Crete, a major Mediterranean biodiversity hotspot, hosts many local endemic, threatened and/or protected plant taxa (species and subspecies). Besides their ecological and conservation significance, these unique phytogenetic resources hold significant economic potential for sustainable utilization. Since DNA barcoding is critical for conservation, taxonomy, and plant-derived product authentication, we studied 15 local Cretan endemic taxa using three chloroplast DNA (cpDNA) regions (rbcL, trnL, trnH-psbA). A comparative analysis against GenBank (NCBI) records revealed significant new data: (i) the first genetic information for five taxa (Centaurea redempta subsp. redempta, Galium fruticosum, Micromeria hispida, Salix kaptarae, Teucrium cuneifolium); (ii) new marker-specific sequences for seven taxa (Helichrysum heldreichii, Scutellaria hirta, Sesleria doerfleri, Staehelina petiolata, Teucrium alpestre, Campanula pelviformis, Phlomis lanata); and (iii) novel genotypes of already represented markers for three species (Phlomis lanata, Scutellaria sieberi, Staehelina petiolata). Phylogenetic analyses were performed for all three molecular markers across selected members of Scutellaria section Scutellaria, Teucrium section Polium, and Campanula section Quinqueloculares. The overall results indicated that, amongst the studied species, the trnH-psbA marker is more suitable for species-level identification, whereas the rbcL and trnL markers were more helpful to genus-level identification within Lamiaceae and Campanulaceae. These results enrich the DNA barcoding reference library and form a concrete contribution towards the protection, conservation and traceability of Crete's unique botanical heritage.

BACKGROUND AND AIMS:Plant functional traits link environmental conditions to plant performance and adaptation. Growing evidence suggests that intraspecific trait variation can be as important as differences between species, yet large intraspecific studies of in-situ variation remain rare. While most studies have focused on plant morphological traits, the concentrations of elemental nutrients in seeds have received much less attention so far. METHODS:We conducted a large-scale in-situ study of the widespread annual ruderal grass Hordeum murinum. We sampled 2070 individuals from 207 populations across a large part of its native range in Europe and North Africa. We measured seed ripening phenology and growth-related traits in-situ and analyzed concentrations of elemental nutrients in the seeds. KEY RESULTS:We found that Hordeum murinum grew larger, produced seeds later, and had heavier seeds in colder and wetter regions. Plants growing in denser vegetation were taller and produced heavier seeds but formed fewer spikes. Concentrations of elemental nutrients in the seed generally declined with seed weight and were primarily driven by climatic variables, whereas soil conditions had only minor effects on plant traits and seed nutrients. Population identity explained a substantial proportion of trait variation, indicating a possible genetic component. CONCLUSIONS:Our findings provide a comprehensive view of how Hordeum murinum responds to environmental gradients across its European distribution. Climatic variables, particularly temperature, are key drivers of reproductive timing and concentrations of elemental nutrients in the seed, whereas local environmental conditions, such as biotic pressures, are more critical for growth-related traits. Together, these patterns indicate that Hordeum murinum modulates its growth and reproductive investment along environmental gradients, balancing phenology, stress tolerance, and limited competitive capacity.

The taxonomic identification of plant species is traditionally based on morphological traits, the use of which may create difficulties in cases of close-related species showing great morphological variability. In such cases, the use of DNA markers for species identification and delimitation can be of great help. Himantoglossum W.D.J.Koch (Orchidaceae) is a genus with notable morphological variability, comprising the clade hircinum-caprinum (Himantoglossum s.s.) with nine taxa, from which H. jankae, H. hircinum, H. montis-tauri, H. caprinum and H. samariense have being reported in Greece. However, a previous morphological study of Himantoglossum s.s. from all over Greece could not verify the presence of these reported species, but of only one highly diverse taxon throughout the country. Here, we studied the genetic variation and differentiation of Himantoglossum s.s. populations from the entire distribution of the genus in Greece employing ISSR markers, to further elucidate the taxonomic status of Himantoglossum s.s. in Greece. High genetic variation was revealed, both in the populations of the “core” distribution and in the peripheral/marginal ones, pointing to their evolutionary potential. This variation is mainly attributed to differences within the populations and, to a lesser extent, among them. No differentiation of the populations proposed to belong to a different taxon was found and no species-specific markers were identified that may discriminate the above populations from the rest. In addition, two cpDNA and one nDNA fragments (accD, psbA-trnH and ITS2, respectively) were sequenced in a number of individuals representative of the whole dataset. All three fragments were conserved, showing restricted polymorphism and having no correlation to the populations or to the taxa of Himantoglossum s.s. in Greece. Overall, the high genetic variation of the populations of Himantoglossum s.s. in Greece, especially of the peripheral/marginal ones, is a valuable asset towards their conservation.

