we do note that the preferred habitat of A. stipes may serve to fragment this species and reduce gene flow (discussed below). Fragmented populations tend to evolve more rapidly due to higher levels of genetic drift , the result of this test in and of itself cannot be used as prima facie evidence of IBD. Significant results of Mantel tests provide evidence of spatial correlation as a rejection of an open gene flow model .The dispersal capabilities of this species, whether by active swimming or drifting in ocean currents, directly impact gene flow within and among island‐groups. The dispersive ability of A. stipes, the ocean currents flow to the north and east from Belize (Shulman & Bermingham, The pelagic larval state of marine fishes can be rather difficult to track due to their small size and the spatial scale of distribution (Mora & Sale, A. stipes in order to elucidate the biological mechanisms that facilitate gene flow.Additionally, high levels of larval dispersal among populations may diminish signals of IBD at smaller spatial scales over time (D'Aloia et al., 5A. stipes is unclear. Unfortunately, the dispersal capabilities between and within island‐groups, in addition to accurate population size estimates, of A. stipes are currently unknown.The Belizean and Floridian island‐groups were found to be highly divergent from each other. Within island‐groups, populations exhibited high numbers of haplotypes and differed significantly from one another though there was no or insufficient evidence for IBD. Populations within both island‐groups were characterized by high percentages of shared haplotypes and high percentages of rare but “unique” haplotypes. Two potential hypotheses were discussed that treat the common haplotypes as evidence of gene flow among populations. They differ by the amount of gene flow required and by the interpretation of the rare “unique” haplotypes. While our data cannot reject either hypothesis, the lack of shared haplotypes among neighboring populations and the very high percentages of rare haplotypes may favor the hypothesis requiring lower gene flow among populations. The results of neutrality tests and the haplotype network provide strong evidence that Floridian populations have undergone recent expansion following a bottleneck. The pattern of evolution among Belizean populations is less clear due to the complexity of their haplotype network and to the limitation of some of the population genetic statistics to discern more historic population patterns. How the population genetic structures can result from dispersive life history traits of A. stipes to mangrove habitats throughout the Caribbean presents an ideal opportunity to examine the potential influences of habitat fragmentation on intraspecific genetic structuring within and among island‐groups. Mangrove ecosystems are critical habitats because they serve as nurseries, feeding grounds, and shelters for many marine organisms (Laegdsgaard & Johnson, A. stipes, which rely heavily on visual cues to search for food in the water column (Thresher, The close association of A. stipes at other island localities throughout the Caribbean will help to illuminate diversification patterns. While this study presents the first genetic analysis of Hardhead silverside populations, a comprehensive examination of the phylogeography throughout the Caribbean is critical for future conservation and management of A. stipes.As this study only analyzed a single mitochondrial gene, there were limitations on the conclusions that we were able to make. It is important that future studies examine additional genomic regions and perform morphological analyses in order to reveal details of gene flow between and within island‐groups. Sampling from other populations of None declared.All authors equally contributed to the preparation of this manuscript.


Triosteum himalayanum (Caprifoliaceae), we sequenced three chloroplast DNA fragments  from 238 individuals representing 20 populations. Nineteen haplotypes (H1–H19) were identified based on 23 single-site mutations and eight indels. Most haplotypes were restricted to a single population or neighboring populations. Analysis of molecular variance revealed that variations among populations were much higher than that within populations for the overall gene pool, as well as for the East Himalayan group (EH group) and the North Hengduan group (NHM group), but not for the Hengduan Mountains group (HM group). Ecoregions representing relatively high genetic diversity or high frequencies of private haplotypes were discovered, suggesting that this alpine herbaceous plant underwent enhanced allopatric divergence in isolated and fragmented locations during the Quaternary glaciations. The current phylogeographic structure of T. himalayanum might be due to heterogeneous habitats and Quaternary climatic oscillations. Based on the phylogeographic structure of T. himalayanum populations, the phylogenetic relationship of identified haplotypes and palaeodistributional reconstruction, we postulated both westwards and northwards expansion from the HM group for this species. The westwards dispersal corridor could be long, narrow mountain areas and/or the Yarlung Zangbo Valley, while the northwards movement path could be south–north oriented mountains and low-elevation valleys.The varying topography and environment that resulted from paleoorogeny and climate fluctuations of the Himalaya–Hengduan Mountains (HHM) areas had a considerable impact on the evolution of biota during the Quaternary. To understand the phylogeographic pattern and historical dynamics of  The present population genetic structure of species carries signals of past dynamics . ExploraIn the early Eocene, the Indian Plate collided with the Eurasian Plate, directly resulting in subsequent uplift of the QTP and the Himalayas and Hengduan Mountains . The sigAlthough geological records show that no unified ice sheets were developed in QTP and adjacent regions during the Quaternary period , arid-coThus, the unique, abundant, and diverse flora of the HHM, resulting from topographical diversity and climate fluctuation, have made this area one of the most prevalent regions for phylogeographic survey. Numerous hypotheses have been provided based on different phylogeographic patterns. However, previous studies mainly focused on the tree  and shruTriosteum himalayanum Wallich (Caprifoliaceae) is a perennial herb with a distribution in the eastern Himalayas, Hengduan Mountains, and central China  at elevations between 1,800 and 4,100 m a.s.l. This species shows a fragmented distribution, occupying relatively wet habitats on mountain slopes, coniferous forests, streamsides, and grasslands , which provided the biogeographic pattern of Triosteum across North America and eastern Asia. However, they presented no details of the intraspecific variation and