Vincetoxicum species using the species distribution model (SDM) and assessed the genetic consequences of secondary contact by combining morphological and genetic approaches. We performed morphometric analysis  and examined 10 nuclear microsatellites  in V. atratum, V. japonicum, and putative hybrid populations.During glacial periods, glacial advances caused temperate plant extirpation or retreat into localized warmer areas, and subsequent postglacial glacial retreats resulted in range expansions, which facilitated secondary contact of previously allopatric isolated lineages. The evolutionary outcomes of secondary contact, including hybrid zones, dynamic hybrid swarm, and resultant hybrid speciation, depends on the strengths of reproductive barriers that have arisen through epistatic and pleiotropic effects during allopatric isolation. The aim of this study was to demonstrate refugia isolation and subsequent secondary contact between two perennial Asclepioid species and to assess the genetic consequences of the secondary contact. We modeled the range shift of two ecologically distinct V. atratum and V. japonicum. High pollen fertility and a lack of linkage disequilibrium suggested that the hybrid populations may be self-sustaining and have persisted since V. atratum and V. japonicum came into contact during the post-glacial period. Moreover, our findings show that the pattern of hybridization between V. atratum and V. japonicum is unidirectional and differs among populations. Geographically-isolated hybrid populations exist as genetically distinct hybrid swarms that consist of V. atratum-like genotypes, V. japonicum-like genotypes, or admixed genotypes. In addition, Bayesian-based clustering analysis and coalescent-based estimates of long-term gene flow showed patterns of introgressive hybridization in three morphologically ‘pure’ V. japonicum populations.Multivariate analysis, model-based Bayesian analysis, and non-model-based discriminant analysis of principal components confirmed the hybridization between V. atratum and V. japonicum came into contact during the post-glacial period. Pattern of hybridization between V. atratum and V. japonicum is unidirectional and differs among populations. We concluded that these differences in the genetic consequences of secondary contact are caused by historical colonization processes and/or natural selection.In this study, we demonstrated that climatic oscillations during the Quaternary period likely led to species range shift and subsequently secondary contact. Hybrid populations may be self-sustaining and have persisted since The online version of this article (doi:10.1186/s12862-016-0587-2) contains supplementary material, which is available to authorized users. In response to climatic oscillations over the last 2 million years (Ma), the geographical distributions of temperate plants have been subject to multiple contractions and expansions . During Assuming that the reproductive isolation between two species coming into contact was incomplete, first generation hybrids may mate with parental species. Frequently backcrossing between hybrids and their parental species will lead introgression of alleles across species boundaries, thereby affecting species history and delimitation . The dynIn recent years, an increasing number of studies have employed species distribution modeling (SDM) or ecological niche modeling (ENM) to support refugia isolation and subsequent range expansion and corroborate phylogenetic approaches. The aim of this study was to demonstrate refugia isolation and subsequent secondary contact by modeling range shifts. This study presents one of the first applications of the species distribution model (SDM) to explain the direction of historical gene flow and its effects on the genetic consequences of secondary contact by predicting potential glacial distribution and postglacial colonization processes.Fagus crenata [Aesculus turbinata [Padus grayana, Euonymus oxyphyllus, Magnolia obovata, Carpinus tschonoskii, C. japonica, C. laxiflora [Platycrater arguta [Rubus palmatus, and R. grayanus [Vincetoxicum (Apocynaceae-Asclepiadoideae), V. atratum (Bunge) Morren et Decaisne and V. japonicum Morren et Decne., provide a suitable study system. Because the different habitat preferences of the species may result in different responses to climate change [V. atratum and V. japonicum may have retreated into separate refugia during the glacial period and subsequent postglacial expansion may have facilitated secondary contact, resulting in hybridization.The Japanese Archipelago is thought to have been free of massive ice sheets during the last glacial maximum (LGM) with the exception of some high altitude areas . Previou crenata , Aesculuurbinata , Padus gaxiflora , Platycrr arguta , Rubus pgrayanus . Two ecoe change , 29–31, Vincetoxicum atratum and V. japonicum are perennial herbs. Vincetoxicum atratum is distributed across the Sino-Japanese region including the Japanese Archipelago, Korean Peninsula, and China, whereas V. japonicum is distributed in the Japanese Archipelago and the Korean Peninsula [V. atratum is found in grasslands or meadows across northern to southern regions of the archipelago, while V. japonicum is found in sunny meadows or on rocky beaches near seashores of central and southern regions of the archipelago [V. atratum populations have decreased rapidly. Morphologically, the two species can be distinguished based on specific characteristics. Vincetoxicum atratum has a purple corolla, which is pubescent outside and glabrous inside, while V. japonicum has a yellowish white corolla, which is glabrous outside and pubescent inside. Leaves of V. atratum are villous on both sides while those of V. japonicum are densely pubescent [V. atratum and V. japonicum [eninsula . In Japahipelago . Becauseubescent . The floaponicum . The lowaponicum .V. atratum and V. japonicum by combining morphological and genetic approaches with paleoclimatic analysis. First, we examined the hybrid status of individuals from seven morphologically putative hybrid populations using a morphometric approach. Second, we examined the genetic structure of morphologically ‘pure’ V. atratum, V. japonicum, and putative hybrid populations. In particular, we examined deviation from linkage equilibrium for nuclear microsatellite markers because linkage disequilibrium would be expected in a hybrid zone as a result of ongoing gene flow between the parent species [V. atratum and V. japonicum and to infer past opportunities for secondary contact between the species. In this report, we address the following three questions. (1) What are the genetic and phenotypic patterns of morphologically ‘pure’ V. atratum, V. japonicum, and putative hybrid populations? (2) What is the outcome of secondary contact between V. atratum and V. japonicum? (3) What is the genetic consequence of hybridization between the two species?In this study, we examined past hybridization and introgression between  species –37. ThirV. atratum and V. japonicum from almost the entire ranges of the two species in Japan , and 0.2 μM each of forward and reverse primers. The reaction parameters were an initial denaturation step at 95 °C for 5 min followed by 26 cycles at 95 °C for 30 s, 60 °C for 90 s, and 72 °C for 60 s, and a final step at 60 °C for 30 min. PCR products were run with an internal size standard GeneScan LIZ-600 (Applied Biosystems) on an ABI 3100 Genetic Analyzer (Applied Biosystems). Allele sizes were scored using GENEMAPPER v3.7 software (Applied Biosystems).Total DNA was extracted from approximately 100 mg of leaf tissue based on the method of Maki, Horie and Yokoyama . Ten nucMorphological measurements were performed on the same individuals that were genotyped. The four largest mature leaves and four largest mature flowers of each individual were subjected to measurement of morphological characteristics and the averages of the measurements per individual were used for morphometric analyses. A total of 13 quantitative and four semi-quantitative characteristics were measured Fig. ; Table 1V. atratum and V. japonicum were tested using one-way analysis of variance (ANOVA). Pearson correlation coefficients were calculated to eliminate highly correlated (Pearson’s correlation > 0.9) characteristics from further analyses. A Kolmogorov-Smirnov test was used to determine whether the data met the assumption of normal distribution, while a Levene’s test was used to test for homoscedasticity. For four ordinal variables, Kruskal-Wallis one-way ANOVA was performed to estimate differences between V. atratum and V. japonicum. Principal coordinate analysis (PCO) was used to visualize morphological variation among morphologically ‘pure’ V. atratum, V. japonicum populations and morphologically hybrid populations. All statistical analyses were performed using R programming language [For 13 continuous variables, significant differences between allopatric language .V. atratum (A01–A03) and V. japonicum (J01–J03) and in all putative hybrid populations following the procedure of [Pollen fertility was examined in three populations of edure of .Na), observed heterozygosity (HO), and expected heterozygosity (HE) were calculated using the program GenAlEx version 6 [FST, [For each population, the mean number of alleles per locus  greater than 0.9 into one of the parental lineages and classified those with values less than 0.9 as hybrids. For analysis using STRUCTURE, different probable numbers of genetic clusters (K) were estimated for all samples under the admixture model with correlated allele frequencies. Ten replicate runs were performed for each value of K ranging from 1 to 10. Each run included a burn-in of 10,000 steps followed by 100,000 Markov chain Monte Carlo (MCMC) simulations. The optimal value of K was calculated using the STRUCTURE HARVESTER program [K were averaged using the CLUMPP program [A Bayesian model-based approach implemented in the program STRUCTURE version 2.2.3  and TESS program . The res program  and were program .K value (the number of clusters) between 2 and 10. The optimal value of K was determined based on the lowest value of the deviance information criterion (DIC). The results from the 10 replicates with the lowest DIC values were averaged using CLUMPP [TESS version 2.3 was used to estimate population structure by incorporating geographical coordinates of individuals as prior information to detect discontinuities in allele frequencies . The anag CLUMPP  and wereg CLUMPP .To detect complex genetic structure, a non-model-based discriminant analysis of principal components (DAPC) using the adegenet version 1.2.8 package  in R 4141 was co‘pure’ V. atratum (A01–A07 and A09), ‘pure’ V. japonicum (J01–J08), south group of putative hybrid population (H01–H05), north group of putative hybrid population (H06–H07) and introgressed V. japonicum (J09–J11). We estimated bidirectional long-term migration rates among the five categories based on 10 nrSSR loci with a coalescent-based model implemented in the LAMARC 2.1 program [M = m/μ) and the mutation-scaled effective population size (Θ = 4Neμ). Ne is the effective population size, m is the migration rate between two populations, and μ is the mutation rate per generation at the locus considered. We used logarithmic prior distributions for migration rate , and effective population size . We conducted Bayesian analyses under the Brownian motion model [i → j), the number of immigrants from population i into population j (Nemi → j) was estimated from Nem = Θi*Mi → j). Results are reported as most probable estimates (MPE) with 95 % confidence intervals (CIs). We used Tracer v1.6 [We divided studied populations based on the genetic constitution of each population defined by STRUCTURE, namely,  program . We randon model . Search cer v1.6  to confiV. atratum and V. japonicum during three periods, namely, the present, the mid-Holocene , and the last glacial maximum . The maximum entropy algorithm , based on presence-only data, and the associated environmental covariates were used to model species distribution. Species occurrence data were collected from the S-Net data portal (http://science-net.kahaku.go.jp/) and our sampling locations  to confirm that each set represented a reasonable location. A total of 19 environmental variables  were used for subsequent analyses  to identify the range of suitable habitats of um LGM; 2,000 ybp.um LGM; 2,000 ybp.um LGM; 2,000 ybp.um LGM; 2,000 ybp.lim.org; . We extrlim.org;  and examlim.org; . After eum LGM; 2,000 ybp.r > 0.9), and thus HBL and HCO were eliminated from subsequent multivariate analyses. Three vegetative characteristics, maximum leaf blade