Salvia species served as a study model. In the widespread S. officinalis no population genetic disturbances were detected, in the endemic S. brachyodon evidence for clonality and a genetic bottleneck were found while the results of the S. fruticosa population indicated high inbreeding levels and hybridization with S. officinalis. As many findings regarding demography of individual population or species can be reached only through their comparison with closely related taxa, this study demonstrates the importance of the multi-species approach.To understand the historical circumstances that shape populations of sympatric and closely related taxa, microsatellite markers were used, while populations of three  Salvia species  served as a study model. In the populations of widespread S. officinalis, located in the central parts of this species' distribution area, no population genetic disturbances were detected. The narrow endemic S. brachyodon showed heterozygote excess, clonal reproduction and a genetic bottleneck. Because the genetic bottleneck was likely caused by the disappearance of suitable open-type habitats, the recent wildfire that cleared the terrain probably saved the S. brachyodon population from gradual deterioration and extinction. At the same time, clonal reproduction could serve as a valuable mechanism in the preservation of genetic variability. The results of the disjunct S. fruticosa population indicated heterozygote deficiency, inbreeding, hybridization with S. officinalis and population expansion. The hybridization with S. officinalis along with the abandonment of the agro-pastoral system are likely the main drivers of the strong expansion of S. fruticosa in the studied location. As many relevant findings and conclusions regarding historical and contemporary demography of individual populations or species can be reached only through their comparison with closely related taxa, this study demonstrates the importance and advantages of such a multi-species approach.Gene flow, natural selection and genetic drift are processes that play a major role in shaping the genetic structure of natural populations. In addition, genetic structures of individual populations are strongly correlated with their geographical position within the species distribution area. The highest levels of genetic variation are usually found in the centre of a species' distribution and tend to decrease beyond that point. Additionally, narrowly endemic taxa are expected to be characterized by lower levels of genetic variation than their widespread congeners. To understand the historical circumstances that shape populations of sympatric and closely related taxa, microsatellite markers were used, while populations of the three closely related and sympatric Mediterranean  There are three processes that are believed to play major roles in shaping the genetic structure of natural populations: gene flow, natural selection and genetic drift .Differences and contrasting patterns in genetic structures across core, edge and disjunct populations are the source of never ending debates and research   . Because this population is located ∼500 km from the nearest population of the same species in southern parts of the Republic of Albania, its origin is unclear, and it is uncertain whether this population is indigenous or naturalized, especially considering that in the 4th century BC, a well-organized Greek colony called Issa was founded at this location.With over 900 species distributed worldwide, the genus e family . Among ties rank , of whicic coast , in the ic coast , and a fic coast . The ranS. officinalis and S. fruticosa, in the coastal Adriatic region, a single additional member of the section Salvia can be found: Salvia brachyodon Vandas, or short-toothed sage , and Mt. Orjen, located 120 km to the southeast, in the border area of Bosnia and Hercegovina and Montenegro (http://hirc.botanic.hr/fcd) and a highly vulnerable species in Montenegro  and can consequently serve as a ‘control group’ in contrast to populations of the other two species, S. brachyodon and S. fruticosa), (2) a population of narrow endemic and endangered S. brachyodon is characterized by lower levels of genetic diversity than its widespread congener, i.e. S. officinalis, (3) a presumably non-native and disjunct population of S. fruticosa is genetically severely impoverished and suffers from population bottleneck, inbreeding and the absence of gene flow.To perform comparative population genetic research on selected species growing in an area characterized by intriguing climatological and ecological history and under strong and long-lasting anthropogenic influence , recentlS. officinalis was collected from both sites, S. brachyodon from the Pelješac Peninsula and S. fruticosa from the Island of Vis. It is important to note that in the studied locations, S. officinalis occurs sympatrically with both taxa and even has an overlapping flowering period with S. fruticosa (late April through early May). The flowering period of S. brachyodon occurs in August and does not overlap with that of S. officinalis . Voucher specimens of studied populations have been deposited in the Herbarium Croaticum (ZA) of the Department of Botany, Faculty of Science, University of Zagreb.Leaf tissue from a total of 96 individuals from four populations  was collected in two localities: Pelješac Peninsula and the Island of Vis. S. officinalis and tested for cross-amplification in S. fruticosa and S. brachyodon, but with limited success . The amplification was performed with a GeneAmp PCR System 9700  using a two-step PCR protocol with an initial touchdown cycle. The cycling conditions were as follows: 94 °C for 5 min; five cycles of 45 s at 94 °C, 30 s of annealing, beginning at 60 °C and lowered by 1 °C in each cycle, and 90 s at 72 °C; 25 cycles of 45 s at 94 °C, 30 s at 55 °C, and 90 s at 72 °C; and an 8-min extension step at 72 °C. The amplification products were run on an ABI 3730XL analyzer (Applied Biosystems) and the results were analysed with GeneMapper 4.0 software (Applied Biosystems).Genomic DNA was extracted from silica gel-dried leaf tissue by using a GenElute Plant Genomic DNA Miniprep Kit . A microsatellite analysis using 13 sequence-tagged SSR loci  and to test the population genotypic frequencies across all loci for their conformity to Hardy–Weinberg expectations (HWE) (multilocus test). Estimates of Na, HO and HE in each population were compared using the Kruskal–Wallis test in SAS version 8.02 . MICRO-CHECKER v.2.2.3 (HE) was compared with the expected gene diversity at mutation-drift equilibrium (HEQ) and calculated from the observed number of alleles under an intermediate two-phase model (TPM) assuming 30 % infinite allele model (IAM) and 70 % stepwise mutation model (SMM)  using the round-robin method of To determine the power of the marker set to discriminate among clones, the unbiased probability of identity  ranging from 1 to 6 were carried out on the Isabella computer cluster at the University of Zagreb, University Computing Centre (SRCE). Each run consisted of a burn-in period of 200 000 steps followed by 106 MCMC (Monte Carlo Markov Chain) replicates assuming admixture model and correlated allele frequencies. No prior information was used to define the clusters. The most likely number of clusters (K) was chosen by comparing the average estimates of the likelihood of the data, ln[Pr(X|K)], for each value of K  and four hybrids . The program was run without any prior information about the hybrid status of collected individuals, and with the uniformative Jeffreys prior option for both mixing proportions and allele frequencies. The results were based on the average of five independent runs consisting of a burn-in phase of 100 000 steps, and 500 000 MCMC sweeps. Following the suggestions of qT) of 0.50.The Bayesian method implemented by NEWHYBRIDS 1.1.  was usedS. officinalis population from the Island of Vis showed no significant deviations from HWE, while the population from the Pelješac Peninsula was characterized by an overall FIS value that was significantly different from zero . However, a closer inspection revealed that this difference was largely explained by a highly significant FIS value at only one locus (SoUZ020). No significant differences were noted when comparing the overall genetic parameters of these populations , and the FIS value was significantly different from zero  (Table P(E) = 0.002)  or among 1.286 individuals by assuming that a population was composed only of sibs (PIsibs = 7.77 × 10−4). The probabilities of obtaining the same multilocus genotypes through distinct sexual recombination events were low in both cases , and even when using a more conservative estimate [Psex(f)] that takes into account possible departures from HWE, the test yielded extremely low values .In S. officinalis population from the same location, no significant differences between observed and expected heterozygosity were found.In comparison with the S. fruticosa, the observed heterozygosity was lower than expected , which resulted in a significantly positive FIS value . Under the TPM, the Wilcoxon test revealed a pattern of heterozygosity deficiency (P(D) = 0.027)  , while the second highest ΔK was at K = 4 (9.62). The presence of two well-differentiated populations belonging to parental taxa was strongly supported by the above results, as was the existence of individuals of hybrid origin.In the 1 hybrids with very similar posterior probabilities ranging from 0.762 to 0.773. Interestingly, posterior probabilities suggesting the possible origin of these individuals through backcrossing were relatively high and ranged from 0.186 to 0.212.The hybrids assignment by NEWHYBRIDS was in concordance with the results from the STRUCTURE Fig. . All indS. officinalis from Pelješac, most loci were in HWE, with the exception of locus SoUZ011, which was characterized by a significantly positive FIS value , S. officinalis populations can be treated as reference populations in contrast to populations of the other two species that probably experienced some of these disruptive factors.Being a typical heliophyte and one of the most abundant plant species in open habitats along the eastern Adriatic coast, S. officinalis from the same location, S. brachyodon was characterized by no significant differences between the observed and expected levels of heterozygosity. There are several possible explanations for such high heterozygosity levels and heterozygosity excess . First, during the sampling expedition, a large number of bumblebees (Bombus sp.) were observed to visit S. brachyodon flowers, supporting the expectation that this species, like most sage species with large flowers  forest  is common and inevitable throughout the region. Being a typical heliophyte, S. brachyodon requires clear, open space to thrive and the abandonment of the traditional pasture management may result in a loss of a suitable habitats. The ecological succession at this location prior to the 1998 fire is the most likely reason for this population bottleneck and recent wildfire likely saved this population from deterioration and possibly even extinction. To re-establish and maintain the desired open habitat and the associated species community that emerged from the wildfire, grazing cattle could be reintroduced in such areas  are not unprecedented. Clonal reproduction can serve as a valuable mechanism to preserve genetic diversity and in spite of the detected population bottleneck, high levels of genetic variability can indeed be found in this species.Although narrow endemic species in general are considered to be characterized by low levels of genetic diversity e.g. , on someFIS value, which suggests high inbreeding levels, and the significant heterozygosity deficiency as revealed by BOTTLENECK. The results additionally confirm the hypothesis of the non-native origin of S. fruticosa in this location. As is often the case with anthropogenic introductions, this population was most likely founded from a very limited number of individuals who were completely isolated