The challenge of species delimitation in the diploid-polyploid complex Veronica subsection Pentasepalae

Nathalie Machon

A reliable taxonomic framework and the identification of evolutionary lineages are essential for effective decisions in conservation biodiversity programs. However, phylogenetic reconstruction becomes extremely difficult when polyploidy and hybridization are involved. Veronica subsection Pentasepalae is a diploid-polyploid complex of ca. 20 species with ploidy levels ranging from 2x to 10x. Here, DNA-ploidy level estimations and AFLP fingerprinting were used to determine the evolutionary history, and species boundaries were reviewed in an integrated approach including also previous data (mainly morphology and sequence-based phylogenetic reconstructions). Molecular analyses were performed for 243 individuals from 95 populations, including for the first time all taxa currently recognized within the subsection. Phylogenetic reconstruction identified four main groups corresponding almost completely to the four clusters identified by genetic structure analyses. Multiple autopolyploidizat...

Accepted Manuscript Editor’s Choice Article The challenge of species delimitation in the diploid-polyploid complex Veronica subsection Pentasepalae. Nélida Padilla-García, Blanca M. Rojas-Andrés, Noemí López-González, Mariana Castro, Sílvia Castro, João Loureiro, Dirk C. Albach, Nathalie Machon, M. Montserrat Martínez-Ortega PII: DOI: Reference: S1055-7903(17)30406-2 https://doi.org/10.1016/j.ympev.2017.11.007 YMPEV 5968 To appear in: Molecular Phylogenetics and Evolution Received Date: Revised Date: Accepted Date: 2 June 2017 15 November 2017 15 November 2017 Please cite this article as: Padilla-García, N., Rojas-Andrés, B.M., López-González, N., Castro, M., Castro, S., Loureiro, J., Albach, D.C., Machon, N., Montserrat Martínez-Ortega, M., The challenge of species delimitation in the diploid-polyploid complex Veronica subsection Pentasepalae., Molecular Phylogenetics and Evolution (2017), doi: https://doi.org/10.1016/j.ympev.2017.11.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. The challenge of species delimitation in the diploid-polyploid complex Veronica subsection Pentasepalae. Nélida Padilla-Garcíaa,b,c,*, Blanca M. Rojas-Andrésa,b,1, Noemí López-Gonzáleza,b, Mariana Castrod, Sílvia Castrod, João Loureirod, Dirk C. Albache, Nathalie Machonc, M. Montserrat Martínez-Ortegaa,b a b Departamento de Botánica, University of Salamanca, E-37007, Salamanca, Spain Biobanco de ADN Vegetal, University of Salamanca, Edificio Multiusos I+D+i, Calle Espejo s/n, 37007 Salamanca, Spain c Centre d’Ecologie et des Sciences de la Conservation (CESCO, UMR7204), Sorbonne University, MNHN, CNRS, UPMC, CP51, Paris, France d CFE, Centre for Functional Ecology, Department of Life Sciences, University of Coimbra, Calçada Martim de Freitas s/n, 3000-456 Coimbra, Portugal e Department of Biology and Environmental Sciences (IBU), AG Biodiversity and Evolution of Plants, Carl von Ossietzky Universität Oldenburg, Oldenburg 26111, Germany * Corresponding author at: Departamento de Botánica, University of Salamanca, E-37007, Salamanca, Spain E-mail address: nelidapg@gmail.com (N. Padilla-García) Abstract A reliable taxonomic framework and the identification of evolutionary lineages are essential for effective decisions in conservation biodiversity programs. However, phylogenetic reconstruction becomes extremely difficult when polyploidy and hybridization are involved. Veronica subsection Pentasepalae is a diploid-polyploid complex of ca. 20 species with ploidy levels ranging from 2x to 10x. Here, DNA-ploidy level estimations and AFLP fingerprinting were used to determine the evolutionary history, and species boundaries were reviewed in an integrated approach including also previous data (mainly morphology and sequence-based 1 Present address: Department of Molecular Evolution and Plant Systematics & Herbarium (LZ), Institute of Biology, Leipzig University, Leipzig, Germany phylogenetic reconstructions). Molecular analyses were performed for 243 individuals from 95 populations, including for the first time all taxa currently recognized within the subsection. Phylogenetic reconstruction identified four main groups corresponding almost completely to the four clusters identified by genetic structure analyses. Multiple autopolyploidization events have occurred in the tetraploid V. satureiifolia giving rise to octoploid entities in central Europe and north of Spain, whereas hybridization is demonstrated to have occurred in several populations from the Balkan Peninsula. Furthermore, our study has established the taxonomic status of taxa, for the most part recovered as monophyletic. Cryptic taxa within the group have been identified, and a new species, Veronica dalmatica, is fully described. This study highlights the implications of polyploidy in species delimitation, and illustrates the importance to conserve polyploid populations as potential sources of diversification due to evolutionary significance of genome duplications in plant evolution. Keywords AFLPs, autopolyploidy, hybridization, Pentasepalae, phylogeny, Veronica dalmatica 1. Introduction The delimitation of species boundaries is a classic problem for biologists. Until about seventy years ago, taxonomists have focused on morphological differences between species rather than on coherence with the evolutionary history of the species for their circumscription. However, since the 1940’s, a wider interest in the evolutionary history of organisms arose (Huxley, 1940). In 1950, Hennig published his theory of phylogenetic systematics giving rise to the origin of cladistics, which revolutionized the field of taxonomy (Hennig, 1950). Despite originally considered for the analysis of morphological characters, it is equally suitable for other types of characters that have been used with taxonomic purposes during the last decades. Currently, molecular phylogenies, complementing morphological characters, are the key 2 instruments for biologists and biosystematists who try to understand the evolutionary processes that shape the history of species. Nevertheless, evolutionary histories involving radiations or complex processes such as hybridization, introgression and/or polyploidization, complicate phylogenetic reconstruction (Naciri and Linder, 2015). This, together with a lack of morphological differences and uncertainties over reproductive isolation among polyploids and their diploid progenitors, has resulted in taxonomic biases within polyploid complexes (Soltis et al., 2007, Barley et al., 2013). Here, the importance of the species concept is fundamental. The biological concept of species proposed by Mayr (1942) is difficult to apply when working with closely related species in which hybridization and introgression are common. Most plant taxonomists have traditionally relied on morphology to delimit species boundaries (i.e., morphological species concept), whereas others adopted in the last decades a concept based on genetic differences and monophyly (i.e., genetic and phylogenetic species concepts). However, in species groups with frequent hybridization and polyploidization, the general lineage concept (de Queiroz 2005, 2007) may be more appropriate. According to this concept, species are defined as separately evolving metapopulation lineages, which can be identified by different properties that species accumulate during the process of speciation (e.g., reproductive isolation, morphological or genetic differences, monophyly, etc.). This general lineage concept has been broadly adopted and promoted the development of an integrative taxonomic approach in which multiple and complementary methods are integrated to delimit species boundaries (Dayrat, 2005). This approach argues that taxonomic inference should be based on congruence across different types of characters and analyses. When results from different sources of data are incongruent, caution to delimit species is preferable since taxonomic conclusions may have significant implications (Carstens et al., 2013). For example, regarding conservation and sustainable use of natural resources, in accordance with the Convention on Biological Diversity (https://www.cbd.int/gti/importance.shtml), taxonomy is necessary for effective decision making, because it provides basic understanding about the components of biodiversity. In a 3 world where wild species are increasingly under threat, the conservation status of a taxon can only be correctly evaluated under the light of a clear taxonomic framework (Mace, 2004). The identification and preservation of evolutionary processes is also essential in conservation programs, especially for endangered, rare and endemic species. In the present study, complementary methodologies are used to address the taxonomic challenges of a study group with a complex evolutionary history, Veronica subsection Pentasepalae Benth. This subsection is a monophyletic lineage within Veronica subgenus Pentasepalae (Benth.) M.M. Mart. Ort., Albach & M.A. Fisch. (Rojas-Andrés et al., 2015). It has a recent origin (mean crown age 2.8 Mya., Meudt et al., 2015) and is composed of ca. 20 closely related perennial species distributed in Eurasia and North Africa. Interestingly, the group comprises five species and three subspecies endemic to single countries or sometimes only a small area within one country. Some of them are included in regional, national and/or international Red Lists (Peñas de Giles et al., 2004; Cabezudo et al., 