length (LL), maximum leaf blade width (LW), and hair density on the adaxial leaf surface (HDL), and eight floral characteristics, pedicel width (PW), corolla diameter (CLD), hair density on the corolla inside (HCI), corona diameter (CND), gynostegium length (GYL), gynostegium diameter (GYD), corpusculum length (COL), and pollinia length (POL) that exhibited significant differences (P < 0.05) among morphologically ‘pure’ V. atratum and V. japonicum populations , hair density on the adaxial leaf surface (HDL), and hair density on the corolla outside (HCO) were highly correlated with each other  analysis accounted for 55.94 and 10.26 % of the total variance, respectively Fig. . The PCOV. atratum populations (A01–A03), 96–100 % in hybrid populations (H01–H07), and 100 % in V. japonicum populations (J01–J03).The minimum, maximum, and median values of pollen fertility are summarized in Additional file V. atratum populations compared with 165 alleles for V. japonicum. The mean number of alleles per locus (Na) within a population was 5.30–8.10 in V. atratum and 2.60–8.90 in V. japonicum  . Genetic differentiation between all pairs of V. atratum and V. japonicum populations were moderate or large with FST values ranging from 0.18 to 0.52  determined from STRUCTURE analysis was 2 based on ∆K values . Because we focused on hybridization between the two species in this study, we adopted K = 2 a priori, even when using TESS. With K = 2, all morphologically ‘pure’ V. atratum populations and the J01–J08 V. japonicum populations clustered into two separate groups  were distributed over intermediate positions near V. japonicum. In the H01, H06, and H07 putative hybrid populations, individuals were distributed over intermediate positions near V. atratum, whereas individuals of the H02–H05 populations were distributed over intermediate positions near V. japonicum.The best value for the number of genetic clusters  was much higher than 4 NmV. japonicum→‘pure’V. atratum‘pure’ (1.18)  data at the time of the LGM is shown in Fig. V. atratum and V. japonicum predicted by SDM suggested that these two ecologically distinct species responded to climate changes in different ways across the Japanese Archipelago  , V. japonicum-like genotypes H02–H05 and admixed genotypes (H01). The DAPC of all individuals again showed two genetically distinct groups as did analysis based on STRUCTURE  Fig. . In the V. atratum and V. japonicum. In addition, biases in offspring fitness unlikely exist, because both V. atratum-like genotypes (H06 and H07), V. japonicum-like genotypes H02–H05 and admixed genotypes (H01) were detected. Alternatively, the unidirectional backcrossing might result from ‘pollen swamping’ by more abundant local species over later-colonizing species [V. atratum were already distributed in the area where the current H06 and H07 populations are located. After the glacial period, V. japonicum may have colonized this area from its southern refugia and been subject to substantial pollen flow from V. atratum. If backcrosses to V. atratum are selectively favored and do not exhibit reduced fitness relative to V. japonicum and V. atratum, they may ultimately displace ‘pure’ populations of V. japonicum and V. atratum.Most hybrid populations (H02–H05 and H06–H07) consist with parental-like genotypes. These genotypes may be generated by frequent backcrossing to one of parental species, because highly backcrossed individuals are difficult to distinguish from parental individuals due to the relatively small number of loci . Unidire species –68. The V. atratum colonized the area where the current H01–H05 populations were located earlier than V. japonicum  surrounded by much larger populations of V. atratum, resulting in directional introgression from the latter species.The Bayesian-based clustering analyses showed signs of introgressive hybridization Fig. . In the V. atratum and V. japonicum .


Bantu speech communities expanded over large parts of sub-Saharan Africa within the last 4000–5000 years, reaching different parts of southern Africa 1200–2000 years ago. The Bantu languages subdivide in several major branches, with languages belonging to the Eastern and Western Bantu branches spreading over large parts of Central, Eastern, and Southern Africa. There is still debate whether this linguistic divide is correlated with a genetic distinction between Eastern and Western Bantu speakers. During their expansion, Bantu speakers would have come into contact with diverse local populations, such as the Khoisan hunter-gatherers and pastoralists of southern Africa, with whom they may have intermarried. In this study, we analyze complete mtDNA genome sequences from over 900 Bantu-speaking individuals from Angola, Zambia, Namibia, and Botswana to investigate the demographic processes at play during the last stages of the Bantu expansion. Our results show that most of these Bantu-speaking populations are genetically very homogenous, with no genetic division between speakers of Eastern and Western Bantu languages. Most of the mtDNA diversity in our dataset is due to different degrees of admixture with autochthonous populations. Only the pastoralist Himba and Herero stand out due to high frequencies of particular L3f and L3d lineages; the latter are also found in the neighboring Damara, who speak a Khoisan language and were foragers and small-stock herders. In contrast, the close cultural and linguistic relatives of the Herero and Himba, the Kuvale, are genetically similar to other Bantu-speakers. Nevertheless, as demonstrated by resampling tests, the genetic divergence of Herero, Himba, and Kuvale is compatible with a common shared ancestry with high levels of drift, while the similarity of the Herero, Himba, and Damara probably reflects admixture, as also suggested by linguistic analyses. Bantu languages started to diffuse from their homeland in the Grassfields of Cameroon around 4,000–5,000 years ago, reaching the southernmost areas of the continent in only a few thousand years Southern Africa represents the last phase of the Bantu expansion. Archaeological data reveal traces of an agricultural way of subsistence in Namibia, Zambia and Botswana around 2000–1200 years ago The Bantu-speaking populations nowadays inhabiting southern Africa are quite diverse linguistically and culturally, comprising pastoralists, agro-pastoralists, and agriculturalists who speak languages belonging to several different subgroups of both Eastern and Western Bantu; these populations share the same territory and are often involved in trade. From a genetic perspective, these populations appear to be relatively homogenous, with little differences even among linguistically distinct populations Among the culturally distinct populations in southern Africa are the Herero, Himba, and Kuvale from northern Namibia and southern Angola, who speak dialects of the same Bantu language and practice intensive semi-nomadic cattle pastoralism. The Herero and Himba appear genetically distinct from other Bantu-speaking populations of the area, including the culturally similar Kuvale In this study we analyze complete mtDNA genome sequences of 944 Bantu-speaking individuals from Angola, Zambia, Namibia, and Botswana to investigate the maternal genetic history of Bantu speakers of southern Africa. We also include 38 Damara mtDNA genome sequences to further investigate the close genetic relationship between the Herero, Himba, and Damara that emerged in previous research This study was approved by the Ethics Committee of the University of Leipzig, the Research Ethics Committee of the University of Zambia and Namibe’s Provincial Health Department. Published samples from Botswana and Namibia come from a sample collection authorized by the governments of Botswana and of Namibia (Research permit CYSC 1/17/2 IV (8) from the Ministry of Youth Sport and Culture of Botswana, and 17/3/3 from the Ministry of Health and Social Services of Namibia). Samples from several populations of southwestern Angola were collected as described by www.mbio.ncsu.edu/BioEdit/bioedit.html) and read alignments were screened with ma Details on the samples and DNA extraction are given in One hundred and ninety seven sequences from Botswana, Namibia, and Angola were previously included in studies focusing on haplogroups L0d and L0k as well as on the prehistory of Khoisan populations http://glottolog.org/). While we were able to group most of the samples into 17 ethnolinguistically homogenous populations that correspond to the identification of donors' maternal grandmothers, some ethnolinguistic groups were represented by only a few individuals. In these cases, we united samples in five aggregate “populations” of speakers of closely related varieties based mainly on linguistic criteria, but ensuring that the resulting populations were genetically homogeneous as shown by non-significant between-population variance in AMOVA analyses and non-significant ΦST distances  and visualized in Network publisher 1.3.0.0. For the networks of haplogroups L3d and L3f all sites were given equal weights, while for the network of haplogroup L0d the hypervariable positions were downweighted by one third in order to reduce the effect of reticulation. A Mantel test was performed between genetic (Φst) and geographic distances with the R package vegan Haplogroup assignment was performed with the online tool Haplogrep −8 from Soares et al. BEAST v1.7.2;  was usede = 2000 and a number of marked individuals corresponding to a range of frequencies of the haplogroup of interest. This ancestral population was split into two daughter populations with Ne = 1000 . From these we consecutively sampled the same number of individuals with replacement (i.e. the population size was kept constant) for a number of generations proportional to 500, 1000, or 2000 years (with a generation time of 25 years). After the final resampling step, a number of individuals corresponding to the population samples of interest  were sampled 100 times from the two daughter populations, and the probability of having a frequency of the haplogroup of interest within the range of the respective confidence intervals for both populations simultaneously was recorded. The entire process was repeated 10,000 times for each initial haplogroup frequency tested and each of the three split times considered, and the average probabilities were recorded in a table. No migration was considered in any of the simulations. The procedure is further described in the Results section, and the R script used to perform the simulations is available upon request from the authors.Resampling tests were performed in R to investigate the possible shared ancestry of the Herero, Himba, and Damara on the one hand, and the Herero, Himba, and Kuvale on the other, notwithstanding the absence of haplogroup L3f in the Damara and the absence of L3d in the Kuvale and the concomitant high frequencies of these haplogroups in the Herero and Himba. In both cases we proceeded as follows: the Herero and the Himba were considered a single population with a sample size of 51 individuals, while for the Damara and Kuvale we used the actual sample sizes included in the study . We then created a series of hypothetical ancestral populations with NAs can be seen from There is very little discernible structure in the maternal genepool of the Bantu-speaking populations of southern Africa, as shown by a three-dimensional MDS analysis. Only two distinct groups of populations emerge : the HimHaplogroups L0d and L0k are known to be an introgression from autochthonous populations As can be seen in the CA plots displayed in Figure S2 in The difference of the Himba, Herero, and Damara from the other populations included in this study also becomes apparent from measures of genetic diversity : while dAn Analysis of Molecular Variance  demonstrThe mtDNA haplogroups L0d and L0k have been convincingly shown to be characteristic of autochthonous populations of southern Africa The Himba and Herero stand out among the Bantu-speaking populations of southern Africa in having very high frequencies of haplogroups L3d  and L3f , while in their cultural and linguistic relatives, the Kuvale, L3d is absent and L3f has a frequency of only 6% . In contWhile haplogroup L3d is found across Africa at low frequency . This caIn contrast to L3d, L3f is found in frequencies >20% not only in southwestern Africa, but also in some populations of the Cameroon/Chad border areas . NeverthThe Damara, who have the highest frequency of L3d and who share a highly frequent L3d haplotype with the Herero and Himba, entirely lack L3f. This finding is compatible with two scenarios: 1) The mtDNA pool of the Damara, the Himba, and Herero is derived from a single ancestral population, and haplogroup L3f was lost in the Damara due to genetic drift. 