from the source population and from any other population of their kind. The resulting genetic drift, bottleneck and inevitable breeding between close relatives subsequently led to increased homozygosity, which had a significantly higher value when compared with the sympatric S. officinalis population . It is likely that over time and within a population of this size, the frequency of new alleles, and consequently heterozygosity, will increase.Two parameters that are interesting for this isolated population are the positive and highly significant on Table . Furtherday Fig.  supportsS. fruticosa. As already mentioned, S. officinalis and S. fruticosa occur sympatrically in this location with overlapping flowering periods. It is also important to note that a putative hybrid between these species has been recorded   and the calyx trichome length and density . However, because this species is characterized by exceptionally high levels of morphological variability  are the hybrids fertile, (ii) is backcrossing to either parental species indeed present and (iii) if backcrossing is present, is it symmetrical or unilateral etc. As hybridization between species is generally considered to be rare on an individual basis, a very limited number of individuals participate in hybridization events. Consequently, hybrids are rare in the population, but even a few hybrids can provide a bridge that allows a trickle of alleles to pass between species  that the Salvia species were only partially confirmed. In accordance with expectations, S. officinalis populations, as true core populations, were characterized by high levels of variability, while the disjunct and isolated population of S. fruticosa contained significantly less genetic variation and struggled with high levels of inbreeding. The natural hybridization between S. officinalis and S. fruticosa was confirmed for the first time. To assess the nature and direction of hybridization, more extensive research including a significantly larger number of samples and additional methods should be conducted. Although morphometrical analyses alone are considered to be of limited value in hybrid identification and characterization  as numerous questions dealing with this species' historical and contemporary demography could be answered.In contrast to expectation, the population of the endangered and narrow endemic Salvia populations could also be a result of certain human activities or their absence. However, before reaching any conclusions, caution is needed, as some detected genetic traits could easily be a result of specific differences in species life history traits, rather than a consequence of human activities. To more reliably address this and similar topics, a different approach is needed.Some of the detected fluctuations in genetic structures of the studied This research demonstrates the importance and advantages of a multi-species approach in population genetics studies, as some relevant findings and conclusions about each species can be reached only if a comparison among different, yet similar and closely related species is performed. The selection of the ‘control’ group should be carefully considered, as it will set the frame of the entire research. Presumably, widespread species with no significant population genetic disturbances, highly specific life traits or large-scale demographic fluctuations, should be considered as the right choice for such a purpose.Croatian Science Foundation within the framework of the Project No. 09.01/246 .This study was supported by the I.R. performed the experiment, wrote and edited the manuscript and created all the figures. Z.S. conducted statistical analyses. Z.L. conceived the idea for the research and extensively edited the manuscript. All authors were equally involved in sample collection.None declared.


In the last half century, gene flow has moved from relative obscurity to a well‐recognized component of evolution. Gene flow, the successful transfer of alleles from one population to another, is now known to vary considerably among species, populations, and individuals as well as over time. It frequently occurs at rates sufficient to play an important evolutionary role for populations of both animals and plants  has considerable applied and evolutionary significance in prokaryotes and eukaryotes  landraces was the result of introgression from the wild Z. m. ssp. mexicana (Hufford et al. The recognition of gene flow's magnitude and variation has led to increased attention to gene flow in applied evolutionary biology. As noted above, breeders have long been acutely aware of gene flow. Intentional anthropogenic gene flow has long been practiced by breeders to deliver desirable traits to domesticated species via wide crosses followed by repeated backcrossing (e.g. Stalker But gene flow involving domesticated species is not universally beneficial. Pollen contamination from conspecifics and congeners growing within cross‐pollination distance can frustrate progress under artificial selection as well as jeopardize the genetic purity of seed multiplication programs (e.g. Kelly and George The foregoing scenario involving a traditionally bred crop represents one of the primary concerns associated with the release of transgenic crops (Ellstrand a priori. The presence of transgenes sometimes correlates with increased relative fitness, decreased relative fitness, or no change in relative fitness. Likewise, enough studies have accumulated to demonstrate that genetic background and environment can sometimes play a key role in determining the direction and magnitude of transgene fitness effects.One such study is presented in this Special Issue. Xia et al.  measuredTwo decades after the commercial release of genetically engineered crops, gene flow has enabled crop transgenes to move into and apparently persist in free‐living populations. The known cases are relatively few and mostly involve feral canola (Ellstrand The evolution of novel weeds and invasives need not involve domesticates at all. Natural gene flow and subsequent admixture involving only non‐domesticated taxa are known to have preceded the evolution of dozens of new weedy and invasive lineages (Schierenbeck and Ellstrand Salsola ryanii, evolved in California as recently as a few decades ago and is certainly no more than a century old. An allohexaploid derivative of hybridization between Salsola australis and Salsola tragus, its known range expanded from a few isolated populations in California's Central Valley to a broad distribution in that Valley as well as California's coastal valleys in roughly a decade. Despite its recent origin and rapid spread, molecular analysis by Welles and Ellstrand (One such example is in this Special Issue. The tumbleweed, llstrand  demonstrThe rise of conservation genetics in the 1980s started with a focus on fragmentation and small populations, featuring the hazards associated with drift, inbreeding, and lack of variation in the face of environmental challenges (Schonewald‐Cox et al. The intentional introduction of new genetic diversity for the purposes of sustaining a population is called “genetic rescue” (Tallmon et al. Of course, the addition of genetic diversity almost always includes the introduction of new individuals. Thus, genetic and demographic relief can be confounded. In this Special Issue, Fitzpatrick et al.  estimateAs conservation genetics began to mature, gene flow was no longer perceived solely as a panacea for the ills of small populations. Significant genomic or eco‐genetic differences between source and recipient populations can result in a fitness drop in interpopulation hybrids, often deemed “outbreeding depression.” If differences are primarily genomic, the fitness drop will tend to be environment independent. If the differences are eco‐genetic, immigrant gene flow will disrupt local adaptation (Price and Waser Gene flow from common species has put rare species at increased risk of extinction by genetic swamping, where the local genotypes are replaced by hybrids, or by demographic swamping, where population growth rates are reduced due to outbreeding depression. In this issue, Todesco et al.  conduct Hybridization involving rare populations also has important practical implications. Should the hybrids be protected for the sake of preserving important pools of genetic diversity or should they be destroyed to slow the process of genetic assimilation? Regardless of the answer to these questions, it is critical that we have robust methods for detecting hybrids (Payseur and Rieseberg Gene flow is now the subject of study beyond the field and the laboratory. When appropriate, decision‐makers are incorporating information and questions about gene flow in policy. Ridley and Alexander's  article


Odorrana schmackeri species complex, mountain stream-dwelling odorous frogs, endemic to southern China. We obtained mitochondrial sequences  of the complete ND2 gene and two flanking tRNAs of 511 individuals from 25 sites for phylogeographic analyses. Phylogenetic reconstruction revealed seven divergent evolutionary lineages, with mean pairwise (K2P) sequence distances from 7.8% to 21.1%, except for a closer ND2 distance (3.4%). The complex geological history of southern China drove matrilineal divergence in the O. schmackeri species complex into highly structured geographical units. The first divergence between lineage A+B and other lineages (C-G) had likely been influenced by the uplift of coastal mountains of Southeast China during the Mio-Pliocene period. The subsequent divergences between the lineages C-G may have followed the formation of the Three Gorges and the intensification of the East Asian summer monsoon during the late Pliocene and early Pleistocene. Demographic analyses indicated that major lineages A and C have been experienced recent population expansion (c. 0.045–0.245 Ma) from multiple refugia prior to the Last Glacial Maximum (LGM). Molecular analysis suggest that these seven lineages may represent seven different species, three described species and four cryptic species and should at least be separated into seven management units corresponding to these seven geographic lineages for conservation.Paleogeological events and Pleistocene climatic fluctuations have had profound influences on the genetic patterns and phylogeographic structure of species in southern China. In this study, we investigated the population genetic structure and Phylogeography of the  O. schmackeri, a mid-to-low altitude range frog species.Past geological events and climatic oscillations have played important roles in forming the contemporary genetic diversity and population structure of animals across the globe –3. The rO. schmackeri species complex.Uplifting of the Qinghai-Tibet Plateau (QTP) rearranged the drainage systems and these geological events may have influenced the geographic patterns and genetic structure of species . During Global cyclical cooling-warming events during the Quaternary have resulted in periodic expansions and contractions of population size and distribution range of species . For exaChina is known to have hosted many important global Pleistocene refugia for lineages that evolved prior to the late Tertiary and Quaternary glaciations . Most ofOdorrana schmackeri), belonging to the Ranidae family, is widely distributed and occurs in southern and south-central China [O. schmackeri is characterized by clearly visible brown or black round spots on the head and back. It is a sexually dimorphic species, with males and females measuring about 44mm and 80mm respectively, in snout-vent length. The species is higly adapted to mountain environments, inhabiting the moist evergreen broad-leaf forests at 200-1400m altitude [O. schmackeri is variable in body size, skin color and some other morphological traits. In recent years, new cryptic species have been discovered and named: O. yizhangensis occurs in Mangshan of Yizhang County, Hunan Province, at 1000–1200 m altitude, and in Nanling of Ruyuan county, Guangdong Province, at 900–1100 m elevation; O. huanggangensis occurs in Wuyi