IntroductionThe woody angiosperm Styrax officinalis L., primarily occurring in the Near East and South-eastern Europe, has been historically considered a human introduction in the Italian Peninsula.MethodsTo challenge this assumption, we conducted a genetic analysis on a comprehensive sample of individuals across its range, utilizing chloroplast and nuclear microsatellites as well as a genome-wide single-nucleotide polymorphism (MIG-seq) sequencing approach.ResultsAnalysis of 351 individuals revealed clear genetic structure across the species’ range. Most Italian populations form a distinct nuclear genetic cluster, suggesting long-term isolation, while three populations show signs of admixture with Cypriot individuals. Although one rare chloroplast haplotype was unique to Italy, widespread eastern haplotypes were entirely absent from the peninsula, which does not support the hypothesis of a recent human-mediated introduction.DiscussionThe results largely support the indigenous nature of the species in the Italian Peninsula, rejecting the notion of recent human introduction, and elevate S. officinalis to the status of a local relict, probably representing a component of the Late Neogene warm-temperate vegetation in Southern Paleo Europe.

Tuberous orchids are ecologically vulnerable species, threatened by a range of environmental pressures such as overharvesting, grazing and land use change. Conservation efforts require accurate species identification, but are impeded by limited phylogenetic resolution of traditional genetic markers, which is exacerbated by widespread taxonomic conflict regarding the classification of orchids. Target enrichment holds promise to resolve both these challenges by offering a large set of nuclear loci with which to increase phylogenetic resolution and evaluate competing species models. Here, we evaluate the effectiveness of the Angiosperms353 markers for distinguishing over 50 tuberous orchid species native to Greece and we explore the possibility of narrowing these markers to a smaller set that could function as a minimal probe set. Our methodology consists of a three-tiered approach: 1) generating a species-level phylogeny using all Angiosperms353 loci with sufficient target recovery, 2) evaluating competing species models based on "splitter" and "lumper" classifications through Bayes Factor species delimitation, and 3) ranking the potential of Angiosperms353 loci to discriminate representatives of lineages with different divergence times based on their phylogenetic informativeness. While the inferred multi-species coalescent phylogeny had overall high support, Bayes Factor delimitation revealed mixed outcomes, favouring splitting in Serapias, while favouring splitting in basal clades and lumping in more recently diverged clades in Ophrys. A molecular clock analysis of Ophrys confirms rapid and recent radiation in clades marked by phylogenetic uncertainty, suggesting the need for additional loci to fully resolve this genus. Finally, we found 30 loci to be highly phylogenetically informative across four epochs of Orchidinae evolution; we suggest these are promising candidates for future marker development. Our findings enhance the Plant Tree of Life (PAFTOL) by contributing additional phylogenomic data for species that were previously underrepresented in trees built with these markers, while shedding light on the ongoing "splitter"-vs-"lumper" debate and offering new directions for species identification of tuberous orchids, a group with distinct taxonomic and conservation challenges.

Evolutionary radiation, a pivotal aspect of macroevolution, offers valuable insights into evolutionary processes. The genus Pinus is the largest genus in conifers with c.$$ c. $$ 90% of the extant species emerged in the Miocene, which signifies a case of rapid diversification. Despite this remarkable history, our understanding of the mechanisms driving radiation within this expansive genus has remained limited. Using exome capture sequencing and a fossil-calibrated phylogeny, we investigated the divergence history, niche diversification, and introgression among 13 closely related Eurasian species spanning climate zones from the tropics to the boreal Arctic. We detected complex introgression among lineages in subsection Pinus at all stages of the phylogeny. Despite this widespread gene exchange, each species maintained its genetic identity and showed clear niche differentiation. Demographic analysis unveiled distinct population histories among these species, which further influenced the nucleotide diversity and efficacy of purifying and positive selection in each species. Our findings suggest that radiation in the Eurasian pines was likely fueled by interspecific recombination and further reinforced by their adaptation to distinct environments. Our study highlights the constraints and opportunities for evolutionary change, and the expectations of future adaptation in response to environmental changes in different lineages.