2) The Damara mtDNA lineages stem from a different ancestral population than the Himba and Herero, and the Himba and Herero incorporated large amounts of haplogroup L3d sequences through gene flow from Damara  these populations stem from a common ancestral population, and differential gene flow led to their strong divergence; 2) these populations have distinct maternal ancestors and their cultural and linguistic relationship is due to a shift in language and culture. These two alternatives were also assessed with a resampling test, assuming a frequency of 31% L3f  19–46%) and 43% haplogroup L3d (C.I. 29–58%) in the Himba/Herero and a frequency of 5.7% L3f (C.I. 1–16%) and a lack of haplogroup L3d (C.I. 0–7%) in the Kuvale. As can be seen from Overall, the Bantu-speaking populations of southern Africa are genetically quite homogenous, with a few exceptions such as the Herero and Himba or Tswana and Kgalagadi. While linguistically the populations can be divided into those speaking Western Bantu languages and those speaking Eastern Bantu languages, this division is not detectable in the maternal genepool, with none of the variance among populations corresponding to this linguistic grouping . FurtherNevertheless, within the homogenous mtDNA landscape of southern African Bantu-speakers some populations do stand out. The most notable outliers are the Herero and Himba (discussed in detail below); apart from these, the Kgalagadi and Tswana as well as Northeast Zambia are separated in the MDS and CA analysis, respectively. The Tswana and Kgalagadi are characterized by very high frequencies of haplogroup L0d, which provides good evidence for extensive admixture in the maternal line with autochthonous populations  and rCRS  are indicated with a black line. Dashed lines indicate nodes with low posterior probability for which the topology could not be resolved. The width of the triangles is proportional to the frequency of a given haplogroup in the dataset. Haplogroups L3d and L3f, in yellow, are described in more detail in Figure S4A and S4B. Figure S2: CA plots based on basic haplogroup frequencies. Color coding by linguistic affiliation as specified in Figure S3: Haplogroup frequencies of important haplogroups in the populations studied here. A: Haplogroups L0d and L0k. B. Subhaplogroups L3d3a1 and L3f1b4a. The size of the circles is proportional to the sample size. Figure S4A: Bayesian tree of all African complete mtDNA sequences belonging to haplogroup L3d, and S4B: belonging to haplogroup L3f. The sequences included in these trees are the same as those included in the networks in Figure S5: Bayesian Skyline Plots for the Himba, Herero, Kuvale and Damara. X axis: time in years ago. Y axis: effective population size per generation time.(PDF)Click here for additional data file.Table S1Information on country of sampling, language and population affiliation with assigned population as well as GenBank accession numbers for individual samples included in the study.(XLSX)Click here for additional data file.Table S2Frequency of L3d and L3f in the African populations included in the Surfer maps and their geographical coordinates.(XLSX)Click here for additional data file.Table S3Basic haplogroup frequencies in the populations studied here.(XLSX)Click here for additional data file.Table S4L0d haplotypes shared between Bantu speakers and Khoisan.(XLSX)Click here for additional data file.


Helicoverpa zea (Boddie) captured during two different seasons were assessed for genetic structure using microsatellite markers and for host plant type using stable carbon isotope analysis. Individuals (N = 568) were genotyped and divided into 13 putative populations based on collection site and time. Fixation indices (F‐statistics), analysis of molecular variance (AMOVA), and discriminant analysis of principal components (DAPC) were used to examine within and among population genetic variation. Mean number of alleles per locus was 10.25 (± 3.2 SD), and allelic richness ranged from 2.38 to 5.13 (± 3.2 SD). The observed and expected heterozygosity ranged from 0.07 to 0.48 and 0.08 to 0.62, respectively. Low FST (0.01 to 0.02) and high FIS (0.08 to 0.33) values suggest captured migrants originated from breeding populations with different allele frequencies. We postulate that high genetic diversity within migrant populations and low genetic differentiation among migrant populations of H. zea are the result of asymmetrical immigration due to the high dispersal and reproductive behavior of H. zea, which may hinder the adaptation and establishment of H. zea to peripheral habitat. These findings highlight the importance of assessing peripheral population structure in relation to ecological and evolutionary dynamics of this and other highly reproductive and dispersive species.Seasonal climatic shifts create peripheral habitats that alternate between habitable and uninhabitable for migratory species. Such dynamic peripheral habitats are potential sites where migratory species could evolve high genetic diversity resulting from convergence of immigrants from multiple regionally distant areas. Migrant populations of  Migration is the primary mechanism species use to avoid competition, find resources and, in sexual systems, locate mates   is a polyphagous pest, feeding on a wide range of C3  and C4  plant species  in seasonal time of pest presence, population levels, and degree of crop damage it causes in northern latitudes, which typically do not observe infestation until late summer  and from July through September 2005 at the Southeast Research Center at Landisville, PA  were collected and stored at −20°C for additional laboratory analysis. Wings dissected from each individual were preserved in 70% ethanol, and genomic DNA from thorax and head of each individual extracted using the Qiagen DNeasy Kit  at Penn State were sent to the USDA‐ARS Southern Insect Management Research Unit (SIMRU) for stable carbon isotope analysis and for microsatellite genotyping, respectively.Adult 2 was done at SMRT using micro‐Dumas combustion using a Costech ECS4010 Elemental Analyzer  coupled to a Thermo Finnigan Delta plus Advantage Mass Spectrometer using a Conflo II Interface . The isotope standard reference material was bovine muscle powder, from the National Institute of Standards and Technology (NIST‐RM 8414). Moths were then determined to either feed primarily on C3 (δ13C ≤ −20) or C4 (δ13C ≥−15) plants based on previous assessment of stable carbon isotope analysis of H. zea ‐labeled universal primer, 1 μL of 10 mM dNTP mix, 0.5 μL of 10× titanium Taq polymerase buffer, 0.1 μL of titanium Taq polymerase , and 1.6 μL of DNase free water for a 5 μL reaction. Cycling conditions included an initial denaturation step for 2 min at 95°C, an initial annealing step of 1 min at 60°C, followed by 30 denaturing and annealing cycles . Reactions were then diluted 1/9 and amplicons separated on an ABI 3700xl genetic analyzer with ROX‐labeled markers. Peaks were scored as genotypes using the software GeneMapper  and confirmed manually.Prior to microsatellite genotyping, DNA samples were quantified using the Quanti‐iT PicoGreen assay kit  for double‐stranded DNA and then adjusted to 20 ng/HE) and observed (HO) heterozygosity, within (FIS) and among (FST) population fixation indices, pairwise FST and allelic richness (Ar).All loci were checked for null alleles and allelic drop out using MICROCHECKER 2.2.3  a set of prior putative H. zea populations, (2) assuming no prior population assignment, and (3) C3 versus C4 plant preference. Prior putative populations were determined as peak abundance periods during the collection periods, which signaled recent mass immigration of H. zea  was then used to evaluate the relevance of different K values to population structure. Assignment values for the selected number of clusters were then generated for each individual using DAPC . Analysis of molecular variance (AMOVA) was performed using Arlequin v3.5   not significantly deviating from zero .The eight loci used in the final genetic analyses had no linkage disequilibrium, allelic dropout, or potential null alleles. Mean number of unique alleles per locus was 10.25 (±3.2 SD). Observed heterozygosity ranged from 0.07 to 0.48, while expected heterozygosity ranged from 0.08 to 0.62 . Specifically, individuals from each putative population were generally found across all DAPC clusters with a mean of 2 to 14 (SD = 1.89 to 4.22) individuals from each putative population grouping in each of the DAPC cluster. Analysis with STRUCTURE showed even less support for genetic differentiation among populations compared to the DAPC analysis . STRUCTURE clusters had mean coefficient of ancestry ranging from 0.17 (SD = 0.01) to 0.29 (SD = 0.08) with 28 to 121 individuals assigned to one of seven clusters. Molecular variance analysis (Excoffier and Lischer 4 or C4), or by collection site/year, indicated that the greatest molecular variance (>87%) occurred in individuals within the total population. Genetic variation among populations was minimal and not significant (Table S3).Results of the DAPC analysis using prior assignment of putative populations Fig.  revealed Jombart . This adH. zea sampled suggest that peripheral, northern populations are panmictic with asymmetrical immigration likely dominated by immigrants from southern populations occurring throughout the growing season. That there was no temporal or spatial relationships associated with the putative population clusters suggest immigrants are highly admixed possibly originating from multiple source populations. This tendency is potentially reflected in high FIS estimates due to capturing of multiple immigrating populations with differing allelic frequencies per trapping event. The continuous mass pooling and asymmetrical dispersal of H. zea is likely influenced by variation in emigration from different southern population generation cycles, which are known to be locally influenced by abiotic  and biotic  factors, and occur more frequently in southern (5 to 7 generations/year) than northern (1 to 2 generations/year) regions  suggest that temporal and spatial genetic differentiation may exist in Australia  to winter peripheral environments, low number (1 to 2) of generations occurring at peripheral sites, high reproductive rate, and rapid mass dispersal behavior , another long distance migratory lepidopteran species, does utilize southerly airflows to return migrate to the Gulf Coast of the USA from the northern USA each autumn  phenotypes  and low genetic differentiation observed. This process could have direct implications for management practices and future assessment of H. zea ecology at range limits likely to be affected as climate change is expected to extend species ranges northward due to increased mean annual temperatures.In summary, this study revealed that peripheral northern populations of Microsatellite genotype data and pertinent metadata used in this manuscript have been included in the supplementary Data file S01. Accession numbers of previously published microsatellite loci used in this analysis are listed in Table None declared.Figure S1. Simulation saturation for the allelic richness.Figure S2. DAPC results using no prior assignment of putative population showing the first two axes of the analysis (depicted in the insert plot).Figure S3. Outcome from STRUCTURE analysis assuming K = 7. Each vertical bar represents a unique individual (x‐axis) with their corresponding assignment score (y‐axis) for inclusion in a particular potential population (color).Click here for additional data file.Table S1. Pairwise FST estimates (upper triangle) for putative population pairs.Table S2. Summary of population level genetic parameters for the non‐prior DAPC analysis; the non‐prior populations cluster (Pop), number of individuals genotyped (N), mean number of alleles/locus (A), observed heterozygosity (Hobs), expected heterozygosity (Hexp), FST and FIS with its 95% confidence interval (FISCI).Table S3. The results of AMOVA of Helicoverpa zea populations grouped by collection time to calculate ΦST (between populations when there was only one group), ΦCT (among groups), or ΦSC (among populations within groups).Click here for additional data file.Data S1. Microsatellite genotypes and collection data for Helicoverpa zea adults collected from Landisville and Rock Springs, PA. Size of each microsatellite allele (in Bp) in an individual is given in two consecutive sets of 3‐digits in the 6‐digit genotype.Click here for additional data file.