Mts., Fujian and eastern Jiangxi provinces, at 200–800 m elevation; O. tianmuii occurs in Tianmu Mts., northern Zhejiang, at 200–800 m [O. schmackeri species complex.The Chinese piebald odorous frog  the orogenesis of the mountains in southeastern China drove the formation of lineage divergence of O. schmackeri species complex; (2) the drainage rearrangement of the Yangtze River was important driving force to shape the current genetic structure of O. schmackeri species complex; (3) lineages experienced recent population expansion prior to LGM.The present study investigated the phylogeography of The capture, measurement, toe clip, and release procedures of the 511 Chinese piebald odorous frogs were approved by the Chinese Wildlife Management Authority which had the right to consent to such investigation work. At the same time, this study was approved by the Ethics Committee of Anhui Normal University and conducted following the Law of People’s Republic of China on the Protection of Wildlife. A part of the sampling was carried out in protected areas and approved by the Wildlife Management Stations of Daiyunshan National Nature Reserve, Nanling National Nature Reserve and Guniujiang National Nature Reserve. The most of sampling was conducted outside protected areas whithout any requirements for specific permission. According to the regulation for the Collection of Genetic Resources (HJ 628–2011), all frogs were captured by hand, then measured for morphometric characters. Toe-clip tissues (up to 1 cm length) were collected and preserved in 95% ethanol immediately after removal; live frogs were subsequently released at the capture point after treating wounds with antiseptic.O. schmackeri species complex (O. margaratae from Shennongjia Mts. (SNJ) and one individual of O. graminea from Huangshan Mts. (HS) were used as outgroups based on the results of Chen et al. [We analyzed a total of 511 individuals from 25 localities covering most of the distribution range of  complex ; Fig 1. n et al. .O. schmackeri from Li et al. [2 2μl, 2mmol/L dNTPs 2μl, 10μmol/L each primer 1μl, 1 unit of Taq DNA polymerase  Co., Ltd., Dalian, China), and 20–60 ng template DNA. PCR procedure consisted of an initial denaturation at 94°C for 5 min, then 32 cycles with 94°C for 40 s, 60°C for 40 s and 72°C for 50 s, followed by a final extension at 72°C for 10 min. The PCR products were separated by electrophoresis in 1.5% agarose gels, then were purified by DNA Gel Extraction Kit  with ABI 3730 . The DNA products were purified, and then sequenced in both directions on an ABI3730 with an ABI PRISM BigDye terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer Biosystems).Tissue samples were digested using proteinase K, and total DNA was subsequently extracted following the standard phenol/chloroform method . A 1151 i et al.  , O. margaratae (KP167579), O. ishikawae (AB511282), Rana dybowskii (KF898355), R. chensinensis (KF898356), R. temporaria (AF314018), R. boylii (AF314019), R. sylvatica (AF314017), Pelophylax plancyi (EF196679), P. nigromaculata (AB043889), P. cretensis (GU812136), P. bedriagae (GU812075), P. lessonae (JN627426).To estimate divergence time, we used an uncorrelated lognormal relaxed molecular clock model as implemented in BEAST v1.75  based onPelophylax cretensis and Pelophylax bedriagae  based on geological data (5–5.5 Ma) [Rana sylvatica and Rana boylii  was estimated based on a multiple gene/calibration analysis [Divergence age estimates were established in this study for –5.5 Ma) . Divergeanalysis .We applied a GTR+I+G model of evolution based on MODELTEST 3.7, and a Yule process for speciation. Three independent MCMC runs were conducted for 10 million generations, each with a burn-in of 1 million generations, and sampled every 1,000 generations. These three runs were then combined in Tracer 1.6 to determine burn-in and convergence of the chains.h), haplotype diversity (Hd) and nucleotide diversity (π) for both overall and each population using the program DnaSP 5.10. To investigate the level of genetic variation between populations, analyses of molecular variance (AMOVA) [FST values among all the geographical populations using ARLEQUIN 3.5. Divergence between the matrilines was estimated by Kimura’s two-parameter (K2P) model [We calculated the number of haplotypes ( (AMOVA)  were per (AMOVA) . We alsoP) model  as impleP) model .FST values) and geographical distance was tested and the significance level was estimated using Mantel permutation procedures in TFPGA 1.3 [To examine the hypothesis of isolation by distance (IBD), the correlation between pairwise genetic  was estimated using the formula τ = 2ut [τ was the mode of mismatch distribution, and u was the mutation rate per generation for the entire sequence under study. The value of u was calculated using the equation u = μk, where μ was the mutation rate per nucleotide per generation and k was the number of nucleotides assayed.If the sudden expansion model was not rejected, the time of population expansions  gene and the flanking transfer RNA (tRNA) genes tRNA-Met , and tRNA-Trp (entire) (hereafter referred to as ND2+tRNA).All of ND2 sequences  successfully translated to amino acids without premature stop codons, and the complete tRNA-Trp had stable secondary structures, indicating that the sequences were obtained from functional genes. Moreover, light strand sequences showed an expected bias against guanine . These data indicated that only the mitochondrial genes were sequenced.O. schmackeri species complex ND2+tRNA sequences  of aligned ingroup nucleotides, 359 sites were variable and 348 sites were parsimony informative. There were 3 sites with alignment gaps or missing data. We identified 94 unique haplotypes among 511 equences . The seqO. schmackeri species complex was composed of seven geographically structured clades  were broadly distributed in the middle and down tributaries of the Yangtze River.O. schmackeri species complex ND2+tRNA sequences were shared among different sampling sites from same regional group. Eighty-one (86.17%) were unique haplotypes which were restricted to a single sampling locality and we found no shared haplotype among different regional groups (Only thirteen out of 94 haplotypes (13.83%) generated by 511 l groups .FCT) when numbers of population groups were assumed to be 3, 5 or 7. The seven groups (A-G) were recognized as the most parsimonious geographic subdivisions because the grouping resulted in the highest values of among-group variance   revealed significant values P < 0.001) of among-group variance  and nucleotide diversity (π = 0.10087 ± 0.00882) showed high values  and geographical distance was not significant , indicating that the genetic differentiation within O. schmackeri species complex did not fit the IBD model.A Mantel test revealed that the correlation between genetic distance  during Late Miocene to Early pliocene. The divergence time between Lineage A and Lineage B was estimated to be 2.21 Ma  and lineages C-G diverged from 1.02 to 5.92 Ma .The estimated divergence times between the major lineages were mapped on the BI tree . The estThe statistical parsimony network was constructed for O. schmackeri species complex with haplotypes connected by six or less mutational steps at a 95% confidence level. The rule resulted in six separate networks, corresponding to the six clades A-F in the BI tree, as well as one separate single haplotype (SH-SNJ1) which was concordant with the clade G . In cladIn cladogram 3–3 (Lineage B), 21 haplotypes connected in the network formed a more diffuse structure, containing 7-one-step clades, 2-two-clades and the total cladogram. Two haplotypes (SH-E1 and SH- E3) showed a widely geographical distribution in East China.In cladogram 2–9 (Lineage C), 17 haplotypes generated a typical star-shape network, and a shared haplotype (SH-C1), located in the central position, was implied to be the ancestral haplotype.P > 0.05, Fs and Tajima’s D statistics (P < 0.01), also indicating that the lineages A and C had undergone a sudden demographic expansion. Based on the estimated mean rate of 0.957% [Unimodal distribution, indicative of demographic expansion, was observed for both lineages A and C , and ragf 0.957%  substituO. schmackeri species complex was genetically structured. About 86% of the total haplotypes occurred just in one location. No haplotypes were shared among all the 25 populations of O. schmackeri species complex. The AMOVA analysis also revealed a high level of geographic structuring. Moreover, there was no unambiguous link among the six haplotype networks and the separate haplotype (SH-SNJ1). The results showed significant genetic differentiation among the species complex.All analyses unambiguously showed that O. schmackeri species complex, roughly corresponding to seven regional groups (Phasianus colchicus) [Gekko swinhonis [The results of this study showed seven main mitochondrial haplotype lineages (A-G) within l groups . The sevlchicus)  and an ewinhonis .Drainage evolution have been revealed as an important driving force for shaping current geographic patterns of stream-associated amphibians ,35,36. HO. schmackeri species complex. Within lineage A, MS has substantial genetic diversity, a large number of private haplotypes, and the ancestral haplotype (SH-S2) is present, suggesting that the Nanling Mts. have serviced as a glacial refugium for this lineage. Similarly, Huangshan, Hupingshan, Wugongshan Mts. and northwestern Guizhou Plateau, might have represented the main refugia for lineage B, C, D, E, respectively.Suitable habitats and relatively stable microclimates in refugia may permit species to persist for long periods of time and even to speciate. Unique genotypes and substantial genetic diversity often occur in these locations . AccordiCephalotaxus oliveri [Pachytriton [Eurycorymbus cavaleriei [Some refugia proposed by our study are corroborated by other case studies; for example, Hupingshan Mts. was considered as a refugium for the three sharp cedar  oliveri , Huangshhytriton , and Nanvaleriei . More exFs and Tajima’s D and the patterns of genetic diversity reported here strongly supported the idea of recent expansion of the lineages A and C.The smooth and unimodal mismatch distributions, significantly negative Fu's 18 Ο value demonstrated that the most substantial glacial extension occurred during the Marine Isotope Stages 16–18  in China [However, unlike temperate species in North America and Europe which commonly expanded in the post-Last Glacial Maximum (LGM) era ,85, the in China . After tO. schmackeri populations in Lineage C dispersing northward to the two localities SNJ and FNS may be suggested by the phylogenetic tree.Secondary contact of previously isolated populations in North America and Europe were well established ,87. In tO. schmackeri species complex by analyses of the mitochondrial ND2+tRNA sequences, with mean pairwise (K2P) sequence distances from 7.8% to 21.1%, except a closer genetic distance (3.4%) between Lineage C and Lineage D. The seven-clade divergence is also supported by AMOVA  and/or evolutionary significant units (ESUs), two commonly used designations for endangered taxa [O. schmackeri species complex would be considered at least MUs. These seven regional populations may represent important components in the evolutionary and adaptive structure of the species complex, and thus any conservation policy should concentrate on protecting these regional populations.Our study has provided a means for assessing the evolutionary distinctiveness of populations of red taxa . Managemred taxa . ConsideS1 Tableh), sample size (N) and coordinates (longitude/latitude) are given. Haplotype diversity (Hd) and nucleotide diversity (π) for each population with sample size > 3 are presented.For each population sampled, geographic origin, identified lineage, number of haplotypes ((DOC)Click here for additional data file.S2 TableN), “SH” represents shared haplotype, and the other codes are abbreviations for geographical locations corresponding to Sample size ((DOC)Click here for additional data file.