2005; Alcántara de la Fuente et al., 2007; Petrova and Vladimirov, 2009; Bilz, 2011; International Union for Conservation of Nature, 2016), although there is a clear lack of information for numerous species that have not yet been carefully evaluated. The most important diversification center of V. subsection Pentasepalae is the Balkan Peninsula. The group is characterized by the presence of a pentapartite calyx with the fifth sepal being significantly smaller, by a capsule usually rounded at the base, and a base chromosome number of x = 8. However, although the group is well defined within Veronica (Albach et al., 2008), the existence of morphologically intermediate forms within the subsection due to overlapping morphological character states makes V. subsection Pentasepalae one of the taxonomically most complicated groups within the genus (Albach and Meudt, 2010). Since Bentham described V. subsection Pentasepalae in 1846, numerous taxonomic treatments have been proposed (for a historical review of monographs and Floras, see Rojas-Andrés and Martínez-Ortega, 2016), and several studies based on morphological, karyological or molecular data have tried to elucidate the evolutionary history of 4 the group (e.g., Martínez-Ortega et al., 2000, 2004, 2009; Andrés-Sánchez et al., 2009). In the most recent molecular study, Rojas-Andrés et al. (2015) used nuclear and plastid DNA sequence data to perform a phylogenetic analysis of the subsection. Despite the contributions of that study to the understanding of the evolutionary history of the group, a high degree of incongruence was found between the ITS and plastid DNA trees, probably caused by hybridization and incomplete lineage sorting (ILS). Hence, some questions about the monophyly and the relationships among species remained unresolved. Such questions are unlikely to be answered using a few loci alone, especially considering the prevalence of hybridization and polyploidization in the genus (Albach and Chase, 2004). The variety of ploidy levels in the subsection, ranging from diploid to decaploid (data previous to 2008 summarized by Albach et al., 2008; Rojas-Andrés et al., 2015), indicate that polyploidy has been a crucial process in the evolution of the group. Polyploidy or wholegenome duplication (WGD) is a frequent mechanism of evolution and speciation in flowering plants (Stebbins, 1950; Grant, 1971; Soltis et al., 2004, 2009, 2015; Mayrose et al., 2011; Kellog, 2016). Despite ongoing research regarding the distinction between the types of polyploids (Levin, 2002; Soltis et al., 2010; Husband et al., 2013; Doyle and Sherman-Broyles, 2017), two main categories are generally recognized based on their origin: i) autopolyploids that arise within a species, via intraspecific hybridization and duplication of similar genomes (homologous) and ii) allopolyploids formed by interspecific hybridization and chromosome doubling of genomes that are more or less divergent (homeologous). The prevalence of different types of polyploids in nature has been intensively discussed (Müntzing, 1936; Stebbins, 1947; Lewis, 1980; Parisod et al., 2010), and recent studies suggest a parity in the incidence of autopolyploidy and allopolyploidy (Barker et al., 2016 but see Doyle and Sherman-Broyles, 2017). The differentiation between these processes is fundamental to evaluate the importance of polyploidization and hybridization in plant evolution. In this context, the diploid-polyploid 5 complex Veronica subsection Pentasepalae is an excellent model to gain deeper insights into the contribution of these mechanisms to the evolutionary history of angiosperms. The aim of this study is to clarify the phylogenetic relationships of V. subsection Pentasepalae by analyzing the nuclear genome using Amplified Fragment Length Polymorphism (AFLP). In addition to its use in phylogeographic studies, the AFLP technique is now widely used to infer phylogenetic relationships and to identify hybridization and polyploid events in recently evolved polyploid non-model groups (Meudt, 2011; Reberning et al., 2012; Himmelreich et al., 2014; Zozomová et al., 2014). Compared to the previous study by RojasAndrés et al., (2015), in addition to using AFLPs, a molecular tool for which markers are distributed throughout the genome, we expand the study to include for the first time all taxa currently recognized within the subsection. Also, we added individuals that are difficult to identify to species and of different ploidy level from mixed-ploidy populations. AFLPs were generated to address the following specific points: i) The role of auto- and allopolyploidization processes in the evolutionary history of V. subsection Pentasepalae; ii) Implications of these processes in species delimitation and classification; iii) A review of the taxonomic status of V. subsection Pentasepalae taxa. 2. Materials and methods 2.1. Plant material Samples were collected in the field during 2009–2015 except for one population of V. satureiifolia and one population of V. tenuifolia subsp. fontqueri that were collected in 2002. Localities, initial taxonomic assignment, and further information about samples are given in Table S1. Fresh leaf material was collected and stored in silica gel. For V. krylovii, three individuals were included of which two were selected from herbarium material and one from a cultivated specimen in the Botanical Garden of Oldenburg (Germany). Veronica orientalis, which belongs to V. subsection Orientales (Wulff) Stroh of V. subgenus Pentasepalae (Benth.) 6 M.M. Mart. Ort., Albach & M.A. Fisch. was chosen as outgroup. The complete data-set comprises 243 individuals from 95 populations (outgroup included) covering the geographic distribution of each taxon (Fig. 1). From each location, 2–3 individuals were included, except for populations with mixed-ploidy levels. In these exceptional cases, two individuals per cytotype were analyzed whenever possible. Initial plant identification was based on the most recent taxonomic treatment (Rojas-Andrés and Martínez-Ortega, 2016), with the exception of some taxa that were not recognized by those authors, but whose names are used here to test their status [i.e., V. crinita f. bosniaca and V. thracica were included as synonyms of V. crinita, and V. macrodonta as a synonym of V. austriaca subsp. austriaca in that taxonomic treatment]. Material that was difficult to identify was initially catalogued using morphological affinity to other species (e.g., V. affinis linearis). Additionally, V. austriaca subsp. jacquinii / V. orbiculata indicates individuals of intermediate morphology between both species. Vouchers are deposited in the herbaria ALTB, GDA, MGC, OLD, SALA, VANF and WU (herbarium acronyms follow Thiers, 2017). 2.2. DNA-ploidy level estimation using flow cytometry DNA-ploidy levels were estimated by flow cytometry using silica gel dried leaves. Individuals from each sampled population were measured separately. Nuclear suspensions were prepared following the method described by Galbraith et al. (1983) in which the leaf tissue of each individual was chopped together with leaf tissue from an internal standard using a sharp razor blade in a Petri dish containing a buffer solution, namely Woody Plant Buffer (WPB; Loureiro et al., 2007). Solanum pseudocapsicum L. (2C = 2.589 pg; Temsch et al., 2010), Zea mays L. ‘CE-777’ (2C = 5.43 pg; Lysak and Dolezel, 1998), Pisum sativum L. ‘Ctirad’ (2C = 9.09 pg; Doležel et al., 1998) and Pisum sativum L. ‘Kleine Rheinländerin’ (2C = 8.84 pg; Greilhuber and Ebert, 1994) were used as internal standards depending on the C-value and standard availability. The suspension of isolated nuclei was filtered through a 48 µm nylon mesh, incubated with RNase to degrade double stranded RNA and stained with a saturating 7 solution of propidium iodide following Loureiro et al. (2007) and Rojas-Andrés et al. (2015). For each individual, one run of 5,000 counts was made on a CyFlow SL (Partec GmbH, Münster, Germany; equipped with a 488 nm solid-state laser) or a CyFlow Space (Partec GmbH, Münster, Germany; equipped with a 532 nm solid-state laser). Results were acquired using Partec FloMax software v2.4d (Partec GmbH, Münster, Germany). A proxy of the holoploid genome size (2C) was calculated as follows: Veronica 2C nuclear DNA content (pg) = (Veronica G1 peak mean/internal standard G1 peak mean)*genome size of the internal standard. The DNA-ploidy level was estimated for each sample based on the values of the genome size proxy and the available chromosome counts for the studied species (Martínez-Ortega et al., 2004; Albach et al., 2008). 