Lutzomyia umbratilis is the main vector of Leishmania guyanensis, one of the species involved in the transmission of American tegumentary leishmaniasis. In Brazil, L. umbratilis has been recorded in the Amazon region, and in the state of Pernambuco, Northeastern region, where an isolated population has been identified. This study assessed the phylogeographic structure and size and shape differences of the wing of three Brazilian populations.In South America, L. umbratilis were collected from Rio Preto da Eva , from Manacapuru (south of the Amazon River), and from the isolated population in Recife, Pernambuco state. These samples were processed to obtain sequences of the Cytochrome Oxidase I mitochondrial gene. Geometrics morphometry analysis of the right wing shape of the three populations was made using discriminate canonical analysis.Samples of Phylogenetic analysis revealed the presence of two distinct monophyletic clades: one clade comprised of the Recife and Rio Preto da Eva samples, and the other clade comprised of the Manacapuru samples. Comparing the Manacapuru population with the Recife and Rio Preto da Eva populations generated high indices of interpopulational divergence. Geometric morphometry analysis indicated two distinct groups between the studied populations. Canonical variate analysis of wing shape indicated that Rio Preto da Eva population is significantly closer to Recife population, and both populations were genetically distant from Manacapuru.L. umbratilis species complex in the North and Northeast regions.The polymorphic sites and geometric morphometry analysis indicate that the distance, lack of continuity and environmental differences have not modified the ancestral relationship between Recife and Rio Preto da Eva populations. The genetic and morphological similarities shared by the Recife and Rio Preto da Eva populations suggest that these populations are more closely related evolutionarily. These results confirm the existence of an  Lutzomyia umbratilis (Diptera: Psychodidae) is the main vector of Leishmania guyanensis, one of the pathogenic agents of American tegumentary leishmaniasis (ATL) [L. umbratilis is widely distributed in the Amazon basin and there is an isolated population in remnants of the Brazilian Atlantic rain forest in the state of Pernambuco, Northeastern Brazil [L. umbratilis naturally infected with Leishmania guyanensis has been recorded on the east side of the Negro River and the north side of the Amazon River, but not on the south side [L. umbratilis is recognized as the most significant vector [is (ATL) –3. This is (ATL) , 5. In Bn Brazil –6. Of thn Brazil . On the uth side . In the t vector , 10.L. umbratilis populations of the North and Northeast Regions. The low dispersal capacity and geographic isolation of sand flies may result in population structuring, which is amplified by the presence of abiotic barriers [L. umbratilis populations of the North and Northeast Regions are separated by distance and abiotic barriers significant enough to suggest that these individuals may have evolved into genetically differentiated populations.Considerable geographic distance separates the barriers –13. The e.g. fecundity, fertility, and duration of larval development) and genetic differences between L. umbratilis populations on the north and south sides of the Amazon River, suggest that these populations might represent a species complex comprised of at least two incipient species [L. umbratilis from the states of Pernambuco and Amazonas.Bionomical  (2°50’50”S/59°56’28”W), from Manacapuru (south of the Amazon River) (3°12’41”S/60°26’20) municipalities, located in the Amazonas State, North Region of Brazil, and in the Atlantic Forest Ecological Reserve of Dois Irmãos (8°03’14”S/34°52’52”W) in Recife Municipality, Pernambuco State, Northeast Region of Brazil.Adult specimens were collected by suction in tree trunks using CDC light traps. The samples were conserved in 95 % alcohol, at -20 °C. The genitalia of the individuals were slide mounted and stored as a voucher, and the sand flies were identified using the keys of Young and Duncan .L. umbratilis were used in the experiments geometric morphometry and molecular biology. For molecular analysis, 129 specimens of L. umbratilis were used: 36 from Manacapuru ; 38 from Rio Preto da Eva ; and 55 from Recife .In all, 201 specimens of et al. [et al. [Genomic DNA extraction was carried out using Chelex®100 , according to Lima Costa-Junior et al. . A fragm [et al. . Amplifi [et al. . All newLutzomyia longipalpis, Lutzomyia migonei and Phlebotomus papatasi were used as the outgroup.Nucleotide sequences were aligned using Muscle , incorpoYulebirth process were used. The tree with the higher clade Bayesian Posterior Probabilities (BPP) was obtained with TreeAnnotator v. 1.7.5 (available in BEAST package), using the meanhights option and 10,000 trees as burnin to assure stationarity.The time at which the two main clades diverged (into the Manacapuru and Rio Preto/Recife isolates) was estimated with a relaxed molecular clock using BEAST v. 1.7.5 . The anahttp://weblogo.berkeley.edu/logo.cgi).Intra-population genetic diversity was measured by the haplotype and nucleotide diversity, K value (number of genetic groups), number of polymorphic sites, and number of transitions and transversions using DnaSP v. 4.0  and ArleFs statistics, and the Mismatch distribution were performed using Arlequin v. 3.5 [SSD) were used in order to validate the model of recent expansion obtained using the Mismatch distribution. Genetic differentiation was assessed with the pairwise fixation index Fst using Arlequin v. 3.5 [Tajima’s D neutrality test, Fu’s n v. 3.5 . The Ragn v. 3.5 .Dxy), the total number of substitutions per site among populations (Da), the number of shared polymorphisms among populations (Ss), and the number of fixed differences among populations (Sf) were calculated using DnaSP v. 4.0 [The average number of substitutions per site among populations (P v. 4.0 .www.fluxus-engineering.com) using the Median-joining method [The haplotype network was created with NETWORK v. 4.6 , Manacapuru (n = 27) and Recife (n = 27) were dissected with forceps and mounted between microscope slide and cover slide using Berlese fluid. Images of each wing were obtained at 4× magnification using a JVC KY-F55 digital camera coupled to a Leica DM 1000 optical microscope.The right wings of adult females of tpsRelW v. 1.46. MOGwin v. 0.82 was used to analyze the shape variables by the generalized Procrustes analysis superimposition algorithm (GPA) [A total of eight ‘type I’ landmarks (LM) were identified Fig. , all in hm (GPA) .Fig. 1PhThe GPA algorithm was used to extract the centroid size; allowing size and shape to be analyzed separately. Size variations were analyzed using the Kruskal-Wallis test. The effect of allometry was analyzed by regression analysis using the coordinates of the landmarks and the centroid size for the three populations. Discriminant analysis of the canonical variables was performed to compare the shape with the overall mean wing size of each population, and the Mahalanobis distances were calculated using 10.000 permutations. To the morphometric statistical analyses we used the softwares TpsUtil 1.60, TpsDig2 v 2.18 (QSC - James Rohlf), TpsRelw 1.54 (QSC - James Rohlf), Tet, MorphoJ 1.06 and Past Program  v. 2.00.L. umbratilis were analyzed. Across the region of sequence analyzed (597 bp), 66 (12.2 %) polymorphic sites observed; these were comprised of 42 (~63.6 %) parsimony-informative sites and 24 (~36.4 %) singletons. Among the polymorphic sites, 84.9 % of the nucleotide substitutions were transitions and 15.1 % were transversions. Analysis of the polymorphic sites identified 13 fixed single nucleotide polymorphisms (SNP) within the 597 bp fragment of COI used in our analyses  for all populations, which shows deviation from the neutrality model. This result could be explained by the high number of rare haplotypes in these populations, which would reinforce the possibility of recent expansion or positive selection. Fu’s Fs was also negative and significant (P < 0.01) for all populations, which confirms the hypothesis of recent expansion (Table Tajima’s on Table .Table 3Nr) did not reject the null hypothesis of recent demographic expansion. The sum of square deviation test (SSD) was not significant. The haplotype fixation index (Fst) was significant (0.89677 – 0.89357) when comparing the Rio Preto da Eva and Recife populations with the Manacapuru population , but not from the Recife population . Also, no significant difference in the mean centroid size was observed between Recife and Manacapuru populations . The allometric effect was 17.18 % (p < 0.0001), and it was removed from the shape analysis.Differences in wing size were observed among the three populations studied. The median centroid size of Rio Preto da Eva specimens was larger than specimens from Manacapuru and Recife Fig. . The meap < 0.0028 in all comparisons using 10.000 permutations).The canonical variate analysis for the wing shape showed that individuals were clustered into distinct groups in the morphospace according to each population Fig. . ManacapL. umbratilis: Manacapuru (Clade I), and Rio Preto da Eva and Recife (Clade II). Naturally infected populations of L. umbratilis have been identified in Rio Preto da Eva [L. guyanensis could be capable of infecting the Recife population. This highlights the necessity of studies that seek to ascertain the vector capacity of the Recife population of L. umbratilis.Analysis indicated the presence of two distinct clades in o da Eva . The facCholoepus didactylus) in the Atlantic forest could act as a limiting factor in the biological cycle of L. guyanensis. Due to the absence of records of this etiological agent in the state of Pernambuco, L. umbratilis has not been incriminated as vectors of ATL despite the high number of cases [The lack of a main natural reservoir  and Manacapuru (south of the Amazon River) populations in this study could be related to changes in the course of the Amazon River . The anao Haffer , separato Haffer , 15. Thio Haffer .L. umbratilis populations from the central Amazonian, the populations from Rio Preto da Eva (north of the Amazon River) and Recife (Northeast region) remained possibly linked by Amazon and Atlantic forests until the last glacial period. The presence of a clade formed by Rio Preto da Eva and Recife individuals suggests that these populations diverged more recent, possibly during Pleistocene, as occurred in Lutzomyia whitmani [et al. [L. whitmani populations of North and Northeast regions suggesting that there was a continuum of intercrossing between the Amazon and Atlantic forests. This data is supported by our geometric morphometry analysis of wings. These results were unexpected because of the distance, lack of continuity and environmental differences between Amazon and Atlantic forest. The occurrence of several Amazon species of sand flies in Pernambuco reinforce the idea of a continuum between the Amazon and Atlantic forest [During this long period of separation among whitmani . Ready e [et al.  assessedc forest , 37. Howc forest .L. whitmani and L. longipalpis species [Fluctuations of climate and vegetation could have modified the distribution of tropical forests during the Cenozoic, resulting in the retraction and expansion of Amazon vegetation . This pr species , 40.L. umbratilis complex, phylogenetic analysis showed that the COI marker used had a high discriminatory capacity; thereby demonstrating the presence of two clades within the Central Amazon and Northeast populations. This gene was used previously to study the L. longipalpis, L. umbratilis, Anopheles albitarsis and Triatoma brasiliensis complexes with considerable efficiency [L. umbratilis.In the taxonomic analysis of the ficiency , [41, 42ficiency –45. The The divergence time estimate indicates that the two clades diverged approximately 0.89 Mya (0.79 – 1.68 Mya), during the Pleistocene. Scarpassa and Alencar  obtainedL. umbratilis inhabiting Manacapuru and Recife/Rio Preto da Eva populations. Similar studies using GM and molecular analysis using Lutzomyia shannoni individuals showed divergence between Mexico and USA populations [As with molecular data, GM analysis indicated two distinct groups between the studied populations, with some homogeneity among them. Also, the canonical variate analysis of wing shape indicated that Rio Preto da Eva population is significantly closer to Recife population, and otherwise Manacapuru and Recife populations were more distant. These results indicate divergence between ulations , 47.L. umbratilis population found in the Atlantic Forest remnant in the state of Pernambuco, Northeastern Brazil.The markers used in this study (phenotypic and genotypic) allowed the two clades to be differentiated: Manacapuru (Clade I), and Rio Preto da Eva and Recife (Clade II). The genetic similarities shared by the Recife and Rio Preto da Eva populations suggest that Recife individuals could have a vector capacity similar to individuals found north of the Amazon River. However, we cannot be certain that the Recife population possesses vector competence for the transmission of ATL etiological agents. Our evolutionary analysis highlights the necessity for novel studies and for constant surveillance of the Ethical approval was not required for the current study.