Ooencyrtus pityocampae (OP), an important egg parasitoid of the pine processionary moth (PPM), is considered a generalist parasitoid. OP emerges from PPM eggs after winter hibernation, mainly in spring and early summer, long before the eggs of the next PPM generation occurs. The occurrence of OP in eggs of the variegated caper bug (CB) Stenozygum coloratum in spring and summer suggests that OP populations alternate seasonally between PPM and CB. However, the identity of OP population on CB eggs seemed uncertain; unlike OP-PPM populations, the former displayed apparently high male/female ratios and lack of attraction to the PPM sex pheromone. We studied the molecular identities of the two populations since the morphological identification of the genus Ooencyrtus, and OP in particular, is difficult. Sequencing of COI and ITS2 DNA fragments and AFLP analysis of individuals from both hosts revealed no apparent differences between the OP-PPM and the OP-CB populations for both the Israeli and the Turkish OPs, which therefore supported the possibility of host alternation. Sequencing data extended our knowledge of the genetic structure of OP populations in the Mediterranean area, and revealed clear separation between East and West Mediterranean populations. The overall level of genetic diversity was rather small, with the Israeli population much less diverse than all others; possible explanations for this finding are discussed. The findings support the possibility of utilizing the CB and other hosts for enhancing biological control of the PPM.The increased use of molecular tools for species identification in recent decades revealed that each of many apparently generalist parasitoids are actually a complex of morphologically similar congeners, most of which have a rather narrow host range.  The term "host alternation" is often used to describe situations in which species are required to change hosts constantly in order to survive, as in the case of true parasites that exhibit complex life cycles that require two different host species , or in tOoencyrtus pityocampae Mercet (OP) (Hymenoptera: Encyrtidae) is a well-studied egg parasitoid of the pine processionary moth (PPM) Thaumetopoea pityocampa (Den and Schiff)/T. wilkinsoni Tams species complex (Lepidoptera: Notodontidae). The moth is a major defoliator of pines throughout the Mediterranean basin [Although most parasitoid species are assumed to have rather narrow host ranges, some apparently cover wide ranges and are able to switch regularly between different host species –9. Howevan basin –20. OP ian basin –30. OP ian basin .Stenozygum coloratum (Hemiptera: Pentatomidae), which is found on caper plants growing within and on the edges of pine forests in Israel [Brachynema signatum (Hemiptera: Pentatomidae), a pest of pistachio orchards in Iran [Ooencyrtus species were present, therefore this report should be further validated.The PPM egg masses are deposited on pine needles in late summer and autumn, i.e., from late August to November, depending on the location, and can support two or three generations of OP. Individuals mainly of the last generation undergo winter diapause inside host eggs, as last-instar larvae ,29,31,32n Israel ,40; its n Israel , and eggn Israel . As far  in Iran , althoug in Iran , indicatIn Israel, the CB stops reproducing around the beginning of autumn, i.e., September-October , which cNevertheless, doubts concerning the identity of the parasitoid population on the CB eggs had emerged, which raised questions regarding the host alternation hypothesis; in general, several different factors could prevent a parasitoid from being able to exploit a specific host . Even inOoencyrtus spp. (These data is about to be published separately). Furthermore, the identification of individuals emerging from PPM and CB eggs, as well as from other known hosts, was based on morphological characteristics, which may be unreliable in this species . As a result, doubts concerning the identity of the parasitoid population on the CB eggs were raised.The ability of OP individuals that parasitize PPM and CB to develop in and switch successfully between these two hosts was confirmed in laboratory trials , which iOoencyrtus spp in the CB eggs ; one of the congeners was genetically identical to OP populations occurring in PPM eggs. These findings supported the alternation hypothesis, and accounted for the high male/female ratio of the CB parasitoid population, because the males were shown to belong to the other Ooencyrtus spp. [The question was whether the "two" OP populations were in fact a single one whose individuals alternated seasonally between the PPM and CB, or whether these individuals represented two distinct populations (or separated species), each specializing on a different host. Our preliminary study, which compared COI sequences of OP individuals from both host species in different areas in Israel, revealed the presence of five closely related, morphologically similar tus spp. .However, the overall level of genetic differences between OP individuals was extremely low, practically zero, according to the DNA fragment used—therefore it could not be determined whether the lack of genetic differences between the two populations obtained from the two respective host species was because they were a single population, or whether the resolution of the genetic analysis was not high enough to detect differences between closely related populations, possibly because the chosen DNA segment was too conserved. This apparent lack of genetic diversity also raised the question of whether the low polymorphism found is unique to the Israeli OP populations, or is a general phenomenon in this species. To answer these questions three different approaches were taken. First, we extended our sample size and sampling area to include other regions in the Mediterranean basin, focusing on areas where both hosts are found, i.e., Israel and southern Turkey. Secondly, another DNA fragment (ITS2) was added to the analysis. Finally, a different genetic analysis, i.e., Amplified Fragment Length Polymorphism—AFLP  was alsoThus, the objectives of the present study were as follows: (1) to confirm the presence or absence of host alternation in OP, by determining the level of genetic similarity between OP populations from PPM and CB; and (2) to determine whether the apparently low genetic diversity in the Israeli OP population is unique to this area, or is a general phenomenon in this species. To address these issues, data on the genetic structure of several OP populations from several locations around the Mediterranean basin were obtained and analyzed.Capparis spp., and PPM egg masses from the various pine species that were present on each site. Each egg mass was placed in a separate glass tube sealed with cotton wool. The emerging OP individuals were collected in Eppendorf tubes filled with 96% ethanol and stored at -20°C. It was attempted to obtain individuals from both hosts and in each sampling area, but this was not always possible, for several reasons. First, the CB is found in the East Mediterranean area, and its distribution only partially overlaps that of the PPM [Ooencyrtus spp. were also parasitizing the eggs, usually substantially outnumbering OP; in some locations no OP individuals were found in the CB eggs sampled, e.g., the Negev desert and Jordan Valley  according to the manufacturer's protocol, or with Lysis buffer (40 μL reaction) made with the following reagents: 5 μL Tris 1M (pH 8), 1 μL 0.5M EDTA, 5 μL Igepal, 50 μL Proteinase-K (20 mg/mL) and 939 μL of double-distilled (dd) HTwo DNA fragments were analyzed: a 946 bp fragment of the mitochondrial Cytochrom oxidase 1 (COI) gene, and a nuclear 931- to 933 bp fragment that comprised a partial (65 bp) fragment of the ribosomal 5.8S unit, a complete (523- to 525 bp) sequence of the ribosomal Internal Transcribed Spacer 2 (ITS2) and a partial (342 bp) fragment of the 28S ribosomal unit.The initial primer sequences used for COI were kindly provided by Marie-Anne Auger-Rozenberg, of INRA. They were:forward—5' CGAATAAATAATATAAGTTTTTG 3' andreverse—5' CAACATAAATAAGAATCTGGA 3').However, the forward primer often attached to the middle of the COI segment and thus only the second half of the segment was obtained. To solve this problem, another forward primer was designed based on the previous one—5' CTCGAATAAATAATATAAGATTTTG 3'. The primers for ITS2 sequences were based on an alignment of ITS2 sequences of species of Chalcidoidea, which were available in NCBI. The forward and reverse primers were, respectively,5' GAACTGCAGGACACATGAACA 3' and 5' CTTGTTCGCTATCGGTCTCGTGGT 3'.Amplification of the COI and ITS2 used the following conditions: Initial denaturation at 95°C for 3 min, followed by 36 cycles of 95°C for 30 s, 54°C for 30 s, and 72°C for 75 s (COI) or 1 min (ITS2), followed by final extension at 72°C for 5 min.2  and 22.05 μL of dd H2O. The PCR for ITS2 was performed with the same reagent concentrations except for the MgCl2, which was at a final concentration of 2 mM.The PCR for COI was performed in a 30-μL reaction volume containing a 3-μL sample, 0.3 μL (30 pmole) of each primer, 0.6 μL of dNTPs (10 mM of each), 0.15 μL (0.75 units) of Taq polymerase , 0.6 μL of MgClPCR products were electrophoresed through 1% Agarose gel, and the specific segment was cut from the gel and later extracted and purified with the RBC-YDF HiYield Gel/PCR DNA Fragments Extraction Kit . Sequencing was done by Macrogen Cor. . The PCR products were sequenced with both forward and reverse primers, and the sequences were aligned by means of the ChromasPro software Version 1.6  and checked manually for errors.Since the sequencing of ITS2 did not reveal a significant level of polymorphism, the sequence analysis was based on the COI sequences alone. The COI sequences were aligned with Mafft (version 7) software  and tranhttp://www.fluxus-engineering.com), under default parameters.The DNASP v5.10.1 program  was usedΦst values were computed by means of the Arlequin v.3.5.1.3 software [Φst values, we also computed Jost’s [Pairwise software , with 10software  was appld Jost’s  distanced Jost’s .An analysis of molecular variance (AMOVA) was conducted with Arlequin, in order to determine the distribution of molecular variance among populations from the various studied areas. It was of particular interest to test for host related differences in the Turkish population, which unlike the Israeli