2.3. DNA extraction and AFLP genotyping Total genomic DNA was extracted from silica gel dried material following the CTAB protocol of Doyle and Doyle (1987). The quality of the extracted DNA was checked on 1% TAE-agarose gels and the amount of DNA was estimated using a Nanodrop 2000C Spectrophotometer (Thermo Scientific). All extractions are stored at -80 ºC at the “Biobanco de ADN Vegetal” (University of Salamanca, Spain). The AFLP procedure followed the method described by Vos et al. (1995) with slight modifications. Genomic DNA (ca. 100 ng) was digested with MseI (New England BioLabs) and EcoRI (Fermentas) and ligated to doublestranded adaptors with T4 DNA-Ligase (Thermo Scientific) in a single restriction-ligation reaction for 3h at 37 ºC. Products were diluted and pre-amplified using primers EcoRI-A (5' GAC TGC GTA CCA ATT CA - 3') and MseI-C (5' GAT GAG TCC TGA GTA AC - 3'). Taq DNA Polymerase (BIOTOOLS BandM Labs. S.A) was used in the following PCR conditions: 2 min at 72 ºC, 29 cycles of 30 s at 94 ºC, 30 s at 56 ºC and 2 min at 72 ºC, and a final extension of 10 min at 72 ºC. At this step, the pre-selective amplified fragments were visualized on 1% TBE-agarose gel. After dilution of pre-selective products, selective amplifications were performed with the following PCR profile: 10 min at 95 ºC, 13 cycles of 30 s at 94 ºC, 1 min at 8 65 ºC (decreasing 0.7 ºC in each cycle) and 2 min at 72 ºC, followed by 24 cycles of 30 s at 94 ºC, 1 min at 56 ºC and 2 min at 72 ºC, with a final extension of 10 min at 72 ºC. All PCR reactions were performed on an Eppendorf-Mastercycler-Pro thermocycler. Twelve individuals from ten different taxa representing the whole diversity of the final dataset were used to screen a total of eight different combinations of selective primers. Four primer combinations were finally selected (Table S2) based on the number and clarity of the peaks, and the polymorphism level observed among individuals, which was checked to be sufficiently variable (i.e., overall genetic similarity among individuals from the same population was higher than that found among individuals from different populations, and much higher than the similarity detected among individuals from different taxa). Final selective PCR products were multiplexed for genotyping using the internal GeneScan 500 LIZ Size Standard (Applied Biosystems) in a multi-capillary sequencer ABI Prism 3730 (Applied Biosystems). Negative controls were run at each step of the process and 4.5% (= 11) of the samples were replicated in each independent run of PCR from the same extracted DNA to assess genotyping errors (Bonin et al., 2004, 2007; Pompanon et al., 2005). Final error rate was estimated after automated scoring according to Bonin et al. (2004) by comparing the 1/0 matrices obtained for the replicated samples. Differences detected here could be due either to technical causes and/or to the automated scoring process. The degree of reproducibility of the data set was also investigated analyzing the placement of replicates in a Neighbor-Joining tree. 2.4. Optimization Procedure of Automated AFLP Scoring Two different protocols were tested for the optimization of scoring parameters: the protocol of Holland et al. (2008) and the open-source software optiFLP version 1.54 developed by Arthofer et al. (2011). The results obtained with these methodologies did not show incongruence or significant differences between them. OptiFLP was chosen for our analyses because of its greater flexibility, faster analysis and the possibility to run the program with its “unsupervised mode”, which uses phylogenetic tree’s robustness to find settings that maximize 9 the differences between groups of profiles. To use the software designed by Arthofer et al. (2011), electropherograms were first visualized in the software PeakScanner v.1.0 (Applied Biosystems) with all default settings except for a “light smoothing”. Samples with poor quality profiles were discarded and AFLP data were exported to the open-source software optiFLP v.1.54 for the optimization of scoring parameters. Subsequently, fragments were automatically scored with tinyFLP v.1.30 (Arthofer, 2010) using the parameters optimized by optiFLP software (Table S3) and the data matrices from the different markers were concatenated using tinyCAT v.1.2 (Arthofer, 2010). A single scoring procedure was run to create data matrices to be used in subsequent genetic structure analyses. 2.5. Phylogenetic analyses AFLP data were analyzed in a phenetic framework (i.e., distance based clustering), due to the limitations reported for alternative methods commonly used for phylogenetic reconstruction (Albach, 2007; Himmelreich et al., 2014). With the aim of understanding the phylogenetic relationships between closely related taxa of the complex, and investigating the possible occurrence of hybridization and/or polyploidization processes, a NeighborNet (NNet) was calculated based on Jaccard distances using SplitsTree4 v.4.13.1 (Huson and Bryan, 2006). Additionaly, Neighbor-Joining (NJ) trees based on Jaccard and Nei-Li distances were also built using SplitsTree4 v.4.13.1 and PAUP* 4.0b10 (Swofford, 2003), respectively, to assess the influence of the distance measure on the results. Bootstrap values (1000 replicates) from the NJ tree based on Jaccard distance were transferred to the NeighborNet graph. 2.6. Genetic Structure Analyses The genetic structure was investigated in the entire AFLP dataset, as well as in data subsets using the same conditions. Data subsets were obtained from the partition of the entire dataset according to the four main K = 4 clusters identified during the initial analysis. Since we are not able to corroborate if the populations under study follow the Hardy-Weinberg model, the genetic structure was initially investigated using two different approaches: non-hierarchical K- 10 means clustering (Hartigan and Wong 1979), which does not assume Hardy-Weinberg equilibrium, and Bayesian clustering analysis based on the MCMC algorithm using Structure v.2.3.4 (Pritchard et al., 2000). Non-hierarchical K-means clustering was performed using the R script of Arrigo et al. (2010). Numbers of K from 1 to 21 were tested and 100,000 independent runs starting from random seeds were performed for each K. To determine the optimal number of genetic clusters, the method of Evanno et al. (2005) was followed as adapted in Arrigo et al. (2010). Bayesian clustering analyses were performed in Structure adopting an admixture model and assuming correlated allele frequencies among populations (Falush et al., 2003) according to a methodology for dominant markers (Falush et al., 2007). Twenty replicates were run for each K from 1 to 21 with a burn-in length of 100,000 generations followed by 1,000,000 additional sampled generations. Structure analyses were run on the computer cluster developed by the UMS 2700 OMSI at the MNHN (Muséum National d’Histoire Naturelle, Paris). The optimal K value was determined using Structure Harvester (Earl and vonHoldt, 2012) following the method of Evanno et al. (2005). The output files were exported to CLUMPP v.1.1.2b (Jakobsson and Rosenberg, 2007) to perform an alignment of cluster assignments across the replicate analyses that we visualized afterwards using Distruct v.1.1 (Rosenberg, 2004). The results obtained with both approaches were independently displayed on a Principal Coordinates Analysis (PCoA) (Krzanowski, 1990) based on the Jaccard distance index using NTSYSpc 2.2 (Exeter Software, Setauket, NY; Rohlf, 2005). The percentages of variance explained by the two different clustering methods were also compared by AMOVA analyses (Excoffier et al., 1992) performed in Arlequin v3.5 (Excoffier et al., 2005, Excoffier and Lischer, 2010). Furthermore, PCO-MC (principal coordinate-modal clustering; Reeves and Richards, 2009) was implemented to test the significance of clusters found in PCoA using PCO-MC software (https://www.ars.usda.gov/plains-area/fort-collins-co/center-for-agricultural-resourcesresearch/plant-germplasm-preservation-research/docs/reeves-pco-mc/). The P-value cutoff was 11 set to 0.9999 and the stability cutoff to 15% to maximize sensitivity to subtle population structure while minimizing type I error (Reeves and Richards, 2009, 2010). 3. Results 3.1. DNA-ploidy level determination DNA-ploidy level estimations according to flow cytometric analyses are shown in Table S1 and Fig. 1. Ploidy was determined for most samples (94%), but for a few (6%) this was problematic likely due to the age of leaf material. In general, our results were in accordance with previous data with the group harboring diploids (2x), tetraploids (4x), hexaploids (6x) and octoploids (8x). Heterogeneity in DNA-ploidy level within a taxon were found only for V. austriaca subsp. austriaca (4x, 6x), V. austriaca subsp. jacquinii (2x, 6x), V. orbiculata (2x, 4x) and V. rosea (2x, 4x). In most cases, only one DNA-ploidy level was observed per population, with the exception of one population of V. rosea in Algeria (2x, 4x), and two populations of V. orbiculata (2x, 4x) in Bosnia and Herzegovina and Croatia, where two DNA-ploidy levels were observed. All populations initially determined as V. austriaca subsp. jacquinii / V. orbiculata were tetraploid except for one population from Montenegro (pop. 19), which was shown to be diploid and was finally ascribed to a new species which is described here (i. e., V. dalmatica sp. nov., see section 5). Similarly, a population from Bosnia and Herzegovina initially identified as V. affinis jacquinii (finally ascribed to V. dalmatica) was confirmed to be diploid (pop. 20), as well as one population of V. affinis linearis from FYROM (pop. 42, with an a posteriori identification as V. linearis). Most of the individuals initially catalogued as V. affinis kindlii (pop. 32, 33, 35) or V. affinis orsiniana (pop. 51, 52, 53, 54, 55) were diploids, except for two populations labeled as V. affinis kindlii (from Greece and Montenegro, pop. 31 and 34 respectively) that were found to be tetraploids. 3.2. Automated scoring of the AFLP data and degree of reproducibility 12 A total of 1127 polymorphic fragments were scored with the software tinyFLP (Table S2). The error rate per locus obtained for our final data-set optimized with the optiFLP software was on average 2.55%. In the NJ analyses, six of the eleven replicated samples were placed with their respective original samples, with a bootstrap value > 98%. The other five replicated samples were recovered at least in the same cluster as their respective original samples and others of the same population. 