Panthera leo) from five main areas in Zambia has uncovered haplotypes which link Southern Africa with East Africa. Phylogenetic analysis suggests Zambia may serve as a bridge connecting the lion populations in southern Africa to eastern Africa, supporting earlier hypotheses that eastern-southern Africa may represent the evolutionary cradle for the species. Overall gene diversity throughout the Zambian lion population was 0.7319 +/- 0.0174 with eight haplotypes found; three haplotypes previously described and the remaining five novel. The addition of these five novel haplotypes, so far only found within Zambia, nearly doubles the number of haplotypes previously reported for any given geographic location of wild lions. However, based on an AMOVA analysis of these haplotypes, there is little to no matrilineal gene flow (Fst = 0.47) when the eastern and western regions of Zambia are considered as two regional sub-populations. Crossover haplotypes  appear in both populations as rare in one but common in the other. This pattern is a possible result of the lion mating system in which predominately males disperse, as all individuals with crossover haplotypes were male. The determination and characterization of lion sub-populations, such as done in this study for Zambia, represent a higher-resolution of knowledge regarding both the genetic health and connectivity of lion populations, which can serve to inform conservation and management of this iconic species.Analysis of DNA sequence diversity at the 12S to 16S mitochondrial genes of 165 African lions ( Panthera leo) is broadly but irregularly distributed across approximately 167,000 km2 of managed habitats comprised of national parks (NP) and game management areas (GMAs). Recent estimates propose the total number of wild lions in Zambia to be between 1000 to 2000 individuals  and a western [Kafue (KF) and Sioma Ngwezi (SI)] sub-population , resulteulations . Within Eight haplotypes were found; three haplotypes  described by Antunes et al.  and fiveposition 1801). The nucleotide differences by position and the number of base pair differences among haplotypes are shown in Z1, found countrywide, differs from H10 by only one base pair. H10 is a haplotype seen only in Kruger National Park and is found with H2  and H11 . Haplotype Z2, which only appeared once, was verified through re-sequencing. Haplotype Z2 differs from haplotype Z1 by only one polymorphic site. This polymorphic site is also the only transversion, an adenine (purine) to a thymine (pyrimidine) substitution, seen at any polymorphic site between all haplotypes (as shown in Average number of pairwise differences and Nei’s distance (d) are shown in Panthera leo–Asia/Central/Northern Africa, East Africa, Southern Africa and Southern/East Africa. The Southern/East Africa group consists of two branches, one containing south central Africa (Botswana and Zambia) and the other eastern Africa from South Africa northwards to Kenya. The same regional clusters could be found in the haplotype network  while Fst calculated within a regional sub-population was 0.05. Values of Fst greater than 0.25 suggest there is a high level of genetic differentiation between populations; a result of low gene flow [st and distance values between the eastern and western sub-populations coupled with low Fst and distance values between areas within sub-populations suggests there to be little to no matrilineal gene flow between the eastern and western sub-populations while there is considerable movement within the eastern and western sub-populations.While gene diversity countrywide was high, matrilineal gene flow between regional sub-populations appeared to be low. Fene flow . Furtherene flow . When coPhylogenetic analysis is consistent with previous studies that postulate eastern-southern Africa as being the evolutionary cradle of the lion –29, suppAnalysis of mtDNA data indicates minimal gene flow between Zambia’s two sub-populations; however, because it only establishes matrilineal distribution, whether the two sub-populations have historically experienced greater gene flow through higher levels of dispersal or if geographic separation has always inhibited lion movements between the eastern and western regions is unknown. Limited dispersal may still occur but not at a rate sufficient enough to maintain or increase the frequency of crossover haplotypes (i.e. H9 in LV and H11 in KF). All individuals with crossover haplotypes were male, a pattern consistent with a genetic population structure of high male dispersal and low female dispersal . In AfriThe most widely dispersed haplotype was H11 . PrimariWith translocation becoming a well‐practiced technique to prevent inbreeding within populations closed to dispersal or immigration , it mustFurther research including the addition of microsatellite analysis is being done to better quantify the level of overall genetic diversity within the population. The combination of mtDNA with nuclear markers will give a clearer picture to examine population-wide gene flow, identify evolutionarily distinct populations and calculate effective population size.anthera leo)[Panthera leo ssp. leo [Panthera leo persica [Panthera leo ssp. persica [The findings of this study coincide with range-wide studies that propose lions are structured by region due to a lack of widespread movement of lions , 13, 34.hera leo)–36 or sussp. leo ) level,  persica , 38, Pan persica –44. In W persica –40. The S1 Appendix(DOCX)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Linguistic, cultural and genetic characteristics of the Malagasy suggest that both Africans and Island Southeast Asians were involved in the colonization of Madagascar. Populations from the Indonesian archipelago played an especially important role because linguistic evidence suggests that the Malagasy language branches from the Southeast Barito language family of southern Borneo, Indonesia, with the closest language spoken today by the Ma’anyan. To test for a genetic link between Malagasy and these linguistically related Indonesian populations, we studied the Ma’anyan and other Indonesian ethnic groups (including the sea nomad Bajo) that, from their historical and linguistic contexts, may be modern descendants of the populations that helped enact the settlement of Madagascar.A combination of phylogeographic analysis of genetic distances, haplotype comparisons and inference of parental populations by linear optimization, using both maternal and paternal DNA lineages, suggests that Malagasy derive from multiple regional sources in Indonesia, with a focus on eastern Borneo, southern Sulawesi and the Lesser Sunda islands.Settlement may have been mediated by ancient sea nomad movements because the linguistically closest population, Ma’anyan, has only subtle genetic connections to Malagasy, whereas genetic links with other sea nomads are more strongly supported. Our data hint at a more complex scenario for the Indonesian settlement of Madagascar than has previously been recognized.The online version of this article (doi:10.1186/s12864-015-1394-7) contains supplementary material, which is available to authorized users. Prior to the European colonial period, Austronesian-speaking populations were the most widespread of any language family ,2. WhileDespite considerable research on the eastward Austronesian expansion, there is little equivalent research on the western edge, leaving major issues unresolved regarding the settlement of Madagascar. Although the exact nature and route of this movement is largely unknown, linguistic and anthropological evidence indicates strong Indonesian influences, as recorded in the vocabulary and socio-cultural life of Malagasy, the modern people of Madagascar -9. LinguThese linguistic findings are broadly supported by genetic studies, which emphasize the shared Indonesian and African genetic heritage of Malagasy. A recent study of genome-wide SNP data suggests that the western and central regions of Indonesia (Java/Borneo/Sulawesi) have the closest genetic connections with Malagasy . This isThe westward Austronesian expansion was likely associated with trading activities of the Srivijaya empire, as suggested by the many Malay loanwords present in Malagasy ,27,28, aSimilarly, whether Barito populations such as the Ma’anyan, the closest linguistic siblings to modern Malagasy, share close genetic lineages with the Malagasy also remains unanswered. For the first time, we report genetic data for the Ma’anyan and the Lebbo’ (a population from Borneo with no presumed role in the settlement of Madagascar) to determine whether the Ma’anyan have an especially close genetic connection with Malagasy. We also include Bajo sea nomads from Sulawesi to determine whether there is a common genetic link based on their shared involvement in long distance maritime trading networks. A large data set of published and unpublished Indonesian populations is included for comparative analysis ,39. To iBased on analysis of 96 Y chromosome binary markers   are from regions near Wallace’s line in the west and south of the Sulawesi sea . Populations with highest affinity to Malagasy are Mandar (Sulawesi), Flores (Lesser Sunda), Bajo (Sulawesi), and east Kalimantan Dayak and Lebbo’ (Borneo) , despite being the only Indonesian population that shares Y haplogroup O2a1a with Malagasy. This may indicate that the genetic contribution of Ma’anyan was limited, either due to the recent arrival of this lineage in Ma’anyan, or perhaps O2a1a has since been lost or is still undetected in other Indonesian populations.These geographical regions comprised part of the trading sphere of the Srivijaya empire, including several Javanese kingdoms that played a crucial role in the region: Heluodan (5th century), Tarumanagara (5th century), Walaing , Kahuripan/Kediri (11th century), Singasari (13th century) and Majapahit (13th -15th centuries) . This reMalagasy and Indonesians share mitochondrial haplogroups B4a1a, B4a1a1 (Polynesian motif), E1a1a, F3b, M7c1a4a, M32c and Q1  is considered strong evidence of Indonesian gene flow into Madagascar, where a variant is found at moderate frequency (11-50%). With the exception of Bali  include Sumba and Flores, the Maluku islands and the Bugis of south Sulawesi. As seen on the Y chromosome, the Bajo cluster with eastern Indonesian groups, while the Ma’anyan and other populations from Borneo cluster with western Indonesian groups. Linear optimization results broadly agree with the FST results: Malagasy are most likely derived from a combination of eastern