population, showed relatively high level of diversity. A second AMOVA test was conducted to determine whether the genetic diversity was partitioned between West Mediterranean sites, i.e., Portugal and Tunisia, and East Mediterranean sites, i.e., Cyprus, Greece, Israel and Turkey. The sampled sites were partitioned either into "region", "host within region" and "within population" components, or into "host", "region for each host", and "within population" components. In Israel and Turkey the samples were partitioned into four subgroups based on locality, i.e., Hatay and Mersin-Adana regions (Turkey) and north and south (Israel), and according to their host, i.e., PPM or CB. The significance of the variance component was determined by using 10,000 permutations:Φst and D) were correlated with geographic distances, a Mantel test was performed, using zt [In order to determine whether genetic distances  analysis , mainly Because the total amount of DNA from a single wasp was not sufficient for the analysis, clones of genetically similar individuals were used. Each sampled female (clone founder) was completely isolated from the presence of males, as a safety measure, and was allowed to parasitize 100 silk moth eggs. The daughters of each female were allowed to parasitize additional eggs, until about 30–50 wasps per clone were obtained, which usually required more than one generation. The DNA was extracted jointly from all offspring of each female founder with the Qiagen DNeasy Blood and Tissue kit  according to the manufacturer's protocol. One microgram of total genomic DNA of each clone was suspended in 50 μL of dd water and used for the analysis.EcoRI/MseI restriction enzyme sites), three PCs were selected based on fingerprints quality and polymorphism scores: E12/M55, E13/M50 and E13/M59 (E12 = 5’-(EcoRI)AC-3’, E13 = 5’-(EcoRI)AG-3’, M49 = 5’-(Msel)CAG-3’, M50 = 5’-(MseI) CAT-3’, M55 = 5’-(MSeI) CGA-3’ and M59 = 5’-(MseI) CTA-3’). After generation of the fingerprints the samples were genotyped according to presence or absence of polymorphisms of AFLP markers. Size ranges of markers were 60–560 bp. The genetic information generated was subjected to three different genetic distance analysis procedures. The marker scores were used to carry out the genetic diversity analyses in order to categorize the samples according to their genetic similarity. The NTSYSpc software [After screening of 48 primer combinations (PCs)  cluster analysis with the use of UPGMA  parameters for all three matrices. To evaluate to what extent the dendrograms were good representations of the similarity matrices, the cophenetic value matrix was calculated for each dendrogram and plotted against the original similarity matrix in order to obtain the cophenetic correlation, which indicates the degree to which the dendrograms represent the similarity matrices.OP haplotypes found in the present study have been deposited in GenBank (accession numbers KM485909 to KM485924 (COI) and KM527071 to KM527091 (ITS2)).A total of 16 different COI haplotypes were obtained , comprisIn spite of the large samples, very low genetic variability was detected among the Israeli populations (H = 0.000–0.083) , Fig 3B.st and Jost's D distances) were found between most populations, except among the Israeli populations, between Hatay-PPM and Greece, and between Greece and Cyprus (n = 5 and n = 7 respectively). In addition, Jost's D distances did not exclude the possibility of null distances between the Turkish populations, except for the Hatay-PPM population, which appeared to be significantly different from the other three tested populations.Significant genetic differences (based both on Φd Cyprus . Howeverp = 0.13). Again, half of the genetic variance was attributed to variation among populations. The Mantel test, however, showed significant results: R = 0.239, p = 0.04 when Φst was used; R = 0.402, p < 0.0001 when Jost's D was used—indicating a correlation between geographic and genetic distances. It is worth noting that this result is still significant or marginally significant when the Israeli populations are excluded: R = 0.218, p = 0.09 when Φst is used; R = 0.450, p = 0.008 when Jost's D is used.The AMOVA analyses of the Turkish specimens did not reveal structures among regions or among hosts . In bothp = 0.02), and the negative value may be an indication of a recent expansion (see p = 0.66).Tajima's D test supported the existence of non-random selection processes in the Israeli population  in a specific microsatellite located at the middle of the ITS2 segment (starting from nucleotide 507). This caused variability in sequence length, which varied between 930 and 932 bp . As a reO. corei did not share a sufficient number of fragments with the other samples, they were not included in the analysis. For PC E12/M49, no polymorphic markers were observed. Scoring the fingerprints generated with the remaining three PCs resulted in a data set of 48 polymorphic markers. The dendrograms based on the matrix generated with the use of the Simple matching, Jaccard and Dice coefficients had cophenetic correlations of 0.96, 0.94 and 0.97, respectively, which indicates that the dendrograms provide good representation of the similarities between the samples. It was observed that the individual from Portugal (sample 42) was the most distantly related to all other individuals  were most likely present much earlier, and it is possible that the PPM survived undetected in these small pockets of trees [Some available data on the occurrence of pine forests and the PPM in Israel may further help to account for the current genetic structure of OP. It is suggested that pine was very rare in cis-Jordan areas  until recent centuries . The PPMollected –72, suggollected . Most ofollected . Howeverof trees . The fasof trees .Ooencyrtus species found on PPM eggs, and it is the dominant egg parasitoid on this host, whereas in other hosts' eggs, including those of the CB, OP is quite uncommon, probably as a result of extreme competition with other Ooencyrtus spp. In fact, OP was rarely found outside the PPM distribution range [Interestingly, OP was the only on range . FurtherThe question of whether OP is dependent on PPM for its survival remains open. If OP population occurred in the area before the migration of PPM; its genetic diversity is expected to be similar to that of the Turkish population, unless dramatic bottleneck events had occurred in this area. The other possible scenario is that the occurrence of OP in Israel is a result of a founder effect associated with a relatively recent migration, following the arrival of PPM to Israel. According to Simonato , the IsrWolbachia, which is known to be associated with OP [A strong geographical/genetic structure was also observed in the populations around the Mediterranean, with the populations of the West Mediterranean having different mitochondrial COI haplotypes from those of the East Mediterranean. This separation was further emphasized by the small but consistent difference in the nuclear ITS2. A single individual from Portugal that was used in the AFLP analysis also showed a high level of divergence, both from the Israeli OP individuals and from the one from Turkey . Results with OP , might bIn the present study we focused on the OP population of the East Mediterranean, a region where the geographical ranges of CB and PPM overlap. Drawing a complete picture of OP genetic structure throughout its area of distribution demands that more areas be surveyed. This is especially true for the West Mediterranean countries in southern Europe and North Africa, from which relatively small samples could be obtained in the present study. Preferably, further analysis should include multiple methods or additional gene segments, because the DNA fragments used in the present study, i.e., COI and ITS2, were shown to only partially distinguish between populations of this species from different locations and countries. Combining additional genetic information derived from AFLP, highly polymorphic microsatellites, or sequencing of additional gene fragments, would facilitate better differentiation between some geographically close populations, as was seen in the Italian OP populations  and in tThe scarcity of evidence for OP presence in areas from which PPM is absent, its rarity on other hosts, and, on the other hand, its emergence pattern in spring when PPM eggs are absent, raise fundamental questions for further research: (1) Is OP dependent on alternative hosts for survival until PPM eggs become available, or is its survival possible on PPM alone? (2) How important is the role of CB in particular, in OP conservation? (3) If OP cannot survive in areas where PPM is absent, should the designation of OP as "generalist" be revised?Finally, the present study points to the importance of alternative hosts as a major tool in conservation biological control, and supports the idea that enrichment of pine forests with host plants for CB or other pentatomids is likely to lead to an increase in OP population. The existence of OP on CB and other pentatomids found on caper plants  supports