3.3. Phylogenetic reconstruction Phylogenies reconstructed with different distance methods were congruent with one another and supported the monophyly of most of the previously recognized species by RojasAndrés and Martínez-Ortega (2016), with high bootstrap values (Table 1). However, since only one population of V. rhodopea was included in our study, monophyly of this particular species could not be confirmed. On the other hand, most of the internal nodes of the NJ trees were not supported by bootstrapping (Fig. S1), and the NeighborNet (Fig. 2) showed a high degree of reticulation existing in the group. Nevertheless, four main groups (I, II, III and IV) were identified according to the placement of individuals in the network and are presented below. Group I. This low-supported group comprised five monophyletic diploid species: V. kindlii (BS = 100), V. linearis (BS = 99.9), V. orsiniana (BS = 100), V. rhodopea (BS = 100), and V. teucrioides (BS = 100). Diploid individuals from the south of Italy (ind. 135–140) of uncertain taxonomic identity, which are morphologically similar to V. orsiniana (V. affinis orsiniana), were recovered as monophyletic together with V. kindlii. One tetraploid population initially determined as V. affinis kindlii (ind. 80–82), was recovered as an independent lineage (BS = 100). Group II. The monophyly of this group was clearly supported (BS = 99). It comprised three species recovered as monophyletic with bootstrap values of 100%. The diploid V. tenuifolia with 3 subspecies [subsp. tenuifolia, subsp. javalambrensis, subsp. fontqueri] and the tetraploid V. aragonensis are endemic to the Iberian Peninsula. The third species V. rosea, 13 mostly diploid but comprising some tetraploid individuals in a single population, is endemic to North Africa. Group III. This group included mostly diploid species with highly supported monophyly but there was low support for it as a whole. First, V. krylovii (BS = 100), one of the species representing the subsection in Siberia and Kazakhstan, was recovered as a strongly supported clade. Second, V. prostrata from central Europe was found to be also well resolved (BS = 100), as well as V. turrilliana (BS = 100), an endemic taxon from the border region of Bulgaria and Turkey. Individuals identified as V. crinita were recovered in two distinct lineages (BS = 100). Finally, this group comprised diploid individuals of V. austriaca subsp. jacquinii (BS = 100), and mixed cytotypes (2x, 4x) of V. orbiculata (BS = 71.7), as well as tetraploid individuals of intermediate morphology recorded as V. austriaca subsp. jacquinii / V. orbiculata. Group IV. Following the initial taxonomic classification, this group included four polyploid taxa: i) tetraploid and hexaploid cytotypes of V. austriaca comprising three subspecies: subsp. austriaca, subsp. dentata, and subsp. jacquinii; ii) the tetraploid species V. satureiifolia; iii) the octoploid V. sennenii, endemic from the north of Spain; and iv) octoploid individuals of V. teucrium var. teucrium, and var. angustifolia. The monophyly of this group was well supported (BS = 93.9) but not the monophyly of most of the species within it. Phylogenetic analysis (Fig. 2) only supported the monophyly of hexaploid individuals of V. austriaca subsp. jacquinii but with a very low bootstrap value (BS = 59.6). Veronica satureiifolia and V. sennenii were recovered together with octoploid individuals identified as V. teucrium subsp. angustifolia (BS = 67.5). 3.4. Genetic Structure Following the method implemented in Structure Harvester (Evanno et al., 2005), Bayesian clustering analysis supported an optimal partition of the subsection in three clusters. On the contrary, non-hierarchical K-means clustering analysis of the same dataset estimated K = 2 as the most likely number of genetic clusters. However, PCoA (Fig. 3) and AMOVA analyses 14 (Table 2) demonstrated that the clustering proposed by Structure explained a higher percentage of the variance among groups than K-means (see Table 2). Accordingly, we here focus on results of Bayesian clustering. High levels of admixture were found in Bayesian clustering analyses performed at higher values of K (Fig. S2C). It should be pointed out that most taxa included in our analyses (with the exception of polyploids from group IV and of V. teucrioides from group I) were recovered as independent clusters when Structure analyses were performed at K = 20 (Fig. S2C). In addition, an exclusive cluster was found grouping tetraploid populations of V. austriaca subsp. jacquinii / V. orbiculata and V. orbiculata. The clusters revealed by Structure at K = 4 (Fig. 2 and Fig. S2A) generally concurred with the groups identified by the NeighborNet with the only exception of V. orsiniana (Table 1), and partially corresponded with geographic regions: cluster A included a group of narrow endemics mostly restricted to the south of the Balkan Peninsula; cluster B comprised the three well recognized species from the Iberian Peninsula and North Africa; cluster C recovered most diploids from the Balkan Peninsula, V. krylovii from Russia, and V. orsiniana; cluster D included all the polyploid taxa mainly from central Europe and north of Spain. Additional Bayesian clustering analyses within clusters A, B, C and D estimated an optimal K = 5, K = 3, K =3 and K = 2, respectively (Fig. S2B). According to the results obtained for cluster A, the four diploid species and the tetraploid population of V. affinis kindlii from Mt. Vermion (pop. 31) were recovered in independent clusters. In cluster B, the three species from the Ibero-NorthAfrican group (V. aragonensis, V. rosea and V. tenuifolia) were recovered in independent and homogeneous clusters almost without admixture among them. When analyses were performed within cluster C, one cluster grouped a single species (i.e., V. orsiniana) and the other two clusters divided the taxa from group III in two subgroups. One subgroup included diploids of V. austriaca subsp. jacquinii and V. orbiculata and all intermediate populations displaying high levels of admixture [V. austriaca subsp. jacquinii / V. orbiculata, V. crinita (= V. crinita f. bosniaca) and V. affinis kindlii]. Another subgroup, also 15 with a certain degree of admixture, comprised the remaining diploid species from group III [V. crinita, V. crinita (= V. thracica), V. turrilliana, V. prostrata, V. krylovii]. Within cluster D, there was an obvious geographic pattern in which polyploids form two separate clusters, however, with many individuals forming a continuous gradation of proportion scores between the two clusters (Fig. 4). Hexaploid individuals classified as V. austriaca subsp. jacquinii (pop. 11–16) were included in one cluster with a very high proportion score (> 0.99; data not shown). By contrast, tetraploid individuals identified as V. satureiifolia, octoploid populations assigned to V. senneni and affinis individuals, had a proportion score > 0.99 (data not shown) to be defined in a second cluster. Most individuals of V. teucrium var. angustifolia showed a high genetic affinity to this second cluster with low levels of admixture. PCO-MC analysis recovered ten species as significant independent clusters: V. aragonensis, V. dalmatica, V. kindlii, V. linearis, V. orbiculata, V. orsiniana, V. prostrata, V. rosea, V. tenuifolia, and V. turrilliana (Table 1). 4. Discussion 4.1. The importance of auto- and allopolyploidization in the subsection and the recurrent formation of polyploids The diversity of cytotypes and the existence of mixed-ploidy levels within species and populations in the group reveal that polyploidization has occurred likely continuously since the origin of the subsection ca. 2.8 Mya (Meudt et al., 2015). The pattern of reticulation shown by the NeighborNet (Fig. 2) and the high levels of admixture found by Structure suggest that V. subsection Pentasepalae is composed of species that are in the initial stages of divergence. Furthermore, based on morphological and (phylo-)genetic intermediacy between potential parental species, hybridization is confirmed in V. subsection Pentasepalae. In addition, ILS cannot be excluded as a cause of the lack of resolution observed for internal nodes of the NeighborNet and NJ trees. Nevertheless, phylogenetic analyses demonstrate that polyploid taxa 16 distributed mainly in central Europe (group IV) constitute a very well supported group (Fig. 2). It should be pointed out that the higher number of AFLP fragments present in polyploid individuals may produce a bias in posterior analyses towards the apparent monophyly of most of the polyploids. However, this artificial grouping due to a higher number of AFLP fragments in polyploids can be discarded in our dataset because other polyploid species of the subsection are not recovered within group IV (e.g., V. aragonensis and V. orbiculata). Thus, our study could indicate a common origin of polyploid entities from group IV at the tetraploid level. This hypothesis was previously rejected by Rojas-Andrés et al. (2015) due to the existence of morphological variation among polyploids and to the fact that most polyploid species have a polytopic origin (Soltis & Soltis, 1999). However, a possible explanation is that hexa- and octoploids have emerged (probably several times independently within each lineage) after a previous differentiation of lineages at the tetraploid level. Furthermore, the