Indonesian groups, such as North Maluku, Bugis and Bajo  the Ma’anyan groups sampled here are not directly related to the individuals who settled Madagascar, 2) subsequent demographic events have erased any genetic affinity between them, or 3) the Ma’anyan were just one population of many that contributed to the settlement of Madagascar (a possibility suggested by the exclusive sharing of Y chromosome haplogroup O2a1a). The first hypothesis might suggest that other Southeast Barito groups from southern Borneo (such as the Samihim or the Dusun Witu ) were inThese conclusions are drawn from a suite of complementary analyses, including phylogeography, haplotype sharing and linear optimization approaches. In combination, they paint a picture of the genetic dynamics between Indonesia and Madagascar. Although the geographic distribution of Indonesian populations that most closely reflect Malagasy genetic diversity are remarkably convergent for both male and female lineages, it is noticeable that the regions suggested by these two systems do not overlap perfectly. Our analyses suggest that populations from the south and west of the Sulawesi Sea  best explain Y chromosome diversity, while populations from the south and east of Sulawesi , all in eastern Indonesia, best explain mtDNA diversity. We emphasize that parts of these two regions overlap, thus potentially explaining both paternal and maternal affinity.ST and linear optimization results also highlight different source regions for the Y chromosome and mtDNA. Together, these patterns suggest that multiple regions may have contributed to the settlement of Madagascar, perhaps via one or a few admixed groups.For instance, the distribution of shared lineages favors different source populations for maternal and paternal loci. Y chromosome haplogroup O2a1a is found only in Ma’anyan, while mtDNA haplogroups B4a1a1 and Q1 are found exclusively in eastern Indonesia. . FSea nomads have been active traders along the eastern coast of Borneo, southern Sulawesi, the Lesser Sunda islands and the Maluku islands for at least the last few hundred years ,63. ThesWe propose that the settlement of Madagascar had an Indonesian source location around southern Sulawesi, the Lesser Sunda islands and eastern Borneo. The populations involved may be related to modern sea nomad groups and the ancient Malay Srivijaya trading network. The Indonesian ancestors of Malagasy certainly carried maternal lineages with greater putative contributions from eastern Indonesia, and paternal lineages from both eastern and western Indonesia. The absence of any clear genetic connection between Malagasy and at least some populations speaking their most closely related language, Ma’anyan, raises important questions about the link between genes and language in the Indonesian dispersal across the Indian Ocean. Studying other Southeast Barito and sea nomad groups  and the application of genome-wide genotyping technologies to provide additional genetic resolution promises to bring new insight to bear on these questions.ST < 0.05 and 95% of FST values non-significant (P > 0.05) for both mtDNA and the Y chromosome) [All samples analyzed in this study were collected with informed consent from unrelated individuals. Subjects were surveyed for language affiliation, current residence, familial birthplaces, and a short genealogy of four generations to establish regional ancestry. A total of 205 DNA samples were analyzed from three ethnic groups: 159 Ma’anyan individuals were collected in Tamiang Layang (East Barito), Central Kalimantan, and Banjarmasin , representing the largest ethnically-defined population sample from Borneo to date; 19 Lebbo’ in East Kalimantan; and 27 sea nomad Bajo in Kendari (Sulawesi). Collection and use of these samples was approved by the Research Ethics Commissions at both the Eijkman Institute for Molecular Biology, Indonesia, and the University of Toulouse, France. We also included data for additional Indonesian populations from published ,39,46 anomosome) ,25,24. Ihttp://dnagenotek.com). DNA was extracted from blood using a standard salting-out procedure, and from saliva using the manufacturer’s standard protocol. For paternal lineage analysis, 96 binary markers on the non-recombining region of the Y chromosome were analyzed. We used a nanofluidic dynamic array  high-throughput genotyping system. This system is developed for SNP genotyping assays and able to perform 9,216 real-time polymerase chain reactions (PCRs) (96 primers × 96 samples) on a single chip. The results were analyzed using the BioMark™ HD system  which integrated the Real-Time PCR Analysis software. Each haplogroup was assigned based on the updated ISOGG’s Y-DNA haplogroup tree [http://haplogrep.uibk.ac.at) based on Phylotree v.16 [We collected blood samples for the Ma’anyan, except the Lebbo’ and Bajo, for which saliva samples were collected using the Oragene DNA Collection kit  algorithm in the R package, limSolve [Pairwise Fin v.3.5  with 5,0limSolve . To captlimSolve -79. SharlimSolve . For thilimSolve  were exc


Rhododendron species: two sympatric populations, of which only one contained hybrids, and a further hybrid-dominated population. No fixed differences between the species were found, and only 5.8% of the markers showed some degree of species differentiation. Additionally, 45.5% of highly species-differentiating markers experienced significant transmission distortion in the hybrids, which was most pronounced in F1 hybrids, suggesting that factors conveying incompatibilities are still segregating within the species. Furthermore, the two hybrid populations showed stark contrasting composition of hybrids; one was an asymmetrically backcrossing hybrid swarm, while in the other, backcrosses were absent, thus preventing gene flow.An increasing number of studies of hybridization in recent years have revealed that complete reproductive isolation between species is frequently not finalized in more or less closely related organisms. Most of these species do, however, seem to retain their phenotypical characteristics despite the implication of gene flow, highlighting the remaining gap in our knowledge of how much of an organism’s genome is permeable to gene flow, and which factors promote or prevent hybridization. We used AFLP markers to investigate the genetic composition of three populations involving two interfertile  While many biologists still favor the biological species concept, which relies on complete reproductive isolation to delineate species, several authors highlight the problems that this approach might have with regard to understanding the underlying processes that actually drive speciation . At lower altitudes, it commonly grows to 1.5 m (T. Marczewski pers. obs.), and sometimes 3 m , leaf shape and bark; the easiest character to identify hybrids is the hair structure on the lower leaf surface , hybrids exhibited diverse morphology and were sympatric with both parents. From this site were gathered 33 putative hybrids (hyb-1), 37 R. aganniphum (aga-1), and 18 R. phaeochrysum (pha-1) accessions. Site Two was 6.4 km from Site One  and comprised a morphologically relatively uniform population of hybrids with only very few dispersed R. aganniphum individuals present; from here, 38 putative hybrids (hyb-2) were collected. Site Three was a different mountain massif, Daxue Shan , where the parent species grew sympatrically, but no hybrids were observed. From here, 33 R. aganniphum (aga-3) and 25 R. phaeochrysum (pha-3) accessions were collected.Two hybrid populations were sampled from Baima Shan mountain, Yunnan. At Site One , according to the protocol provided by the manufacturer, but using 1.5× the suggested amounts of buffers. DNA content of the extracts was assessed photometrically , and a minimum of 50 ng of total DNA was used for each digestion–ligation reaction. For generation of AFLP profiles, the method published by Vos et al.  was follTo assess error due to different reactions and scoring, all steps for the generation of the AFLP profiles, apart from the DNA extraction, were duplicated for 14 individuals. For a further two individuals (one from each species), all steps were carried out five times, so that these two individuals were present in each sequencer run performed to generate the AFLP profiles for all individuals.The error was then calculated as the mean mismatch for all possible pairwise comparisons between AFLP profiles of the same individual. Where two different individuals had a mismatch score no larger than the mean error for the above within-individual comparisons, those two individuals were flagged as being putative clones of one another.A total of 344 loci were scored. Afterward, loci for which no population showed allele frequency differences to any other population were removed as uninformative; the function prop.test in R , locality differentiating (type l), population differentiating (type p), or complex (type x).Loci that showed higher than average differentiation were assigned, based on pairwise FST values for the four parental populations  were grouped into (a) comparisons between populations of the same species and (b) comparisons between populations of different species. Loci were categorized as type s if they satisfied both of the following conditions: (1) The locus showed higher differentiation between species than average observed differentiation within species, meaning that all between-species FST values had to be above the overall mean of all within-species FST values  and (2) all between-species FST values being at least twice as high as the highest within-species comparison for the locus in question.Species-differentiating loci were those that differentiated both populations belonging to the same species from those of the other. Pairwise population FST fell above the 90% quantile, the locus was additionally classed as highly species differentiating (subtype sh).If the mean between-species FST values were divided into (a) within-locality  and (b) between-locality comparisons, irrespective of species identity. Loci were then categorized as type l if (1) the locus showed above mean differentiation, meaning in this case that all between-locality FST values had to be above the overall mean of all within-species FST values , and (2) FST values for all within-locality comparisons were lower than the smallest between-locality comparison.Locality-differentiating loci were those that differentiated the two populations at one locality from those at the other. All pairwise FST values were first normalized by dividing all comparisons by the highest observed FST value for the locus. Then, for each population, the mean of all pairwise comparisons not involving the population in question was subtracted from the mean of all comparisons involving that population. A score of 1 would indicate a marker frequency unique to the selected population that showed no differences between the other three, whereas 0 or below would indicate that this population was no more different from the others (or less if negative) than they were from each other. However, a marker that strongly differentiated the selected population but also showed differentiation among the other three would also score less than 1. Based on this, loci were categorized as population differentiating for a given population if their score for that population was above 0.67.Population-differentiating loci were those that differentiated one population from the other three. To detect these, Additional cases were detected where two populations were strongly differentiated from one another, but one or both of the remaining populations were intermediate, leading to no population having an individual score above 0.67. Such markers were categorized as complex type x; ; see supK between one and ten, and then evaluating plots of ΔK, as suggested by Evanno et al. , datasets containing inbred early- to later-generation hybrids (F1–F4) were simulated to investigate the resulting