Cichorium intybus subsp. intybus var. foliosum L.) is a diploid plant species (2n = 18) of the Asteraceae family. The term “chicory” specifies at least two types of cultivated plants: a leafy vegetable, which is highly differentiated with respect to several cultural types, and a root crop, whose current industrial utilization primarily addresses the extraction of inulin or the production of a coffee substitute. The populations grown are generally represented by local varieties  with high variation and adaptation to the natural and anthropological environment where they originated, and have been yearly selected and multiplied by farmers. Currently, molecular genetics and biotechnology are widely utilized in marker-assisted breeding programs in this species. In particular, molecular markers are becoming essential tools for developing parental lines with traits of interest and for assessing the specific combining ability of these lines to breed F1 hybrids.Leaf chicory  marker loci scattered throughout the linkage groups. Information on the genetic diversity across molecular markers and plant accessions was successfully assessed along with descriptive statistics over all marker loci and inbred lines. Our overall data support an efficient method for assessing a multi-locus genotype of plant individuals and lineages that is useful for the selection of new varieties and the certification of local products derived from Radicchio of Chioggia.i.e., heterosis) of experimental F1 hybrids on the basis of the genetic distance and allelic divergence between parental inbred lines. Knowing the parental genotypes would allow us not only to protect newly registered varieties but also to assess the genetic purity and identity of the seed stocks of commercial F1 hybrids, and to certificate the origin of their food derivatives.This method proved to be useful for assessing the observed degree of homozygosity of the inbred lines as a measure of their genetic stability; plus it allowed an estimate of the specific combining ability (SCA) between maternal and paternal inbred lines on the basis of their genetic diversity and the predicted degree of heterozygosity of their F1 hybrids. This information could be exploited for planning crosses and predicting plant vigor traits (The online version of this article (doi:10.1186/s13104-015-1819-z) contains supplementary material, which is available to authorized users. Cichorium intybus subsp. intybus var. foliosum L.) comprises diploid plant species (2n = 18) belonging to the Asteraceae family, subfamily Cichoriodeae, tribe Lactuceae or Cichorieae. These species are biennial or, in the wild, perennial species [i.e., proterandry, wherein the anthers mature before the pistils) and a favorable competition of allo-pollen grains and tubes  [Cichorium spp. are currently among the most important cultivated vegetable crops. They are generally used as components in fresh salads or, more rarely, cooked according to local traditions and alimentary habits [Cichorium  with high variation and adaptation to the natural and anthropological environment where they originated, and have been yearly selected and multiplied by farmers [The materials grown are generally represented by actively cultivated local populations  markers. Among the different PCR-derived molecular systems, SSR markers are suitable for population genetics studies and marker-assisted selection programs because they have several desirable features, such as a high level of reproducibility, co-dominant heredity, and no need for high throughput technology . MoreoveC. intybus [C. intybus was constructed by using SSR markers [A large-scale application of molecular marker techniques, including amplified fragment length polymorphism (AFLP) and random amplified polymorphic DNA (RAPD), were used to construct the first genetic linkage maps of  intybus , 9. In 2 markers . This coi.e., observed degree of homozygosity) and genetic diversity between paternal and maternal lines . Information derived from the application of this method should then be exploited for planning crosses and predicting plant vigor traits  of experimental F1 hybrids of leaf chicory on the basis of the genetic distance and allelic divergence between parental inbred lines. Knowing the parental genotypes would allow us not only to protect newly registered varieties but also to assess the genetic purity and identity of the seed stocks of commercial F1 hybrids, and to certificate the origin of their food derivatives.The aim of our study was to develop a method for the genetic characterization of elite inbred lines of the “Red of Chioggia” chicory using mapped SSR markers with a particular emphasis on the assessment of the genetic stability within  in the SSR loci assayed was 5.9, varying from 3.3 in LG5 and LG7 to 9.3 in LG2 (Table i) proved to be low when the observed number of marker alleles was high and vice versa . At the same time, both the expected heterozygosity (He) and Shannon’s information index of phenotypic diversity (I) were estimated to be high when the observed number of marker alleles was high  , respectively, greatly contributed to this average homozygosity. Wright’s inbreeding coefficients (F-statistics) for single marker loci were also computed , and ranged from 0.521 to 0.993. The marker loci M3.8, M4.11b and M8.22, with observed heterozygosity values of 0.007, 0.017 and 0.021 (m) were also computed for each locus  to 23 (85.2 %) out of the total of 27 marker loci  estimates, can be discriminated from the others on the basis of genotyping data, as shown in Fig. A neighbor-joining (NJ) tree was also constructed on the basis of the genetic dissimilarity matrix whose mean coefficients were computed between all possible pair-wise combinations of inbred lines of the core collection Fig. . Two maii.e., membership <70 %), as expected with the occurrence of genetic recombination and hybridization.The population structure was investigated using the ΔK method , which eA second level of genetic structure was investigated within groups of inbred lines  by assaying microsatellite marker loci selected for the linkage map position and polymorphism information content. The genetic identity and stability of 37 inbred lines and the extent of their genetic diversity, expressed as genetic distance and allelic divergence, were addressed by using a panel of neutral SSR markers. Furthermore, our subset of 27 mapped SSR marker loci  proved to be very informative for the study of the population structure and inbreeding level of C. intybus plant materials. Along with estimates of observed homozygosity, the genetic stability of each breeding stock was addressed by computing Shannon’s estimates of phenotypic diversity and Rohlf’s coefficients of genetic similarity. On the whole, these statistics indicated strong genetic uniformity for the investigated inbred lines, as expected for breeding stocks developed by selfing and full-sibling programs. Furthermore, a marked inbreeding for the majority of the accessions was supported by the high homozygosity observed in nearly all the inbred lines . Also, the Wright’s fixation index indicated that the genetic differentiation between the inbred lines is high (approximately 63 %) and that one-third of the genetic variation (approximately 33 %) is occurring within the inbred lines, due not only to homozygosity for different marker alleles but also for heterozygosity at some of the marker loci. Our data demonstrate that most of the genetic differentiation is occurring among inbred lines; thus, each breeding stock can be considered as genetically uniform and distinguished from the others of the core collection.In this study, we developed a method for genotyping elite breeding stocks of leaf chicory  could be a consequence of the reproductive system of C. intybus, which is naturally characterized by high frequency of allogamy as a result of self-incompatibility. We may also speculate that a fraction of the observed heterozygosity could be a consequence of phenotypic selection  operated by breeders during inbreeding programs.It is worth mentioning that individual inbreeding coefficients, as estimate of the strength of inbreeding for single inbred lines, were shown to be low or negative, indicating that the observed heterozygosity was greater than expected. Maintenance of such levels of heterozygosity in spite of inbreeding reproductive strategies .All mapped SSR markers exploited in this multi-locus DNA genotyping method scored high polymorphism information content, with the exception of marker M8.22 that revealed an almost monomorphic condition. Although its very low or null discriminant ability, this marker locus was taken into account as it showed an allele-specific genotype, which is typical of leaf chicory and allows to identify Radicchio from other i.e., inbred lines that originated from the same local variety) proved to form a very well-defined subgroups of the tree . The STRUCTURE analysis of the population of genotypes (for K = 25) revealed clusters of single individuals in agreement with the grouping of inbred lines shown by the NJ tree analysis. In fact, inbred lines P04, S02 and S03 were grouped in the same cluster of ancestry and associated to the same node of the tree. This is also true for several pollen donor lines, such as SEG111, SE111S6 and SE111S7, and Z34 and SC24. In addition, the inbred lines belonging to the minor clade of the NJ tree were all grouped in different STRUCTURE clusters  starting from individuals selected within a given local variety of leaf chicory, namely Radicchio of Chioggia.PCA allowed for the definition of centroids for all the inbred lines. The first component was positively associated with cycle length, discriminating long cycle accessions from short cycle accessions, with only a very few exceptions per class. It is also worth mentioning that the centroids of inbred lines with a common origin could be plotted in different areas of the quadrants. This finding suggests that it is possible to develop and select genotypically different inbred lines  experimental materials, which not only manifest valuable traits, but also possess applicable uses in modern breeding programs.Our research deals with the implementation and validation of a multi-locus genotyping system in leaf chicory that may prove to be useful for the marker-assisted breeding of new varieties of Radicchio. In particular, the plant materials used in this study cover a core collection of Radicchio of Chioggia  following the manufacturer’s instructions. The quality of the DNA samples was assessed by electrophoresis on 1 % (w/v) agarose gel stained with 1X SYBR® Safe™ DNA Gel Stain (Life Technologies) in Tris–Acetate-EDTA (TAE) running buffer. The yield and purity of the extracted genomic DNA samples were evaluated using a NanoDrop 2000c UV–Vis Spectrophotometer (Thermo Scientific). Following DNA quantification, all the DNA samples were diluted to a final concentration of 25 ng/μl to be used as template for PCR amplifications.The genomic DNA was extracted from 100 mg of fresh leaves with the GenElute™ Plant Genomic DNA Miniprep Kit , 0.25 µM of each tailed primer, 0.75 µM of each non-tailed primer, 0.5 µM of each labeled primer , 25 ng of DNA and distilled water. The amplification reactions were performed in a Gene Amp® PCR System 9700 thermal cycler (Applied Biosystems).The reactions were performed in a total volume of 20 µl including 2X PlatinumThe microsatellite-containing regions were amplified using two different PCR cycles defined to maximize the amplification efficiency of the different primer sets according to their annealing temperatures, as reported in Additional file The cycle termed 'rad-multi 54' consisted of an amplification reaction performed under the following conditions: 95 °C for 5 min followed by 5 cycles at 95 °C for 30 s, 58 °C for 45 s decreasing by 0.8 °C every cycle, 68 °C for 45 s followed by 35 cycles at 95 °C for 30 s, 54 °C for 45 s, 68 °C for 45 s and a final extension of 60 min at 68 °C. Similarly, the cycle 'rad-multi 56' consisted of the following conditions: 95 °C for 5 min followed by 5 cycles at 95 °C for 30 s, 60 °C for 45 s decreasing by 0.8 °C every cycle, 68 °C for 45 s followed by 35 cycles at 95 °C for 30 s, 56 °C for 45 s, 68 °C for 45 s and a final extension of 60 min at 68 °C.