extinction of diploid or some of the tetraploid ancestors within group IV is also likely, which together with the limitations of the available methodologies hampers the obtention of ancestor-derivative patterns within group IV (Stebbins, 1971; McDade, 1992; Buggs et al., 2014). Our results suggest that polyploid species in the subsection may have emerged by different processes. Whereas autopolyploidization appears to be the main evolutionary force for some taxa, allopolyploidization also seems to be common. Evidence for autopolyploidization is found, for example, in group II. Tetraploid individuals have been found in a population of V. rosea from Algeria (Table S1, pop. 63), which cluster together with the rest of diploid individuals of the species (Fig. 2, Fig. S2), thus suggesting a recent autopolyploid event occurring within the population. Analyses further point to an autopolyploid origin of the octoploid V. sennenii from tetraploid V. satureiifolia. The individuals belonging to V. satureiifolia and V. sennenii are recovered in the same group in the NeighborNet without any clear separation between individuals of both species, and they form a homogeneous cluster in the Bayesian clustering 17 analyses (Fig. 4). Flow cytometric analyses (Table S1) have confirmed that both species (and consequently, both ploidy levels) grow in sympatry in the province of Huesca, in the north of Spain (pop. 69, 4x; and pop. 73, 8x). On the basis of all these results, this is another example of autopolyploid speciation in natural populations (Soltis et al., 2007). Likewise, according to the NeighborNet, most octoploid individuals determined as V. teucrium var. angustifolia are nested with V. satureiifolia and V. sennenii and have very similar genetic composition in clustering analyses (Fig. 4). Furthermore, one population of V. satureiifolia (pop. 70, 4x) and one of V. teucrium var. angustifolia (pop. 87, 8x) have been found in close proximity (about 400 m) in the region of Île-de-France. Veronica satureiifolia and V. teucrium var. angustifolia were also shown to share the same cpDNA haplotype (Rojas-Andrés et al. 2015). Consequently, a plausible interpretation is that multiple autopolyploidization events might have occurred in the tetraploid V. satureiifolia giving rise to octoploids that have been identified as V. sennenii in the Iberian Peninsula and V. teucrium var. angustifolia in France. Alternatively, a past continuous distribution area of the octoploid entity and a subsequent fragmentation scenario cannot be discarded, although it seems unlikely considering the distance of 500 km between the French southernmost and the Spanish northernmost populations, and the existence of the Pyrenees in between. Our results also reveal that at least two episodes of polyploidy have occurred within V. orbiculata (Fig. 2; Group III), although the processes seem to be more complex. Autopolyploid formation has been detected in one population from Bosnia and Herzegovina (Table S1; pop. 43) and might be occurring in other, not surveyed, populations. Tetraploid individuals found in this population (ind. 107, 108) are nested within the diploid individuals (ind. 109–116) with a BS value of 100% (Fig. 2). Moreover, individuals of both cytotypes are recovered together as a significant cluster in PCO-MC and Structure analyses. In contrast, tetraploid individuals (ind. 117–119) from another population in Croatia (Table S1; pop. 45) are recovered well separated from the diploids of the same population (ind. 114–116; BS = 99.5) and are not included 18 together within any significant cluster in PCO-MC analyses. Moreover, an exclusive cluster is found in Structure analyses (Fig. S2C) that groups these tetraploid individuals with tetraploid populations recorded as V. austriaca subsp. jacquinii / V. orbiculata. Thus, these tetraploids are probably the result of an allopolyploidization event. Consequently, V. orbiculata is a further example of a diploid-polyploid species with numerous independent origins of polyploid entities as shown in other species (Soltis and Soltis, 1999; Bardy et al., 2010, 2011). There are other strong arguments of recurrent formation of allopolyploids within group III. Specifically, tetraploid individuals from Bosnia and Herzegovina (Table S1; ind. 42–47) recorded as V. austriaca subsp. jacquinii / V. orbiculata due to their transitional morphology, are recovered by the NJ in an intermediate position between these species (Fig. S1). Thus, a hybrid origin of these populations is suggested with diploid V. austriaca subsp. jacquinii and V. orbiculata as putative parental species. In addition, diploid individuals located in Montenegro labeled as V. affinis kindlii (ind. 83–84), are also recovered in an intermediate position together with a population of V. crinita, putatively belonging to f. bosniaca (ind. 64–66). Furthermore, the position of individual 85 (2x) in the NeighborNet suggests that homoploid hybridization could be also an important evolutionary process occurring in the group, which is here demonstrated for the first time and of which V. x gundisalvi may represent an additional example (Martínez-Ortega et al., 2004). Last, hybridization and/or introgression events may have affected to the tetraploid population of V. aff. kindlii located in Mt. Vermion (Greece; Fig. 2, group I, pop. 31). Bayesian clustering analyses show high levels of admixture with the polyploid group IV (Fig. S2A). Veronica austriaca subsp. jacquinii is the only species from group IV distributed in this southern area of the Balkan Peninsula. Thus, the position of population 31 in the NeighborNet could be influenced by hybridization and/or introgression processes involving this species and representatives from group I or its ancestors. 19 4.2. The challenging task of delimitating species within a recently diverged diploid-polyploid complex Species delimitation within recently diverged plant complexes is currently a major challenge for systematists. In general, at this level of lineage separation, phenotypic differences among species may not be evident and a clear phylogenetic signal is not always obtained (Federici et al., 2013). Consequently, other characteristics (e.g., ploidy levels, differences in habitat, pollinators, phenology, etc.) are important lines of evidence when delimiting species in this recently diverged, phenotypically, and phylogenetically complex groups. This situation requires the adoption of the general lineage concept of species in which different species properties (that have been used as criteria under rival species concepts), serve as lines of evidence to assess lineage delimitation (de Queiroz, 2007). Identifying biological diversity at the species level is even more challenging when processes such polyploidy are involved in the evolution of a group. Polyploidy has long been considered a mechanism of direct sympatric speciation (Otto and Whitton, 2000; Schemske, 2000; Rieseberg and Willis, 2007). However, recent studies suggest that polyploid speciation is not necessarily an instantaneous process (Husband et al., 2013). The formation of unreduced gametes and other biological traits are fundamental in initial stages of polyploid emergence and establishment (Rieseberg and Willis, 2007; Fowler and Levin, 2016). Both, the rates of production of unreduced gametes and the successful long-term establishment and spread of new polyploid individuals are affected by genetic and environmental factors (Ramsey and Schemske, 1998; Comai, 2005; Lafon-Placette et al., 2016). Regardless of the timing of the process, it has been estimated that 15% of angiosperm speciation events, and even more in Veronica, are associated with a ploidy increase (Albach et al., 2008; Wood et al., 2009). Indeed, it has been corroborated that the effect of genome duplication on countless features (e.g., reproductive biology, phenotype, physiology, geographical and environmental distributions of cytotypes, genetic, epigenetic and genomic consequences, and so forth; reviewed in Ramsey and Ramsey, 20 2014). Whether caused by ploidy per se, adaptation or founder effects and genetic drift, these changes may maintain polyploids as separately evolving metapopulation lineages from their parental taxa, which justify their treatment as separate taxonomic species (de Queiroz, 2007). Our study has demonstrated that most of the species of V. subsection Pentasepalae are still in the initial stages of divergence. Moreover, auto- and allopolyploids have been identified within the group. In this situation, the taxonomic status of diploid and polyploid taxa within V. subsection Pentasepalae is reviewed adopting the general species concept of de Queiroz (2007) and making use of an integrative taxonomic approach. In our case study, no significant differences in habitat preference are observed, experimental data on reproductive biology are not available, and probably many species share pollinators to a great extent. Thus, we have based the decisions of species delimitation on ploidy level, phylogeny and genetic divergence, but also on information from morphology, distribution, ecology, etc., available in Rojas-Andrés and Martínez Ortega (2016). Furthermore, a conservative approach to taxonomy is preferable when incongruences among different lines of evidence are found (Carstens et al., 2013). When such situation was encountered, we maintained the last taxonomic treatment of Rojas-Andrés and Martínez-Ortega (2016). Moreover, we think that populations identified in this study, for which we have not obtained sufficient evidence to be delimited as species (e.g., tetraploid hybrid populations catalogued as V. austriaca subsp. jacquinii / V. orbiculata that cannot be identified as a different species but could potentially evolve as a distinct lineage) would have to be considered as functional units of biological diversity. In these cases, this recognition would help to address future ecological, evolutionary and taxonomic questions (Ramsey and Ramsey, 2014; Laport and Ng, 2017). Additionally, we consider that further molecular tools (i.e., molecular studies using neutral markers as SSRs; López-González et al., in prep), morphological data (e.g., traits with potential impact on individual fitness), and deeper ecological and biological information (e.g., 21 environmental distribution analyses among cytotypes, crossing experiments to understand reproductive interactions) are needed to re-evaluate whether the species rank is appropriate for some of these taxa (e.g., polyploids from group IV). 