clustering in STRUCTURE. Additionally, backcross populations were simulated for comparison and to combine the simulated hybrid populations for analysis in introgress, to check for deviation of marker clines.For the simulations, allele frequency estimates from the populations aga-1 and pha-1 were used to obtain two parental populations, aga-A and pha-B, comprising 50 individuals each. We decided to use simulated parental populations representing the observed allele frequencies instead of the originally sampled individuals, as for each individual at each locus that has the dominant allele, the second allele was not known. Furthermore, the simulated parental populations would certainly conform to the assumptions (1) that the loci are independent and (2) that the population is in Hardy–Weinberg equilibrium.Genotypes for parental individuals were generated by assigning two alleles to each locus, where the probabilities of obtaining the dominant or the recessive allele were set to the estimated population frequency of the respective allele. Hence, both alleles present at each locus were known for each individual. The two parental populations simulated in this way were used for all hybrid simulations.F1 hybrid individuals were generated by randomly drawing one individual from each parental population, and then genotypes for loci were obtained by choosing randomly one allele from each parent at each locus. A hybrid population was generated by repeating this process, whereby alleles were drawn with replacement, so that individuals could contribute differently to the hybrid population.To generate an inbred F2 hybrid population, an F1 population was created as described above, and then F2s were generated by selfing this F1 population. For later-generation hybrids, this process was repeated, with the resulting hybrid population being selfed.In the case of backcross (BC) populations, a generated F1 or BC population was used as one parent, while the initial parental population (aga-A or pha-B) served as the other.Ten populations each of BC1A, BC2A, F1, F2, F3, F4, BC1B, and BC2B hybrids, all comprising 50 individuals, were generated. All were simulated independently, so that no analyzed F1 population was the parent of an F2 population and so on. However, for all simulations, the same parental populations  were used as the starting point. The size of all populations, including the intermediate-precursor populations used to generate later-generation hybrids, was set to 50 individuals.K between one and six were performed, using the settings for the original data.Each of the 80 simulated hybrid populations was combined with the parental populations , and for each resulting dataset, 16 runs for every R. phaeochrysum are present in any of the hybrid populations, the cline estimation was additionally carried out with all ten datasets, but the backcrosses to pha-B  removed.For the analysis in introgress, the hybrid populations were combined into ten datasets, so that each dataset contained one simulated hybrid population from each of the eight hybrid classes, thereby mimicking a hybrid swarm. The analysis was carried out using the same setting as for the original data. As the original data suggested that no backcrosses to The combined error introduced due to different reaction conditions and the scoring process, as estimated by the mismatch between separate AFLP profiles for the same individual, was 2.6% (SD ± 0.97), based on the 146 loci included in further analyses. Three pairs of hybrid individuals had pairwise mismatch scores ≤2.6%, indicating putative clones or shared parentage. All three putative clone pairs were individuals from the hybrid population hyb-2, and in each case, one individual from each pair was removed from the data, resulting in 177 individuals remaining in the analyzed dataset.K  and R. phaeochrysum ; however 11 individuals from hyb-1 also clustered with R. aganniphum. In addition, a few individuals in the population aga-3 and one individual in pha-3 showed introgression, as indicated by admixture with the respective other species  of Q for admixture from the R. aganniphum cluster (K = 2) were as follows: F1 0.63 (0.60–0.70); backcrosses to aga-A – BC1A 0.95 (0.93–0.97), BC2A 0.98 (0.95–0.99); backcrosses to pha-B – BC1B 0.24 (0.20–0.29), BC2B 0.05 (0.03–0.07); F2 0.55 (0.48–0.59); F3 0.55 (0.50–0.62); F4 0.53 (0.46–0.70)  .K = 2, all individuals of aga-A were assigned to one cluster, and all individuals from pha-B to the respective other cluster, with a few individuals in both populations, showing low levels of admixture . Only two loci were location-differentiating , including 11 that were highly so (type sP < 0.05) deviation from neutral expectations only using the data from hyb-1, did so in the combined dataset  and only the strictly sympatric hybrids (hyb-1) qualitatively agreed; apart from one exception all loci that were significant for hyb-1 were also significant for hyb-1 and hyb-2 combined. However, ten loci for which clines did not show a significant (h), four type p (two differentiating aga-1 and two aga-3), one a type l locus, three of type x, and seven uncategorized. With regard to all analyzed loci, the following percentage of loci in each category showed clines deviating significantly from neutral expectations: 45% of type sh loci , 50% of type l loci (one locus of two), 22% of type p loci that differentiated aga-1 and pha-1, 13% of type x, and 10% without category . This same pattern was also observed in one of the type p loci which showed exceptional differentiation between aga-1 and pha-1 . Two further loci showed significant deviation in several: locus 28 (D2_313) in six of the ten simulated datasets and locus 59 (D3_259) in all ten. Apart from locus 46, all of these loci fall into category x or into no category, and in no case was a locus significant that was significant for the original data.The two hybrid zones show very divergent patterns of composition; one potentially allows unidirectional gene flow, while the other presents a barrier. While most of the genome is unaffected by incompatibilities, nearly half of species-differentiating markers show transmission ratio distortion in hybrids. Transmission ratio distortion is already visible in F1 individuals; therefore, most incompatibility factors are likely to be still segregating in the species.Q were significantly biased toward the R. aganniphum cluster; this was apparent in the F1s as well as the backcrosses to R. aganniphum , the bias being mostly visible in the F1 generation (mean of 0.45), but not being significant in backcrosses. Therefore, the cline analyses should not be affected significantly by this pattern.When analyzed, the sympatric population (hyb-1) and the parapatric population (hyb-2) showed different patterns of hybridization, which agreed with initial morphological observations.Rhododendron  contributed to the observed pattern. Due to the nature of the data, it is not possible to distinguish between a scenario of mostly F1 hybrids or a swarm of inbred later-generation hybrids. F1 hybrids could show particular allele frequencies due to strong transmission distortion, which could, for example, be caused by lethality of certain allele combinations at loci that are polymorphic in the parents, or ecological selection eliminating F1 hybrid offspring that lack the right combination of alleles. On the other hand, the apparent scarcity of individuals from either parental species in the vicinity of hyb-2 could have led to the establishment of an inbreeding hybrid swarm comprising later-generation hybrids. Although this distinction is not possible, the data give strong evidence that backcrosses to either parent are absent from hyb-2, suggesting that this hybrid zone does not contribute to gene flow between R. aganniphum and R. phaeochrysum.The isolated hybrid population hyb-2, which was characterized by uniform morphology, showed a hybrid index between 0.4 and 0.5 , which, in hyb-2 . TherefoR. aganniphum cluster . Pollen competition might contribute, but on its own should allow a few backcross events toward R. phaeochrysum, as mostly paternity bias would be expected . This is a pattern that has been previously reported in other closely related hybridizing species such as Silene  deviate significantly from expectations  and one locality-differentiating locus (type l) showed clines significantly deviating from neutral expectations. Two of the significant type p loci (29 and 36) differentiated aga-3 from the other populations, while aga-1 and pha-1 showed no differentiation. As there is no information about how these loci would behave in hybrids with aga-3 as one parent, it seems unreasonable to make inferences of what caused the significant deviation. For the other two type p loci (32 and 43), the population aga-1 was differentiated from the other populations and hence the sympatric parents (aga-1 and pha-1). The clines of both of these loci showed a pattern similar to the significant type s loci  and H, iFST patterns for the only significant type l locus (22) showed very high differentiation between both localities (Baima Shan, aga-1 and pha-1, and Daxue Shan aga-3 and pha-3 (TableThe a-3 Table. This waa-3 Table. As the R. aganniphum was prevalent, thereby allowing unidirectional gene flow from R. phaeochrysum to R. aganniphum. Contrastingly hyb-2 comprised either F1s, later-generation hybrids, or a mixture of both, but no backcrossed individuals to either of the parental species, hence not providing a bridge for gene flow. Admixture of some individuals in the populations aga-3 and pha-3 suggested that hybrids were once present at Daxue Shan, but are absent in the current population. A large proportion of the genome did not appear to be affected by incompatibilities, and up to 98% of loci are potentially not protected from introgression. In contrast, nearly half of the markers that showed high species differentiation seemed to be linked to a locus causing transmission ratio distortion in early-generation hybrids. Therefore, most factors conveying incompatibilities are likely to be still segregating within species. On the other hand, over half of species-differentiating markers did not show unusual cline patterns in the hybrid population, indicating that the species have accumulated neutral mutations in parts of the genome that are not affected by loci causing incompatibilities.The two analyzed hybrid populations showed a very different composition of admixed individuals. In the morphologically diverse hyb-1, entirely asymmetric backcrossing to Overall, observed patterns conformed to predictions made by the genic species concept, as parts of the genome seemed to be affected by incompatibilities affecting gene flow between species, while other parts do not show such patterns. A strong population-specific genetic component influencing cross-compatibility was not implied by the data, as it seemed more likely that population-differentiating loci experiencing transmission ratio distortion are located within areas of the genome generally affected by incompatibilities. Furthermore, after correcting for experimental artifacts, there seemed to be no clear evidence for preferential introgression of any population-specific marker. The absence of a clear population-specific genetic component influencing hybridization suggests that environmental conditions play a major role in influencing the occurrence of hybrids as well as hybrid swarm composition. Incompatibilities seemed to be affecting the generation of F1 hybrids most; hence, it is plausible that they are only formed if environmental conditions tip the balance in their favor. This might only happen sporadically, but as both species are long-lived, the hybrids would remain present in the population for a long time. This could explain the complete absence of hybrids at Daxue Shan, which seemed ecologically similar to Baima Shan; the conditions could have been different when most of the initial hybrids where formed.