biogeography of T. himalayanum in the HHM and adjacent regions. A phylogeographic study of T. himalayanum would give us a better understanding of how fragmented populations have evolved since species diversification in the context of environmental heterogenization and Quaternary glaciation. Furthermore, a phylogeographic survey of multiple co-distributed species  would be greatly useful for providing a detailed picture of plant diversity and endemism of regional hotspots  to reveal the genetic structure of this species with fragmented distribution and (2) to infer the historical dynamics, especially the dispersal direction between ecogroups and pathways in association with habitat differentiation and Quaternary climate fluctuations. In order to better interpret these issues, we divided all the sampled populations into three groups: the East Himalayan group , the Hengduan Mountains group , and the north Hengduan Mountains group .asslands . Gould &hotspots . Here, wT. himalayanum according to specimen records in the herbaria. However, populations of T. himalayanum that were previously distributed in central China (according to the specimen study) were difficult to collect due to land use changes. Fresh leaves of 4–25 individuals were collected for each population, separated at least 20 m apart. A total of 238 individuals from 20 populations were collected. Among these populations, 19 were from the HHM, and only one population (P20) was not from this region , has been chosen as out group for haplotype phylogenetic analyses.rbcL-accD, rps15-ycf1, and trnH-psbA were chosen for further analysis, since these three intergenic spacers revealed sequence polymorphism. Polymerase chain reactions (PCRs) were performed in 25 μL volume containing 0.6 μL of template DNA (approximately 20 ng), 2.5 μL of 10 × PCR buffer (with Mg2+), 1.0 μL of 10 mM dNTPs, 0.5 μL of 5 pM of each primer, and 0.2 μL (1.5 units) of Taq polymerase . Amplification conditions were: 5 min at 95 °C; followed by 35 cycles of 50 s at 95 °C, 1 min at 58 °C, and 1 min at 72 °C; with a final extension of 6 min at 72 °C. PCR products were purified using a CASpure PCR Purification Kit  following the manufacturer’s protocols. Sequencing reactions and analysis were performed using an ABI 3730xl DNA sequencer . Primers used for sequencing were the same as those used for amplification.Total DNA was extracted from the silica-gel dried leaf materials following a modified CTAB . DNA sequences were aligned in Clustal_X , total gene diversity (HT), GST , GST , and NSTGST  was calculated, and significance was tested.Analysis of molecular variance AMOVA;  for overion 3.11 . The F-sT. himalayanum were generated in PhyML ver. 3.1 (L. trichosantha as the outgroup. We selected BioNJ as the distance-based tree reconstruction method during tree searching. Subtree pruning and regrafting (SPR) moves were relied on to explore the space of tree topologies. The number of random starting trees was five. The tree branch support was estimated by non-parametric bootstrap analysis, and the number of bootstrap replicates was 100. Bayesian inference was conducted using MrBayes ver. 3.2 (The appropriate model of DNA substitution for maximum likelihood (ML) analysis was determined using the AIC in jModelTest ver. 2.1.6 . Best-fiver. 3.1  using L.ver. 3.2 . The MCMver. 3.2 . The numhttp://www.fluxus-engineering.com).To further detect genealogical relationships, a haplotype network was constructed among cpDNA haplotypes using the median‐joining method and MP calculations as implemented in Network ver. 5.0.0.0 , last glacial maximum , and last interglacial maximum  were identified based on polymorphic sites across the concatenated cpDNA sequence data , followed by rps15–ycf1 , and trnH-psbA (three substitutions).The combined cpDNA sequences of nce data . Among tTwelve haplotypes  were relatively rare, each was restricted to a single population . Moreover, all of the twelve haplotypes were distributed in the HM group of five populations, indicating a very high level of molecular diversity . AnotherThe most common haplotype was H14, which occurred in nine populations with variable locations in the HM group (three populations) and NHM group (six populations). Haplotypes H7 and H16 both occurred in four populations but with locations not far from each other .Twelve populations  were fixed for single haplotype, showing no variation within the population. However, the remaining eight populations  in the HM and NHM groups were particularly diverse, with two to five haplotypes in each population ; Fig. 1.Population P19 was located far from P20, isolated by the Sichuan Basin. However, these two populations shared the sole haplotype H19.K values increased from two to seven while FCT values changed irregularly from 0.897 to 0.781. However, each newly defined group was represented by a single population. On the basis of the distribution ecoregions of the populations and the genetic structure of the T. himalayanum populations, we divided the populations into three groups: the East Himalayan group , the Hengduan Mountains group , and the North Hengduan Mountains group . The division backgrounds which can be considered as temporal and biota differences between these three eco-regions are illustrated as follows: (1) distinctions between the Hengduan Mountains and the Himalaya. T. himalayanum and some other species . A significant difference between NST and GST was also found in the EH and NHM groups but not in the HM group. The values of total genetic diversity (HT) were much higher than those of gene diversity within populations (HS) for T. himalayanum in the overall populations , EH group , HM group , and NHM group , suggesting slight range expansion. Fu’s Fs statistics showed positive values or not significant negative values for the whole gene pool and three groups  to numerous peaks above 6,000 m a.s.l., and it is one of the most ecologically diverse areas in the QTP region . FurtherT. himalayanum is a high level of private haplotypes. Twelve out of 19 detected cpDNA haplotypes were private. High total diversity versus a relatively low within-population diversity is another feature of T. himalayanum, and this type of geographic genetic structure is found in many studied alpine species located in the HHM and adjoining regions  to the NHM group (HT = 0.498) could result from the south–north direction postglacial recolonization. This hypothesis was also best supported by the palaeodistributional reconstruction  to both the East Himalaya area (EH group) and the North Hengduan Mountains (NHM group) from Mid Holocene.Even the values of Fu’s ulations  reject tHT = 0.922) to the EH group (HT = 0.714). One of the migration corridors may be the Yarlung Zangbo Valley, which is the largest vapor channel of the QTP  detected among 20 populations of Click here for additional data file.