4.3. Taxonomic considerations This study provides new insights into the systematics of the polyploid complex Veronica subsection Pentasepalae. Our analyses support 20 distinct species in the group. The most recent taxonomic treatment available (Rojas-Andrés and Martínez-Ortega, 2016) has been revised and updated (changes summarized in Table S1). First, the individuals of V. austriaca subsp. jacquinii, included in this study are placed in two separate phylogenetic lineages differentiated by their ploidy levels (diploids vs. hexaploids) (Fig. 2). Monophyly of the diploid individuals, which represent an example of cryptic species in the subsection, is well supported by phylogenetic reconstruction (BS = 99.8), whereas hexaploids are recovered with a low bootstrap value (BS = 59.6). Additionally, PCOMC and Bayesian clustering analysis recover these populations as significant and independent clusters (Table 1 and Fig. S2C). Indeed, after an exhaustive revision of herbarium specimens, morphological characters corresponding to each of these species have been found (see section 5). In addition, the distribution area of the diploid cytotypes is restricted to the Adriatic coast of Albania, Bosnia and Herzegovina, Croatia and Montenegro. Based on all these lines of evidence, we consider that diploid entities of V. austriaca subsp. jacquinii should be recognized at the specific rank as V. dalmatica N.Pad.Gar., Rojas-Andrés, López-González and M.M.Mart.Ort. (see section 5). Second, the analyses presented here allow the recognition of V. thracica at the species level. Veronica thracica was described by Velenovsky (1893) to differentiate plants mainly occurring in Bulgaria characterized by white hairy stems and oval-obovate, deeply cordate, almost amplexicaulus leaves. This name was later combined under V. teucrium as subspecies (Velenovsky, 1898) or variety (Maly, 1908) and has been related to V. crinita by other authors 22 (e.g., Watzl, 1910; Peev, 1995). These individuals were considered within the variation of V. crinita in the most recent taxonomic treatment due to their morphological similarities (RojasAndrés & Martínez-Ortega, 2016). Genetic data now provide evidence that these populations constitute an independent evolutionary lineage differentiated from typical V. crinita described from Hungary (Fig. 2 and Fig. S2C) and it represents an additional example of a cryptic species within the subsection (Martínez-Ortega et al., 2004). Furthermore, after examining the morphological characters of this material, we found that V. thracica has dense tomentose indument on leaves and stems, formed by patent to slightly incurvate hairs that confer a whitish (light green in vivo) color to the plant. In contrast, V. crinita has villous indument on leaves and stems, which is constituted by crooked, generally interwoven hairs that confer a brownish green color to the plant. The leaves are concolor (i.e., upper and lower leaf sides of the same color) and more densely pilose in V. thracica, while they are slightly bicolor (i.e., dark / dive green color of the upper leaf side vs. green color of the underside of the leaf) and comparatively not so densely pilose in V. crinita. We consider that there is enough evidence to recognize these lineages as separate species, and consequently, the recognition at the specific rank of the Bulgarian populations is proposed. Finally, several taxa have been described under V. teucrium, but only two varieties have been recognized in the last taxonomic treatment of the subsection (Rojas-Andrés and MartínezOrtega, 2016): V. teucrium var. teucrium and V. teucrium var. angustifolia. These two mostly allopatric octaploid entities are morphologically distinct. Veronica teucrium var. teucrium is distributed in Germany, Austria, and Bulgaria, while V. teucrium var. angustifolia occurs in France extending towards western Switzerland and northern Italy. Moreover, both varieties are recovered in different subclusters in our molecular analyses (Fig. 4). Based on all available data, we consider that both entities should be recognized at the specific level as V. teucrium L. and V. angustifolia (Vahl) Bernh., respectively. Additionally, apart from geographic differentiation, individuals of V. teucrium var. angustifolia are very similar to V. sennenii in size and 23 appearance. Taken together these data would suggest that V. sennenii and V. teucrium var. angustifolia have the same parental origin, although they could have arisen from the same or different autopolyploid events. If they were considered synonyms, the name V. angustifolia (Vahl) Bernh. would prevail at the specific level, according to the principle of priority. But this taxonomic decision should not be firmly adopted until additional exhaustive analyses including more populations of these taxa are performed. Additionally, the importance of some populations from the south of Italy identified in Flora d’Italia as V. austriaca and their relationship with those from the Balkan Peninsula have been previously highlighted (Fischer, 1982). However, the identity of these plants (ind. 135–140; labeled in this study as V. affinis orsiniana) has remained unclear for many years. Our analyses confirm the identity of these plants as V. kindlii and show that this species should be considered independent from V. orsiniana or V. austriaca (Table 1, Fig. 2). The name V. kindlii has recently been resurrected to designate those populations from the Balkan Peninsula, which were previously known as V. orsiniana (Rojas-Andrés et al., 2015). Thus, a clear amphi-Adriatic distribution of V. kindlii is now demonstrated. Finally, there is no evidence that the individuals initially identified as V. affinis kindlii belong to V. kindlii. Unfortunately, the taxonomic status of these entities remains unresolved. Additional exhaustive field sampling in order to have a good representation of these unresolved entities and posterior molecular studies could shed some light on the taxonomic identity of these individuals. Another important outcome is the corroboration of the genetic distinctiveness and monophyly of V. linearis, a diploid endemic species from FYROM that passed unnoticed for many years. This name did not appear in Floras or monographs of Veronica. The plant was initially described as V. kindlii var. linearis by Bornmüller (1937) and has recently been elevated to the species level based on morphological evidence (Rojas-Andrés and Martínez Ortega, 2016). According to our phylogenetic reconstruction (Fig. 2) and PCO-MC analyses (Table 1), V. linearis is recovered as monophyletic within group I and its closest relatives are V. 24 kindlii and V. teucrioides (BS = 74.7 for [V. linearis + V. kindlii + V. teucrioides]). In addition, one population with dubious morphological characters labeled as V. affinis linearis (pop. 42) is recovered within V. linearis. Nevertheless, clustering analyses (Fig. S2B) showed introgression with V. teucrioides, which is in agreement with the dubious determination based on morphological characters. With regard to the delimitation of varieties and subspecies, the theoretical framework behind their concept is less clear as it is for species. We have attempted to avoid the use of these ranks in the proposed taxonomic changes, but in two cases the data available are not conclusive: i) Within group II, three subspecies are recognized under V. tenuifolia. Their different distribution areas and the divergence found in the NeighborNet and NJ trees between populations corresponding to each subspecies could indicate reduced gene flow among them (Fig. 2, Fig. S1), as previously showed by studies based on AFLP and morphology (MartínezOrtega et al., 2004; Andrés-Sánchez et al., 2009). However, our study does not support the recognition of these taxa at the specific rank (see PCO-MC and genetic structure results in Table 1, Fig. S2). Likewise, phylogenetic analyses based on nuclear and plastid DNA sequences did not differentiate among the three subspecies currently recognized (Rojas-Andrés et al. 2015). Due to the incongruences found among different sources of data, we suggest to maintain their current formal rank as subspecies. ii) The subspecific rank has also been retained for some taxonomic entities belonging to group IV (i.e., three subspecies recognized under V. austriaca). The lack of resolution in our AFLP analyses (Table 1, Fig. 4) as well as in nuclear and plastid DNA trees (Rojas-Andrés et al., 2015) do not support the recognition of current subspecies as different species, nor the unification in a single species. Unfortunately, the phylogenetic relationships among these taxa remain unresolved. These subspecies have been described in the last taxonomic treatment given the morphological and chorological differences among them (Rojas-Andrés and MartínezOrtega, 2016). Consequently, we have retained the subspecies rank within V. austriaca (subsp. 