Salvia officinalis L., Lamiaceae) is a well-known aromatic and medicinal Mediterranean plant that is native in coastal regions of the western Balkan and southern Apennine Peninsulas and is commonly cultivated worldwide. It is widely used in the food, pharmaceutical and cosmetic industries. Knowledge of its genetic diversity and spatiotemporal patterns is important for plant breeding programmes and conservation. We used eight microsatellite markers to investigate evolutionary history of indigenous populations as well as genetic diversity and structure within and among indigenous and cultivated/naturalised populations distributed across the Balkan Peninsula. The results showed a clear separation between the indigenous and cultivated/naturalised groups, with the cultivated material originating from one restricted geographical area. Most of the genetic diversity in both groups was attributable to differences among individuals within populations, although spatial genetic analysis of indigenous populations indicated the existence of isolation by distance. Geographical structuring of indigenous populations was found using clustering analysis, with three sub-clusters of indigenous populations. The highest level of gene diversity and the greatest number of private alleles were found in the central part of the eastern Adriatic coast, while decreases in gene diversity and number of private alleles were evident towards the northwestern Adriatic coast and southern and eastern regions of the Balkan Peninsula. The results of Ecological Niche Modelling during Last Glacial Maximum and Approximate Bayesian Computation suggested two plausible evolutionary trajectories: 1) the species survived in the glacial refugium in southern Adriatic coastal region with subsequent colonization events towards northern, eastern and southern Balkan Peninsula; 2) species survived in several refugia exhibiting concurrent divergence into three genetic groups. The insight into genetic diversity and structure also provide the baseline data for conservation of S. officinalis genetic resources valuable for future breeding programmes.Dalmatian sage ( For thousands of years, people have gathered plant and animal resources for their needs, resulting in changes to genetic structure of populations over the course of cultivation and domestication. This process is particularly manifested in crop species used for food , but is The reductions of gene diversity in domesticated plants vary across species and have usually been examined in crop plants such as soybean , maize  and wheaSalvia officinalis L.) is an outcrossing, insect-pollinated, perennial subshrubby plant of the family Lamiaceae. The genus Salvia is one of the largest genera in the family, with nearly 1,000 species distributed worldwide  and compare it to the original values using Wilcoxon Two-Sample Test in SAS. Pairwise FST values were also estimated after correcting for the presence of null alleles using ENA correction method  as implemented in FreeNA and compared to original values (as above).We used the program MICRO-CHECKER  to checkNar, was calculated as the number of alleles per locus independent of sample size using FSTAT ver. 2.9.3.2 , for each value of K  corrected for the presence of null alleles were only slightly higher than the original values (Tab. HE and HE(null) in any population (PWilcoxon = 0.22–0.50) suggesting that null alleles did not have substantial impact on the results.Indigenous populations exhibited significantly higher values of both observed  with M-ratios (0.493–0.552) far below the critical value of 0.68. Moreover, the analysis of southernmost wild populations from Macedonia  and Greece (P21) also yielded the M-ratios below but close to the critical value (0.550–0.668). Finally, the M-ratios slightly lower than the critical value were also observed in three Croatian populations  ranging from 0.673 to 0.676.The heterozygosity-excess method implemented in BOTTLENECK (Wilcoxon signed rank test assuming a two-phased model) identified significant bottleneck events only in two cultivated populations from Romania  except between two neighbouring indigenous populations from Slovenia (P01/P02), between two neighbouring indigenous populations, one from Croatia (P10) and the other from Bosnia and Herzegovina (P14), and between two neighbouring naturalised populations from Kosovo (P23/P24). The lowest FST value was observed between two indigenous populations from Slovenia , while the highest value was found between two populations from Serbia . Global FST values without correction for null alleles vs. FST values using ENA [FST(null)] were 0.153  and 0.149 (CI = 0.136–0.163), respectively (FST and FST(null)(PWilcoxon = 0.257), suggesting that the presence of putative null alleles did not affect this analysis.All pairwise ectively . No signThe unrooted Fitch-Margoliash tree based on Cavalli-Sforza's chord distance between 30 Dalmatian sage populations is shown in ϕ-value among populations suggested the existence of population differentiation. Two-way AMOVA revealed that only a minority of variations in the genetic diversity was explained by differences between groups of populations  < 0.0001), thus confirming the structuring of populations according to cultivation status. Excluding the seven cultivated/naturalised populations, the among-population component of genetic diversity was 10.80% , suggesting further geographic structuring of indigenous populations.The AMOVA showed that most of the total genetic diversity was attributable to differences between individuals within populations (84.73%) . Howeverln[Pr(X|K)], increased with increased K, as did the standard deviations among different runs for each K. The highest ΔK value was observed for K = 2 (3183.82), followed by that for K = 4 (4.10) (ln[Pr(X|K)]. At K = 2, all the individuals belonging to indigenous populations were assigned to a separate cluster (cluster A) from individuals belonging to cultivated/naturalised populations (cluster B). At K = 4, three subclusters of indigenous populations appeared, thus confirming the geographical structuring of wild populations. Subcluster A1 was predominant in the north-west part of the Adriatic coast and included Slovenian populations (P01 and P02) and five out of eight Croatian populations (P03-P07), as well as a population from Montenegro (P15). Subcluster A2 was found primarily in the southern part of the Adriatic coast and included the most southern Croatian populations (P07-P09), all the populations from Bosnia and Herzegovina (P11-P14), a population from Montenegro (P16) and two Albanian populations (P17 and P18). The indigenous population from Serbia (P26) clearly belongs to the same cluster. The majority of individuals belonging to Macedonian (P19 and P20) and Greek (P21 and P22) populations were assigned to subcluster A3.The results of the STRUCTURE analysis are shown in 4 (4.10) . The aveFST/(1-FST) ratios] and geographical [ln(km)] distances was significant , suggesting that 22.50% of the variance could be explained by isolation-by-distance : BIO1 , BIO3 , BIO7 , BIO8 , BIO9 , BIO14  and BIO19 . Logistic threshold was set according to ‘‘maximum training sensitivity plus specificity” at the value of 0.262. Model selection chose the model with regularization multipliers value 5 as the best model. All resulting models are shown in l of LGM . On the l of LGM .t generation ago. The median values of effective population size of Pop1, Pop2, Pop3, and the ancestral populations were N1 = 4,330, N2 = 7,190, N3 = 2,440, and NA = 8,550, respectively (Na) and mean expected heterozygosity (HE) for each populations as well as Na, HE, FST, mean individual assignment likelihoods and shared-allele distance (DAS) between pairs of populations] had tail-area probability values  lower than 0.05  estimated for scenario 5  was higher than those for scenario 1 (0.166), 2 (0.193), 3 (0.075) and 4 (0.126) with 95% confidence intervals never overlapping those of the other scenarios . Scenariectively . Estimathan 0.05  indicatihan 0.05  providedS. officinalis using eight microsatellite markers revealed a high degree of genetic diversity in 23 indigenous populations and significantly lower genetic diversity in seven cultivated/naturalised populations; the two groups on the Balkan Peninsula could clearly be separated according to their origin. The pattern of genetic diversity between wild and cultivated populations of S. officinalis observed herein was consistent with other domesticated species [S. officinalis populations originated from the same geographic area, i.e., from the northernmost wild populations of the Balkan Peninsula. Such a uniform geographical origin is suggestive of planned human-mediated movement of seeds and cuttings that probably took place quite recently. Salvia officinalis is known to have been cultivated on the Balkan Peninsula since Roman times [ϕST values relative to indigenous populations Click here for additional data file.S2 AppendixMedian values and 95% confidence intervals are given for each parameter. Pop1 represents the Northwestern cluster, Pop2 the Southern cluster and Pop3 Macedonian/Greek cluster.(PDF)Click here for additional data file.S3 Appendix(PDF)Click here for additional data file.S4 Appendix(PDF)Click here for additional data file.S5 Appendix(XLS)Click here for additional data file.S6 Appendix(PDF)Click here for additional data file.S7 Appendix(PDF)Click here for additional data file.S8 Appendix(PDF)Click here for additional data file.