® Safe™ DNA Gel Stain (Life Technologies) using Tris–Acetate-EDTA (TAE) running buffer. The concentrations of the PCR products were estimated with KODAK 1D Image Analysis Software by comparing the intensity of the PCR products to that of a 1 Kb Plus DNA Ladder (Life Technologies). Then, for each PCR reaction and fluorophore, approximately 20 ng of amplification products were pooled and prepared for capillary electrophoresis.The quality of the PCR products was assessed by electrophoresis on a 2 % (w/v) Agarose gel stained with 1X SYBRTm v. 1.0 (Life Technologies).DNA fragment capillary electrophoresis was completed at BMR Genomics . Following electrophoresis, fragment analyses were performed with Peak Scanneri.e., years 1960–1970s), belonging to the same botanical variety , we assumed the absence of homoplasy and adopted an infinite alleles model, hence considering that marker alleles of the same size at a given locus had the same evolutionary history. Statistical analyses of the SSRs were performed with the PopGene software package v. 1.32 [e), Levene’s [is) and between (Fit) subpopulations were determined, as was the fixation index (Fst) according to Wright [Owing to the relatively recent origin of the biotype “Red of Chioggia”  estimates among the subpopulations were derived from the fixation index as described by McDermott and McDonald [The phenotypic diversity of the marker allele profiles was estimated using Shannon’s information index (I) as reported by Lewontin . Gene flMcDonald .The NJ dendrogram of all the accessions was based on Nei’s method . A bootsAll the relevant calculations and analyses were conducted using the appropriate routines in the NTSYS software package v. 2.21c .The population structure of the inbred red Chioggia chicory lines was investigated using the model-based (Bayesian) clustering algorithm implemented in the STRUCTURE software , which g


Dictyocaulus nematode worms live as parasites in the lower airways of ungulates and can cause significant disease in both wild and farmed hosts. This study represents the first population genetic analysis of large lungworms in wildlife. Specifically, we quantify genetic variation in Dictyocaulus lungworms from wild deer  in Hungary, based on mitochondrial cytochrome c oxidase subunit 1 (cox1) sequence data, using population genetic and phylogenetic analyses. The studied Dictyocaulus taxa display considerable genetic diversity. At least one cryptic species and a new parasite–host relationship are revealed by our molecular study. Population genetic analyses for Dictyocaulus eckerti revealed high gene flow amongst weakly structured spatial populations that utilise the three host deer species considered here. Our results suggest that D. eckerti is a widespread generalist parasite in ungulates, with a diverse genetic backround and high evolutionary potential. In contrast, evidence of cryptic genetic structure at regional geographic scales was observed for Dictyocaulus capreolus, which infects just one host species, suggesting it is a specialist within the studied area. D. capreolus displayed lower genetic diversity overall, with only moderate gene flow compared to the closely related D. eckerti. We suggest that the differing vagility and dispersal behaviour of hosts are important contributing factors to the population structure of lungworms, and possibly other nematode parasites with single-host life cycles. Our findings are of relevance for the management of lungworms in deer farms and wild deer populations. Dictyocaulus lungworms live as parasites in the lower airways of ruminants. Worm burden  varies from mild to heavy and can result in severe host pathology, a condition referred to as ʽdictyocaulosis’. For example, Dictyocaulus viviparus  causes severe and frequently fatal bronchitis and pneumonia in cattle (termed ʽhusk’), which is of serious veterinary and agricultural importance due to animal welfare issues, reduced production yields and costs associated with treatment , Dictyocaulus arnfieldi , Dictyocaulus cameli , Dictyocaulus capreolus , D. eckerti, Dictyocaulus filaria  and D. viviparus  sequences  amongst Swedish farms  in particular is frequently used in population genetic studies because it exhibits a relatively high mutation rate, conserved primers are available, and the large amount of data available for other species provides a comparison of genetic variation and population structure. Indeed, the mitochondrial cox1 gene has been employed in a variety of studies on parasite nematodes  assess host relationships amongst the observed species; (3) identify genetic diversity, differentiation, geneflow and demographic history for recovered Dictyocaulus species; and (4) determine whether patterns are similar to those observed for D. viviparus in farmed cattle, which represent the only other Dictyocaulus species for which population genetic data are available.In the present study, we analyse genetic diversity at Dama dama), red deer (Cervus elaphus) and roe deer (Capreolus capreolus). Samples were taken from 23 sites in Hungary and one locality in neighbouring Romania  of the nuclear ribisomal DNA was conducted .Adult lungworms were collected from the trachea and bronchi of the following deer species harvested during hunting: fallow deer  gene fragment was amplified using the universal barcoding primers LCO1490 5′-GGTCAACAAATCATAAAGATATTGG-3′ and HCO2198 5′-TAAACTTCAGGGTGACCAAAAAATCA-3′ , 1 U of Taq polymerase (Fermentas) and varying concentrations of DNA and dH2O depending on the quality of the DNA extraction. Samples lacking genomic DNA were included in each PCR amplification as negative controls, and no products were detected in these negative controls. Amplification was preceded by one cycle of initial denaturation at 94 °C for 120 s, followed by six cycles of 94 °C for 30 s, 50 °C for 90 s and 72 °C for 60 s, and then 36 cycles of 94 °C for 30 s, 55 °C for 90 s and 72 °C for 60 s, with a terminal extension of 72 °C for 5 min. The yield and quantity of DNA were analysed using ethidium bromide staining and agarose gel electrophoresis. PCR products were cleaned using shrimp alkaline phosphatase and Eschericia coli exonuclease I (Fermentas) and sequenced directly on an ABI Prism 3730 Genetic Analyser machine using ABI BigDye Terminator Sequencing chemistry. Purified PCR products were sequenced using the same primers as for the PCR reaction, in both directions to minimise PCR artefacts, ambiguities and base-calling errors. Chromatogram output was checked by eye using Bioedit v.7  DNA. In such cases, these results were eliminated from the study. Since cox1 is a protein coding gene, only specimens for which a single open reading frame (ORF) was identified were included in the analyses. In total, our analyses include 103 new sequences, each derived from a single worm specimen, as well as nine sequences retrieved from GenBank: accession nos. JX519460, KM359418, KM359416, KM359417 for D. viviparus; JX519459 for D. eckerti (cf. red deer); JX519458 for Aelurostrongylus abstrusus; GQ888714 for Metastrongylus pudendotectus; GQ888715 for Metastrongylus salmi; and KF481953 for Protostrongylus rufescens. The last four species are related to Dictyocaulidae within the Strongylida order, and their sequences were included as outgroups in the phylogenetic analysis. All sequences generated in this study were deposited in GenBank under accession nos. KT372244–KT372346 (cox1) and KT438069–KT438071  following the manufacturer’s protocol. Each DNA sample was eluted using 200 μl of EA buffer, as supplied in the kit, and subsequently stored at −20 °C. The v.7 Hall . In a smcox1 were aligned using ClustalX version 2.0  as implemented in MODELTEST , GC nucleotide content, haplotype number (H), haplotype diversity (Hd), average number of nucleotid differences within groups (K) and nucleotide diversity (π), were calculated within species and populations using DnaSP version 5  relative to the entire population , as described by Harpending et al. (t) to the most recent common ancestor (tMRCA) for our samples was also estimated. This estimates the number of generations since the population expanded and was calculated from the peak distribution (τ) using the equation: t = τ/2μ (Li μ is the mutation rate per gene per generation and is obtained by multiplying the mutation rate per site per generation by the number of nucleotides in the studied fragment (657 bp in this case). The mutation rate of substitutions per site per generation was estimated using values for the mitochondrial DNA (mtDNA) of Caenorhabditis elegans: 1.57 × 10−7 ± 3.1 × 10−8  divided by generations per year gives an estimate of time in terms of number of years.The population history of D Tajima  and Fu’sution Fu , were usg et al. . The tim τ/2μ Li . The part Kassai ; howevercox1 Dictyocaulus sequences were amplified. Each sequence originates from a single lungworm specimen. In total, lungworms were collected from 47 individual deer (Table cox1 sequences were of the same length (657 bp) and could be aligned unambiguously. The resultant cox1 alignment corresponds to positions 69–725 bp of the complete mitochondrial genome sequence of D. eckerti cf. red der .A total of 103 Dictyocaulus cox1 sequences grouped into four main clades according to maximum likelihood phylogenetic analysis, revealing that lungworms collected from wild deer belong to three distinct clades  showed high similarity to D. eckerti  from the additional clade did not show close similarity to any currently known Dictyocaulus species; therefore, we consider it an unknown, probably undescribed species and refer to it here as D. sp. S-HU . Additionally, the lungworm sequence collected from a red deer in New Zealand (JX519459) is divergent with respect to the Hungarian D. eckerti samples, and the 0.094 mean pairwise sequence difference between the New Zealand sample and Hungarian sequences within the D. eckerti clade suggests these