25 austriaca; subsp. dentata, subsp. jacquinii), at least until future studies clarify the evolutionary history and taxonomy of these polyploid entities. 5. Conclusions and description of a new species. The exhaustive sampling of V. subsection Pentasepalae, and the use of AFLP fingerprinting together with flow cytometry data provided new insights into the evolutionary history and species delimitation of a taxonomically complex plant group, in which auto- and allopolyploidization appear to be active evolutionary processes, even nowadays. Based on all sources of data currently available, V. subsection Pentasepalae contains at least 20 monophyletic species, five of them narrow endemics. This taxonomic framework is essential to design suitable conservation strategies. Future studies should focus on trying to understand in more detail the role that hybridization has played in the evolution of the subsection, and on ecological factors that make polyploidy so important in plant evolution and speciation. Veronica dalmatica N.Pad.Gar., Rojas-Andrés, López-González & M.M.Mart.Ort., sp. nov. – Type: Holotype: Bosnia and Herzegovina, Republica Srpska: between Brgat and Trebinje, 42.68289 N, 18.28949 E, 283 m, 10/VI/2015, clearings in a forest of Carpinus betulus with Paliurus spina-christi. Leg. M. Martínez Ortega, X. Giráldez, N. Padilla and N. López, MMO6119 (SALA No. 157047!). Next, we provide a diagnosis between V. dalmatica and the morphologically closest taxa, as well as a full description of V. dalmatica, which is parallel to the descriptions provided by Rojas-Andrés and Martínez-Ortega (2016). The indument is described according to Beentje (2010). Two measurements given together refer always to length  width. 26 Diagnosis: V. dalmatica differs from V. austriaca subsp. jacquinii by its smaller plant size (10– 16 vs. 25–50 cm), having shorter stem-hairs (0.3–0.4 vs. 0.8–1.2 mm), smaller leaves (12–16  5–10 vs. 20–30  10–20 mm), shorter styles (3–5 vs. 4–7 mm) and tiny capsules with a less deep sinus (up to 0.5 vs. 1.0 mm). Attending to chromosome number, V. dalmatica is diploid (2n = 16) whereas V. austriaca subsp. jacquinii is frequently hexaploid [2n = (32), 48, (64), (80)]. Overlapping in ploidy level, V. dalmatica (2n = 16) is morphologically differentiated from V. orbiculata (2n = 16, 32) by having the eglandular hairs of the stem not arranged in two opposite lines along it. Apical shoot leaves are opposite in V. dalmatica, whereas in V. orbiculata they can be opposite, alternate or verticillate by three. Description: Stems (6) 10–16 (24) cm long, slightly ascending to erect, covered by eglandular hairs (0.18) 0.30–0.40 (0.81) mm long, incurvate, ± appressed and antrorse, not arranged in 2 opposite lines along the stem; apical shoot bearing (5) 8–11 (16) pairs of leaves. Leaves opposite, (8) 12–16 (20) × (3) 5–10 (16) mm; ovate, obovate, or narrowly to widely trullate; more or less rounded or cuneate at the base; pinnatifid to pinnatisect, with linear-lanceolate to narrowly elliptic segments, variable in width, entire, revolute to subrevolute, subglabrous or pilose, covered by hairs (0.06) 0.10–0.18 (0.23) mm long, sessile to shortly petiolate. Basal leaves pinnatifid to pinnatisect, segments 0.4–1.0 mm wide; medium leaves (i.e., those situated in the central segment of the stem) pinnatipartite to pinnatisect, segments 0.25–1.00 mm wide; uppermost leaves pinnatisect, rarely bipinnatifid, segments 0.25–0.60 mm wide. Leaves of the apical shoot opposite, linear to lanceolate, narrowly elliptic, entire, dentate-serrate or pinnatifid, revolute to subrevolute. Racemes axillary, opposite, exceptionally solitary, bearing (9) 20–40 (48) flowers, loosely to densely arranged; peduncles (2.5) 3.0–7.0 (11) cm long, covered by a non-glandular indument similar to that of the leaves; bracts (1.5) 3.0–5.0 (8.0) mm long, linear, entire, exceptionally pinnatifid to pinnatisect at the base with one or two segments, glabrous or 27 subglabrous, covered by hairs similar to those covering the leaves; pedicels (1.6) 3.0–5.0 (8.5) mm long. Calyx (0.7) 2.0–4.0 (5.0) mm long, with (4) 5 sepals, linear-lanceolate, usually shorter than the capsule, glabrous or subglabrous. Corolla 9–15 mm in diameter, light or dark blue. Capsule (2.0) 3.0–5.0 (6.0) × (2.0) 3.0–4.5 (5.3) mm, glabrous, widely elliptic or widely obovate to very widely depressed ovate-obovate, rounded at the base, slightly emarginated or rounded at apex, sinus up to 0.5 (0.6) mm depth. Style (2.8) 3.0–5.0 (6.0) mm long. Seeds (0.9) 1.3–1.7 × 1.5–1.8 (2.0) mm, ca. 8 per capsule. Chromosome Number. – 2n = 16 Habitat. – Dry and stony meadows, steppes, forest glades and shrublands, rocky slopes; usually on calcareous soils; (50) 200–1,100 (1,400) m above sea level. Distribution. – W Balkan Peninsula; Albania, Bosnia and Herzegovina, Croatia, Montenegro. Etymology. – The epithet indicates geographical distribution of the species. Dalmatia is a historical region of the Adriatic Sea ranging from Rab (Croatia) to the Bay of Kotor (Montenegro) including a small area of Bosnia and Herzegovina. Notes. – The plant is illustrated in Rojas-Andrés and Martínez-Ortega, 2016; Figure 3 (f-i). Specimens examined. – See Appendix B. Acknowledgements We are grateful to X. Giráldez and all other colleagues for collecting material that was used in this study: A. Abad de Blas, A.C. Torres, D. Pinto-Carrasco, E. Rico, G. Calabrese, J. Fuentes, J. Peñas de Giles, L. Gutiérrez, T. Romero, M. Santos-Vicente, R. Peña-García, S. Andrés-Sánchez, S. Rubio, V. Di Donato and V. Lucía. We thank P. Kosachev, B. Frajman and Julio Fuentes for providing material. We would like to express our gratitude to S. Filoche for giving us information to find V. satureiifolia in Fontainebleau. We thank Teresa Malvar for technical support in the lab, P. Reeves for help running PCO-MC analysis, and to our friends E. Rico and X. Giráldez for their continuous support. This work was supported by the Spanish 28 Ministerio de Economía y Competitividad (projects CGL2009-07555 and CGL2012-32574); and the University of Salamanca (Ph.D. grant to NPG cofounded by Banco Santander). FCT with POPH/FSE funds financed SC and MC (staring grant project IF/01267/2013 and SFRH/BD/89910/2012). Appendix A. Supplementary Material (Fig. S1, Fig. S2, Table S1, Table S2 and Table S3) Appendix B. Specimens examined of V. dalmatica. Information listed is country, locality, geographical coordinates, altitude, collection date, habitat, collector names, collector number, and herbarium code (Thiers, 2017). ALBANIA. Lezhë: Lezhë, cerca de Fishte, 41.89112N, 19.67781E, 56 m, 17/VI/2015, pastos secos en flysch, M. Martínez Ortega, X. Giráldez, N. Padilla & N. López, NPG48 (SALA 157035). BOSNIA AND HERZEGOVINA. Republica Srpska: between Brgat and Trebinje, 42.68289 N, 18.28949 E, 283 m, 10/VI/2015, clearings in a forest of Carpinus betulus with Paliurus spina-christi, M. Martínez-Ortega, X. Giráldez, N. Padilla and N. López, MMO6119 (SALA 157047); entre Tjentište y Gacko, 43.18547N, 18.56603E, 1085 m, 13/VII/2010, prados sobre calizas. S. Andrés, X. Giráldez, M. Martínez Ortega & B. Rojas Andrés, MO5552 (SALA 149274); entre Trebinje y Dubrovnik, 42.68992N, 18.297E, 282 m, 14/VII/2010, sobre rocas calizas en zonas aclaradas, S. Andrés, X. Giráldez, M. Martínez Ortega & B. Rojas Andrés, BR108 (SALA 149284); entre Gaccko y Tjentište, 43.1847N, 18.56578E, 1076 m, 10/VI/2015, prados calizos subalpinos, M. Martínez-Ortega, X. Giráldez, N. Padilla and N. López, MO6123bis (SALA 157025). CROATIA. Dubrovnik-Neretva: Dubrovnik, entre Sumet y Gornji Brgat, 42.64408N, 18.14644E, 212 m, 15/VII/2010, prados sobre calizas, S. Andrés, X. Giráldez, M. Martínez Ortega & B. Rojas Andrés, SA384 (SALA 149286); Dubrovnik, Gromača, 42.72444N, 29 18.01778E, 320 m, 14/VII/2010, prados secos sobre calizas, S. Andrés, X. Giráldez, M. Martínez Ortega & B. Rojas Andrés, BR112 (SALA 149039). MONTENEGRO. Andrijevica: Andrijevica, a 2 km en dirección Kolasin, 42.73946N, 19.76141E, 989 m, 8/VI/2015, claros de robledal, calizas, M. Martínez Ortega, X. Giraldez, N. Padilla & N. López, NPG31 (SALA 157015); Andrijevica, a 1 km en dirección Kolasin, pista que sale a la derecha, 42.74523N, 19.77552E, 884 m, 8/VI/2015, prados sobre calizas, M. Martínez-Ortega, X. Giráldez, N. Padilla & N. López, NPG32 (SALA 157016); Bar: entre Sutorman y Karuci, Rumija Planina, 42.16105N, 19.09708E, 738 m, 9/VI/2015, claros de bosque sobre calizas junto a la carretera, M. Martínez Ortega, X. Giráldez, N. Padilla & N. López, NLG136 (SALA 157017); entre Sutorman y Karuči, Rumija Planina, 42.16219N, 19.09794E, 753 m, 16/VII/2010, prados sobre calizas, S. Andrés, X. Giráldez, M. Martínez Ortega & B. Rojas Andrés, MO5556 (SALA 149285); Kotor: Kotor, Lovcen, 42.41802N, 18.79413E, 904 m, 9/VI/2015, claros sobre calizas, M. Martínez Ortega, X. Giráldez, N. Padilla & N. López, NLG137 (SALA 157018); Žabljak: Meždo, 43.16384N, 19.14908E, 1390 m, 12/VI/2015, prados cortos con enebros, calizas, M. Martínez Ortega, X. Giráldez, N. Padilla & N. 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New Phytol. 