The distribution of vascular plant species and assemblages existing in beech (Fagus sylvatica L.) forests was compared with the distribution of beech chloroplast DNA (cpDNA) haplotypes, aiming to identify possible interpretable trends of co-occurrence, on a small geographical scale, and to infer the relevant historical factors. Vegetation and genetic (cpSSR) data were collected from 60 plots on Mt. Menikio (northeastern Greece). Classification and ordination analyses were applied on the vegetation data, while on the cpSSR data, diversity measures and genetic structure analyses were employed. A probabilistic co-occurrence analysis was performed on haplotypes and taxa. The results show that a plant biogeographical border exists on Mt. Menikio which, in addition, has acted both as a refugium and as a meeting point of lineages for more than one glacial cycle. Significant associations of co-occurrence between haplotypes and vascular taxa were found but no common distribution patterns between the former and species assemblages were identified. The combined consideration of the distribution profiles of species assemblages, plant species and cpDNA haplotypes (corresponding to the three levels of biodiversity) provides concrete information on historical events, leading to a comprehensive understanding of the evolutionary and biogeographical processes that have shaped specific spatial patterns of biodiversity.

Scots pine is the foundation species of diverse forested ecosystems across Eurasia and displays remarkable ecological breadth, occurring in environments ranging from temperate rainforests to arid tundra margins. Such expansive distributions can be favored by various demographic and adaptive processes and the interactions between them. To understand the impact of neutral and selective forces on genetic structure in Scots pine, we conducted range-wide population genetic analyses on 2321 trees from 202 populations using genotyping-by-sequencing, reconstructed the recent demography of the species and examined signals of genetic adaptation. We found a high and uniform genetic diversity across the entire range (global FST 0.048), no increased genetic load in expanding populations and minor impact of the last glacial maximum on historical population sizes. Genetic-environmental associations identified only a handful of single-nucleotide polymorphisms significantly linked to environmental gradients. The results suggest that extensive gene flow is predominantly responsible for the observed genetic patterns in Scots pine. The apparent missing signal of genetic adaptation is likely attributed to the intricate genetic architecture controlling adaptation to multi-dimensional environments. The panmixia metapopulation of Scots pine offers a good study system for further exploration into how genetic adaptation and plasticity evolve under gene flow and changing environment.

Abstract Hybridization is a process of great importance in the ecology and evolution of plants. However, identifying hybrids is not an easy task, especially in cases of sympatry. Such cases are the Abies taxa in Greece; Silver fir (A. alba), Greek fir (A. cephalonica) and King Boris fir (A. × borisii-regis). The latter has been characterized as a hybrid between the first two species as its morphological traits are either a combination of the parental ones or intermediate. Besides the morphological traits, A. × borisii-regis has so far been identified only at the population level, with biochemical and DNA markers. Here, we describe the identification of A. × borisii-regis at the individual level by employing a species-specific marker in the cpDNA and another in mtDNA, taking advantage of their uniparental inheritance mode from the paternal and the maternal parent, respectively. In addition, based on these markers, essential information emerges on the distribution and the mtDNA diversity of the Abies taxa in Greece.