sequences may belong to different species.Amplified des Fig. . Sequencdes Fig.  compareddes Fig. , suggestdes Fig. . Additiocox1 tree indicates high genetic differentiation between the three Dictyocaulus species from wild deer, but little or no differentiation within each species according to locality or geographical region . The mean nucleotide differences and nucleotide diversity for D. eckerti were approximately two times higher than those for D. capreolus and D. sp. S-HU . The genetic diversity of D. eckerti within populations was consistent  and much higher than for D. capreolus . Hd values are close to 1 for all examined species, showing a diverse haplotype distribution. The number of haplotypes for D. eckerti, D. capreolus and D. sp. S-HU were 51, 13 and 6, respectively, and there were many polymorphic sites (2.9–14 %). Most haplotypes were represented by a single specimen . The most common haplotype of D. eckerti (HP5) comprises samples distributed through four regions .Interspecific pairwise sequence distances between sequences from separate Dictyocaulus species separately at several study levels. Because D. sp. S-HU is represented by relatively few samples, we focus our analyses on the datasets of D. eckerti and D. capreolus. The lungworm species examined showed different population genetic structures. The genetic structure of D. eckerti was low, with population estimates of FST, GST and NST consistently within the range 0.034–0.050 at all levels (Table Nm) values were high, indicating high gene flow amongst a priori defined populations. Estimated pairwise FST between populations of D. eckerti defined by collecting region ranged from 0.0037 to 0.0598  or locality levels. Based on roughly equal pairwise FST calculations, the three D. capreolus populations are equally isolated from each other (Table Nm = 3.27) suggest that D. capreolus has intermediate genetic structure. The samples of D. sp. S-HU grouped into two populations, revealing limited genetic differences, but this result should be regarded cautiously due to the small sample size.Population genetic analyses were conducted for the 98 Table . The hig98 Table . D. caprer Table . The oveD. eckerti samples collected from three host species . Maximum likelihood phylogenetic analyses revealed that samples of D. eckerti lungworms grouped into subclusters, which were not correlated with the host species  between host species suggest that D. eckerti uses multiple hosts and has well-connected populations in Hungary and with the Carpathian population.Genetic structure was tested for ies Fig. . HaplotyST Table . The lowns Table  and the D neutrality tests showed negative values for all three Dictyocaulus species .Tajima’s um Table . Deviatiape Fig. . The lowD. eckerti since there was no or only weak genetic evidence for differentiated populations. The peak of the unimodal distribution (τ) was 9.529, corresponding to a population expansion approximately 46,000 generations ago, which equals ∼11,500  years ago, placing the event at the end of the last Ice Age, assuming four generations per year. These calculations assume that the D. eckerti population is at equilibrium. Based on the unimodal mismatch distribution, a similar analysis was performed for all samples of D. capreolus collectively, as well as separately for two populations. For all samples collectively, the estimate of τ was 3.669, suggesting an expansion estimate ∼4,500 years ago (3700–5500 CI). Because of evidence of moderate genetic structure for D. capreolus and the observation that eastern (E) and southwestern (SW) populations differ in genetic variability by about five times , and low raggedness (0.0363), indicating an expanded population. The unimodal mismatch distribution for the eastern population . However, the SW population of D. capreolus exhibited a low negative Tajima’s D  and low positive Fu’s Fs  with moderate raggedness (0.23), suggesting a roughly constant population size. The mismatch distribution of the SW population of D. capreolus , lungworms are believed to be pathogenic in farmed or fenced circumstances within temperate regions . Given the patterns of sequence divergence within (<2 %) and between clades (>13 %), our results strongly suggest that these three clades correspond to separate lungworm species in Hungary. Whilst support for our clades of interest is strong, the values amongst major clades are poor, limiting our ability to elucidate evolutionary relationships amongst them. A previous phylogenetic analysis of European Dictyocaulus species using rDNA ITS2 recovered a different pattern from that observed in our results, with D. capreolus more basal, although support amongst clades was similarly low  would be of interest to determine the origin and identity of these worms.Phylogenetic analyses reveal that d et al.  also notD. eckerti is a frequent parasite in wild and semi-domesticated hosts and is recorded from several host species: fallow deer, hog deer (Axis porcinus), Indian muntjac (Muntiacus muntjak), moose , musk ox (Ovibos moschatus), red deer, reindeer (Rangifer tarandus), sika deer (Cervus nippon) and wapiti . We find that red deer is the primary host for D. eckerti within the region that we sampled. Although D. eckerti samples were recovered from all three deer species considered here, the vast majority of worms originated from red deer. However, to some extent, this may reflect sampling bias since 70 % of the deer sampled in this study were red deer. All of the six lungworms collected from fallow deer were identified as D. eckerti. The prevalence and abundance of dictyocaulid worms in fallow deer are very low in comparison to values in red deer (unpublished results). The distribution of fallow deer, which is patchy, may provide only a secondary host for the parasite, but more sampling is required to confirm this and to ascertain the focal host of D. eckerti and if this varies across its large range.Regarding host relationships, D. eckerti, only 1 of 17 D. capreolus worms originated from red deer, with the rest sampled from roe deer. Thus, our data suggest that D. capreolus is a roe deer specialist in Hungary, although in Sweden it was also recovered from moose, suggesting a complex pattern of host associations . We could not identify any ecological factors underlying genetic differentiation between D. eckerti and D. sp. S-HU, and, hence, an interesting question is what factors exist to promote reproductive isolation between them. We did not investigate the closely related cattle lungworm, D. viviparus, here, but studies report that it is widespread in Hungary  shows high population genetic structure far above the critical FST value. It is likely that D. viviparus has very low gene flow as the cattle hosts of D. viviparus are isolated by farms. Wild deer are not suitable hosts for D. viviparus  nematode parasite Mazamastrongylus odocoilei in North America shows high genetic diversity and moderate genetic structure (NST = 0.12 and 0.31; Blouin et al. M. odocoilei was studied using mitochondrial DNA sequences of the ND4 region, which is more variable than the cox1 locus, and also that the sites examined in America were situated at larger distances than those in our study.The estimated distributions of lungworm species examined in this study are larger than the sampling area. Whilst D. eckerti, can migrate large distances, whilst roe deer migrate less and are considered to be a territorial species (Kropil et al. Dictyocaulus in red and roe deer. The prevalence of infection values were 8.3, 13.0 and 46.6 % for forest-based roe deer, field-based roe deer and red deer, respectively (Sugár Dictyocaulus species.Several studies have reported that the most important factor to impact on parasite population structure is the vagility of hosts (Levin and Parker ly Sugár , 1997. PD. eckerti with high gene flow between host species probably switch hosts often and may not experience such extreme population bottlenecks compared to worms restricted to a single host species. D. capreolus is reported to utilise an additional host species to roe deer in Sweden, the moose (Gibbons and Höglund D. capreolus in Sweden may show lower genetic structure than those examined here in Hungary, particularly as the moose is a long-distance disperser (Sweanor and Sandegren The differing levels of gene flow observed in lungworms have consequences for population dynamics and evolutionary potential (Barrett et al. D. eckerti has not experienced a severe recent population bottleneck and that there was a population expansion ∼11,500 years ago (although these results should be interpreted with caution, e.g. see Morrison and Höglund D. eckerti population expansion is likely to be driven by the population expansion of its hosts. The population expansion time estimate is concordant with host migration and population expansion after the last Ice Age since climate warming began approximately 15,000 years ago (Denton et al. D. eckerti across a larger spatial scale, including western and Medierreanean populations, may indicate greater genetic structure, following the main host’s genetic structure. In future studies, it would be interesting to examine whether lungworm genetic structure reflects that of its red deer hosts at larger scales across Europe and to what extent worms arising from different refugial populations have spread across distinct European host populations.Our results suggest that D. eckerti in this study likely reflects a parasite population that extends over a larger spatial scale than our study area. When a population expands, it is expected to gain rare alleles, which we observe here for D. eckerti. The predicted large distribution, high genetic diversity and high gene flow for D. eckerti have important evolutionary consequences and offer the potential for new mutations to spread rapidly. The majority of red deer in Hungary are infected by lungworms during their first year (Sugár D. capreolus is likely to have a much smaller overall population size than D. eckerti. The high population size of D. eckerti could maintain high genetic diversity and an ability to respond quickly to forces of selection, and the impact of genetic drift should be negligible compared to that of natural selection. These predictions have considerable implications for lungworm management, particularly since high gene flow enhances the efficient evolution of resistance to treatment methods.The high gene flow observed for ar Sugár , 1997, w