The consumption of poisonous plants can lead to serious health problems or even casualties due to various factors, including easy access to poisonous plants due to their common distribution, co-occurrence and resemblance with edible plants, and the lack of regulation in the food product supply chain. Clinical diagnosis of intoxications usually relies on the availability of the plant consumed by the patient and on the morphology of the plant parts found in the patient's stomach. Therefore, given the fragmented nature of ingested plant material, species identification may face serious difficulties, can be inaccurate, and time-consuming. This highlights the need for rapid and reliable tools to identify toxic species. In the present study, we developed an ITS2-high-resolution melting (HRM) assay for: (1) the discrimination of common toxic plants and their edible lookalikes, and (2) the detection of toxic plants in digested samples. More specifically, we designed species-specific ITS2 primers for the authentication of poisonous species in simulated mixtures and verified them with Bar-HRM. Moreover, the developed HRM-based molecular tool was capable of quantifying the toxic species Datura stramonium in simulated mixtures with the edible Amaranthus retroflexus down to at least 0.5% v/v. This study shows that species-specific ITS2 primers can amplify the DNA from fragmented and/or artificially digested samples and that Bar-HRM is capable of detecting poisonous plant species in digested samples even after 4 h. The developed Bar-HRM protocol has important implications for application in medicine, forensics, and the agricultural industry, either to accurately detect the cause of plant intoxications or as a tool for quality control in the supply chain. Practical Application In this work, we established a high-resolution melting DNA-based protocol capable of discriminating between phenotypically similar common toxic and edible plant species in mixtures, even at very low quantities. This technology also proved efficient in detecting the toxic species in mixtures digested in artificial gastric acid, as it would be the case after accidental ingestion. This work is expected to have important implications for application in medicine, forensics, and the agricultural industry, either for identifying the cause of plant intoxications or as a tool for quality control in different steps of the supply chain.

Fertilization in angiosperms involves the germination of pollen on the stigma, followed by the extrusion of a pollen tube that elongates through the style and delivers two sperm cells to the embryo sac. Sexual selection could occur throughout this process when male gametophytes compete for fertilization. The strength of sexual selection during pollen competition should be affected by the number of genotypes deposited on the stigma. As increased self-fertilization reduces the number of mating partners, and the genetic diversity and heterozygosity of populations, it should thereby reduce the intensity of sexual selection during pollen competition. Despite the prevalence of mating system shifts, few studies have directly compared the molecular signatures of sexual selection during pollen competition in populations with different mating systems. Here we analyzed whole-genome sequences from natural populations of Arabis alpina , a species showing mating system variation across its distribution, to test whether shifts from cross- to self-fertilization result in molecular signatures consistent with sexual selection on genes involved in pollen competition. We found evidence for efficient purifying selection on genes expressed in vegetative pollen, and overall weaker selection on sperm-expressed genes. This pattern was robust when controlling for gene expression level and specificity. In agreement with the expectation that sexual selection intensifies under cross-fertilization, we found that the efficacy of purifying selection on male gametophyte-expressed genes was significantly stronger in genetically more diverse and outbred populations. Our results show that intra-sexual competition shapes the evolution of pollen-expressed genes, and that its strength fades with increasing self-fertilization rates.

Herbal medicinal products gain increasing popularity. The growing demand for herbal medicine along with a lack of regulation render herbal products subject to intentional adulteration. The substitution of costly ingredients with unlabelled plant-based fillers of inferior quality has been widely reported. Such fraudulent practices erode consumer trust, but can also pose serious health risks. In this work, 71 herbal medicinal products were randomly purchased from Greek markets and analysed using ITS2 metabarcoding for species identification. The aim was to investigate possible adulterations and assess the efficacy of metabarcoding in plant-based product authentication. Of the 131 detected species in our analyses, 87 were not listed on the product labels. This indicates a high level of adulteration and/or contamination during processing and distribution. Two toxic species, Chelidonium majus and Nicotiana tabacum, were also detected as major ingredients of two herbal mixtures for medicinal purposes. Furthermore, the detection of wheat in eight samples raises concerns for people with gluten intolerance. This study stresses the need for stricter quality control of herbal products. In addition, to overcome the limitations of metabarcoding and augment the approach we used Bar-HRM for the first time as a verification tool. The combination of metabarcoding with species-specific Bar-HRM analysis can enhance the reliability of the results.

Plant mating systems have profound effects on levels and structuring of genetic variation, and can affect the impact of natural selection. While theory predicts that intermediate outcrossing rates may allow plants to prevent accumulation of deleterious alleles, few studies have empirically tested this prediction using genomic data. Here, we study the effect of mating system on purifying selection by conducting population genomic analyses on whole-genome resequencing data from 38 European individuals of the arctic-alpine crucifer Arabis alpina . We find that outcrossing and mixed-mating populations maintain genetic diversity at similar levels, whereas highly self-fertilizing Scandinavian A. alpina show a strong reduction in genetic diversity, most likely as a result of a postglacial colonization bottleneck. We further find evidence for accumulation of genetic load in highly self-fertilizing populations, whereas the genome-wide impact of purifying selection does not differ greatly between mixed-mating and outcrossing populations. Our results demonstrate that intermediate levels of outcrossing may allow efficient selection against harmful alleles whereas demographic effects can be important for relaxed purifying selection in highly selfing populations. Thus, both mating system and demography shape the impact of purifying selection on genomic variation in A. alpina . These results are important for an improved understanding of the evolutionary consequences of mating system variation and the maintenance of mixed-mating strategies. Significance Intermediate outcrossing rates are theoretically predicted to maintain effective selection against harmful alleles, but few studies have empirically tested this prediction using genomic data. We used whole-genome resequencing data from alpine rock-cress to study how genetic variation and purifying selection vary with mating system. We find that populations with intermediate outcrossing rates have similar levels of genetic diversity as outcrossing populations, and that purifying selection against harmful alleles is efficient in mixed-mating populations. In contrast, self-fertilizing populations from Scandinavia have strongly reduced genetic diversity, and accumulate harmful mutations, likely as a result of demographic effects of postglacial colonization. Our results suggest that mixed-mating populations can avoid the negative evolutionary consequences of high self-fertilization rates.

Epipactis Zinn. is a highly diverse genus, with numerous taxa naturally occurring throughout Europe. While their morphological variability has resulted in contradictory opinions about their taxonomy, the knowledge of the genetic variation of many of its species is limited. Such an example is E. atrorubens in the Balkan Peninsula. In this work, the cpDNA variation of seven E. atrorubens populations from northern Greece and of one population from Germany has been investigated by means of PCR-RFLP markers. Two regions of the cpDNA were studied (trnH-psbA, trnL intron) and four PCR-RFLP markers were employed (trnH-psbA/HaeIII, trnH-psbA/HinfI, trnH-psbA/VspI, trnL/MboI), which have revealed variation in other Epipactis species. The results demonstrate limited genetic variability in the studied populations, as only one haplotype was present in all the populations (both Greek and German), with the exception of Mt. Menikion (Greece) where a second haplotype was also recorded. This limited variation may be attributed to the properties of the genetic markers involved, to the conserved status of the two cpDNA regions in this species or to factors related to the biology and evolutionary history of E. atrorubens. The presence of a second haplotype only in Mt. Menikion could be attributed to a hybridisation event in the past and/or to the potential existence of a glacial refugium on this mountain.

The genus Pinus L. consists of ca. 110 ecologically and economically important species extending from the arctic zone to the tropics. Nevertheless, there is little information in the literature on DNA-based methods for the identification of pine species. Here, we identified a new cpDNA fragment (trnV-H/x-h) able to differentiate among pine species and correctly depict the phylogeny within the genus. The fragment was identified based on PCR-RFLP profiles and primers designed based on the sequences of six Pinus species naturally occurring in Greece (Pinus brutia Ten., Pinus halepensis Mill., Pinus leucodermis Antoine, Pinus nigra J.F. Arnold, Pinus pinea L, and Pinus sylvestris L). We analyzed 90 highly similar pine sequences retrieved from the GenBank to investigate specificity of our marker and the haplotypes found showed to be specific to Pinus and able to differentiate among 39 different species. The phylogenetic tree constructed using these species, correctly depicted the phylogeny of the genus up to the subsection level. These characteristics together with its relatively small size (376-418 bp) make the trnV-H/x-h marker useful for pine identification even in contexts where DNA is degraded, such as in timber tracing, forensic botany and palaeobotanical investigations. (C) 2016 Elsevier Ltd. All rights reserved.

Background Local adaptation is a key driver of phenotypic and genetic divergence at loci responsible for adaptive traits variations in forest tree populations. Its experimental assessment requires rigorous sampling strategies such as those involving population pairs replicated across broad spatial scales. Methods A hierarchical Bayesian model of selection (HBM) that explicitly considers both the replication of the environmental contrast and the hierarchical genetic structure among replicated study sites is introduced. Its power was assessed through simulations and compared to classical ‘within-site’ approaches (FDIST, BAYESCAN) and a simplified, within-site, version of the model introduced here (SBM). Results HBM demonstrates that hierarchical approaches are very powerful to detect replicated patterns of adaptive divergence with low false-discovery (FDR) and false-non-discovery (FNR) rates compared to the analysis of different sites separately through within-site approaches. The hypothesis of local adaptation to altitude was further addressed by analyzing replicated Abies alba population pairs (low and high elevations) across the species’ southern distribution range, where the effects of climatic selection are expected to be the strongest. For comparison, a single population pair from the closely related species A. cephalonica was also analyzed. The hierarchical model did not detect any pattern of adaptive divergence to altitude replicated in the different study sites. Instead, idiosyncratic patterns of local adaptation among sites were detected by within-site approaches. Conclusion Hierarchical approaches may miss idiosyncratic patterns of adaptation among sites, and we strongly recommend the use of both hierarchical (multi-site) and classical (within-site) approaches when addressing the question of adaptation across broad spatial scales.

Differences in needle traits of coniferous tree species are considered as the combined result of direct environmental pressure and specific genetic adaptations. In this study, diversity and differentiation within and among four Abies cephalonica subpopulations of a marginal population on Mt. Parnitha - Greece, were estimated using needle morphological traits and gene markers. We tested the connection of morphological variability patterns of light and shade needles with possible adaptation strategies and genetic diversity. Six morphological characteristics were used for the description of both light and shade needles at 100 trees, describing needle size and shape, stomatal density and needle position on the twigs. Additionally, six RAPD and three ISSR markers were applied on DNA from the same trees. Light needles were significantly different than shade needles, in all traits measured, apparently following a different light harvesting strategy. All four subpopulations exhibited high genetic diversity and the differentiation among them was relatively low. Differences among populations in light needles seemed to depend on light exposure and aspect. In shade needles, the four subpopulations seemed to deviate stronger from each other and express a rather geographic pattern, similarly to the genetic markers. Two of the subpopulations studied were lost during a wildfire, two years after sampling. Although the subpopulations burnt were most diverse and most differentiated, we expect a large part of the total genetic diversity of the burnt trees to still exist in the surviving subpopulations, since gene flow must have been effective in keeping all subpopulations connected.

Premise of research. Even though the Balkan Peninsula is a biodiversity hot spot in southeastern Europe harboring many endemic plants, very little is known about the temporal extent of the phylogeographic history and the contemporary genetic diversity of the endemics there. Haberlea rhodopensis is one of the European Gesneriaceae species occurring in this region and represents appropriate study material to address these questions.Methodology. We generated fossil-dated molecular phylogenies (atpB-rbcL, trnH-psbA, trnLF, ITS) across the Lamiales to determine the origin and age of H. rhodopensis and conducted phylogeographic (trnH-psbA, ITS) and population genetic (ISSRs) analyses on 17 populations from Bulgaria and Greece, covering the entire distribution range of the species, to investigate their biogeographic history, present-day genetic diversity, and differentiation levels.Pivotal results. The European Gesneriaceae genera have a Tertiary origin in the early Oligocene, while the Haberlea lineage emerged in the late Oligocene. Extinctions appear to have marked the history of the genus for a long period of time, and the extant populations diverged in the late Pleistocene. A significant differentiation was apparent between populations from Bulgaria, that is, the Balkan Mountains in the north and the Bulgarian side of the Rhodopi massif in the south, but there was an even stronger differentiation between the latter and populations from Greece. This might be explained by the Rhodopi massif representing a barrier to gene flow, enforced during the Last Glacial Maximum, during which populations descended on opposing north- and south-facing mountain slopes.Conclusions. Haberlea represents an ancient lineage with recent diversification. The extant populations are of recent origin and indicate glacial refugial areas in Bulgaria and Greece. This study sheds light on historic and current phylogenetic and phylogeographic events that shaped the flora of the Balkan Peninsula, an area that has long been recognized for its species diversity and richness. Our data suggest that it may have acted as a persistent refugial area in southeast Europe since the mid-Tertiary.