161: 173–191. http://dx.doi.org/10.1046/j.14698137.2003.00948.x Soltis, D.E., Soltis, P.S., 1999. Polyploidy: recurrent formation and genome evolution. Trends Ecol. Evol. 14: 348. https://doi.org/10.1016/S0169-5347(99)01638-9 Soltis, P.S., Marchant, D.B., Van de Peer, Y., Soltis, D.E., 2015. Polyploidy and genome evolution in plants. Curr. Opin. Genet. Dev. 35: 119–125. https://doi.org/10.1016/j.pbi.2005.01.001 Stebbins, G.L.Jr., 1947. Types of polyploids: their classification and significance. Adv. Genet. 1: 403–429. https://doi.org/10.1016/S0065-2660(08)60490-3 Stebbins, G.L.Jr., 1950. Variation and evolution in plants. New York, USA: Columbia University Press. Stebbins G.L.Jr., 1971. Chromosomal evolution in higher plants. London, UK: Edward Arnold. Swofford, D.L., 2003. PAUP*: phylogenetic analysis using parsimony (*and other methods). Version 4.0b10. Sunderland, MA: Sinauer Associates. Temsch, E.M., Greilhuber, J., Krisai, R., 2010. Genome size in liverworts. Preslia, 82: 63-80. Thiers, B., (2017, continuously updated). Index Herbariorum: A global directory of public herbaria and associated staff. New York Botanical Garden’s Virtual Herbarium. http://sweetgum.nybg.org/ih/. (last accessed 15 January 2017). 42 Velenovsky, J., 1893. Dritter Nachtrag zur Flora von Bulgarien. Sitzungsber. Königl. Böhm. Ges. Wiss. Mathe.-Naturwisse. Cl. 37: 1–72 Velenovsky, J., 1898. Flora bulgarica. Descriptio et enumeratio systematica plantarum vascularium in principatu Bulgariae sponte nascentium. Supplementum I. Pragae: F. Rivnác. Vos, P., Hogers, R., Bleeker, M., Reijans, M., Van de Lee, T., Hornes, M., Frijters, A., Pot, J., Peleman, J., Kuiper, M., Zabeau, M., 1995. AFLP: a new technique for DNA fingerprinting. Nucleic acids Res. 23: 4407–4414. https://doi.org/10.1093/nar/23.21.4407 Watzl, B., 1910. Veronica prostrata L., teucrium L. und austriaca L. Nebst einem Anhang über deren nächste Verwandte. Abh. K. K. Zool.-Bot. Ges. Wien 5(5): 1–94. http://bibdigital.rjb.csic.es/ing/Libro.php?Libro=5939 Wood, T.E., Takebayashi, N., Barker, M.S., Mayrose, I., Greenspoon, P.B., Rieseberg, L.H., 2009. The frequency of polyploid speciation in vascular plants. PNAS, 106: 1387513879. https://doi.org/10.1073/pnas.0811575106 Zozomová-Lihová, J., Marhold, K., Spaniel, S., 2014. Taxonomy and evolutionary history of Alyssum montanum (Brassicaceae) and related taxa in southwestern Europe and Morocco: diversification driven by polyploidy, geographic and ecological isolation. Taxon 63: 562–591. https://doi.org/10.12705/633.18 43 Neighbor-Net Groups GROUP I GROUP II GROUP III GROUP IV Taxa Ploidy V. kindlii Adamović BS values Clustering NJ Jaccard NJ Nei-Li K-means K=2 Structure K=3 Structure K=4 PCO-MC 2x 100.0 100.0 1/2 A A ✓ V. linearis (Bornm.) Rojas-Andrés & M.M. Mart. Ort. 2x 100.0 100.0 1 A A ✓ V. orsiniana Ten. 2x 100.0 100.0 2 A C ✓ * V. rhodopea (Velen.) Degen. ex Stoj. & Stef. 2x 100.0 100.0 2 A A ─ V. teucrioides Boiss. & Heldr. 2x 99.9 100.0 2 A A ─ V. affinis kindlii 4x 100.0 100.0 1 A A ─ V. aragonensis Stroh 4x 100.0 100.0 1 B B ✓ V. rosea Desf. 2x, 4x 100.0 100.0 2 B B ✓ V. tenuifolia subsp. fontqueri (Pau) M.M. Mart. Ort. & E. Rico 2x 100.0 100.0 2 B B ─ V. tenuifolia subsp. javalambrensis (Pau) Molero & J. Pujadas 2x 100.0 99.0 2 B B ─ V. tenuifolia Asso subsp. tenuifolia 2x 100.0 100.0 2 B B ─ V. dalmatica N. Pad. Gar., Rojas-Andrés, López-González & M.M. Mart. Ort. 2x 99.8 100.0 1 A C ✓ V. austriaca subsp. jacquinii / V. orbiculata 4x 84.3 83.0 1 A C ─ V. crinita Kit 2x 100.0 100.0 1 A C ─ V. crinita (= V. crinita f. bosniaca) 2x 62.9 61.0 1 A C ─ V. thracica Velen. 2x 100.0 100.0 1 A C ─ V. krylovii Schischk. 2x 100.0 100.0 1 A C ─ V. orbiculata A. Kern 2x, 4x 73.0 78.0 2 A C ✓ V. prostrata L. 2x 100.0 100.0 2 A C ✓ V. turrilliana Stoj. & Stef. 2x 100.0 100.0 2 A C ✓ V. affinis kindlii 2x, 4x Not monophyletic Not monophyletic 1 A C ─ V. austriaca subsp. austriaca L. 6x Not monophyletic Not monophyletic 1 C D ─ * V. austriaca subsp. austriaca (= V. macrodonta) 4x 94.4 93.0 1 C D ─ V. austriaca subsp. dentata (F.W. Schmidt) Watzl 6x Not monophyletic Not monophyletic 1 C D ─ V. austriaca subsp. jacquinii (Baumg.) Watzl 6x 59.6 56.0 1 C D ─ V. satureiifolia Poit. & Turpin 4x 63.9 62.0 2 C D ─ V. sennenii (Pau) M.M. Mart. Ort. & E. Rico 8x Not monophyletic Not monophyletic 2 C D ─ V. angustifolia Vahl (Bernh.) 8x Not monophyletic Not monophyletic 2 C D ─ 8x Not monophyletic Not monophyletic 2 C D ─ V. teucrium L. *Only one population sampled for this study Table 1. Overview of the boostrap (BS) values detected by different phylogenetic analyses supporting each taxon included in the study. Taxa are shown according to the four groups identified by the placement of taxa in the Neighbor-Net network. Final taxonomic assignments, DNA ploidy level and classification of individuals in clusters using different methodologies are indicated. Species recovered as independent clusters in PCOMC analyses are indicated by a checkmark (✓). 44 Clustering approach K value Source of variation Sum of squares Variance components Percentage of variation Statistic K-means model K =2 Among clusters 569.761 3.18 3.10 Fct= 0.031 Among populations 16,272.464 52.08 50.75 Fsc= 0.524 Within populations 7,198.333 47.36 46.15 Fst= 0.538 Among clusters 3,310.346 15.78 15.03 Fct= 0.150 Among populations 13,873.399 42.14 40.12 Fsc= 0.472 Within populations 6,971.500 47.10 44.85 Fst= 0.551 Among clusters 2,589.107 16.12 14.96 Fct= 0.150 Among populations 14,594.638 44.50 41.31 Fsc= 0.486 Within populations 6,971.500 47.10 43.73 Fst= 0.563 Among clusters 3,398.245 16.60 15.74 Fct= 0.157 Among populations 13,785.499 41.75 39.59 Fsc= 0.470 Within populations 6,971.500 47.10 44.67 Fst= 0.553 Among clusters 9,018.235 36.00 34.74 Fct= 0.347 Among populations 6,507.650 21.60 20.85 Fsc= 0.319 Within populations 5,889.167 46. 01 44.41 Fst= 0.556 NeighborNet Structure algorithm Structure algorithm Structure algorithm K =4 K =3 K =4 K = 20 Table 2. Analysis of molecular variance (AMOVA) performed with different grouping approaches. Percentage of variation explained by different methodological groupings (K-means model, Structure algorithm and NeighborNet) are shown. 45 Pop. Code Ind. Code Initial taxonomic assignment 1 1 1 2 2 2 3 3 3 4 4 4 5 5 5 6 6 6 7 7 7 8 8 8 9 9 10 10 10 11 11 12 12 13 13 14 14 15 15 16 16 17 17 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 V. aragonensis Stroh V. aragonensis Stroh V. aragonensis Stroh V. aragonensis Stroh V. aragonensis Stroh V. aragonensis Stroh V. austriaca subsp. austriaca L. V. austriaca subsp. austriaca L. V. austriaca subsp. austriaca L. V. austriaca subsp. austriaca L. V. austriaca subsp. austriaca L. V. austriaca subsp. austriaca L. V. austriaca subsp. austriaca L. (= V. macrodonta) V. austriaca subsp. austriaca L. (= V. macrodonta) V. austriaca subsp. austriaca L. (= V. macrodonta) V. austriaca subsp. dentata (F.W. Schmidt) Watzl V. austriaca subsp. dentata (F.W. Schmidt) Watzl V. austriaca subsp. dentata (F.W. Schmidt) Watzl V. austriaca subsp. dentata (F.W. Schmidt) Watzl V. austriaca subsp. dentata (F.W. Schmidt) Watzl V. austriaca subsp. dentata (F.W. Schmidt) Watzl V. austriaca subsp. jacquinii (Baumg.) Watzl V. austriaca subsp. jacquinii (Baumg.) Watzl V. austriaca subsp. jacquinii (Baumg.) Watzl V. austriaca subsp. jacquinii (Baumg.) Watzl V. austriaca subsp. jacquinii (Baumg.) Watzl V. austriaca subsp. jacquinii (Baumg.) Watzl V. austriaca subsp. jacquinii (Baumg.) Watzl V. austriaca subsp. jacquinii (Baumg.) Watzl V. austriaca subsp. jacquinii (Baumg.) Watzl V. austriaca subsp. jacquinii (Baumg.) Watzl V. austriaca subsp. jacquinii (Baumg.) Watzl V. austriaca subsp. jacquinii (Baumg.) Watzl V. austriaca subsp. jacquinii (Baumg.) Watzl V. austriaca subsp. jacquinii (Baumg.) Watzl V. austriaca subsp. jacquinii (Baumg.) Watzl V. austriaca subsp. jacquinii (Baumg.) Watzl V. austriaca subsp. jacquinii (Baumg.) Watzl V. austriaca subsp. jacquinii (Baumg.) Watzl V. austriaca subsp. jacquinii (Baumg.) Watzl V. austriaca subsp. jacquinii (Baumg.) Watzl V. austriaca subsp. jacquinii / V. orbiculata V. austriaca subsp. jacquinii / V. orbiculata Final taxonomic assignment (if changed after the analyses) Unresolved Unresolved Unresolved V. dalmatica N. Pad. Gar., Rojas-Andrés, López-González & M.M. Mart. Ort. V. dalmatica N. Pad. Gar., Rojas-Andrés, López-González & M.M. Mart. Ort. V. dalmatica N. Pad. Gar., Rojas-Andrés, López-González & M.M. Mart. Ort. V. dalmatica N. Pad. Gar., Rojas-Andrés, López-González & M.M. Mart. Ort. V. dalmatica N. Pad. Gar., Rojas-Andrés, López-González & M.M. Mart. Ort. V. dalmatica N. Pad. Gar., Rojas-Andrés, López-González & M.M. Mart. Ort. V. dalmatica N. Pad. Gar., Rojas-Andrés, López-González & M.M. Mart. Ort. V. dalmatica N. Pad. Gar., Rojas-Andrés, López-González & M.M. Mart. Ort. Unresolved Unresolved Collector Number Ploidy Level Voucher Code* Locality NPG18-14 NPG18-1 NPG18-26 NPG28_SD1419B NPG28_SB14005 NPG28_SC1410 BR265-1 BR265-5 BR265-9 NLG129-10 NLG129-7 NLG129-9 MO6106-1 MO6106-24 MO6106-8 BR178-1 BR178-3 BR178-4 BR275-1 BR275-2 BR275-3 BR108-1 BR108-3 BR108-6 BR112-3 BR112-4 SA392-1 SA392-6 SA392-8 BR121-2 BR121-5 MO4595-2 MO4595-4 MO5528-2 MO5528-4 MS1251-1 MS1251-4 SA382-1 SA382-5 SA429-1 SA429-3 BR102-1 BR102-3 4x 4x 4x 4x 4x 4x 6x 6x 6x 6x 6x 6x 4x 4x 4x 6x 6x 6x 6x 6x 6x 2x 2x 2x 2x 2x 2x 2x 2x 6x 6x 6x 6x 6x 6x 6x 6x 6x 6x 6x 6x 4x 4x SALA 154410 SALA 154410 SALA 154410 SALA 93529 SALA 93529 SALA 93529 SALA 153002 SALA 153002 SALA 153002 SALA 157058 SALA 157058 SALA 157058 SALA 157051 SALA 157051 SALA 157051 SALA 149043 SALA 149043 SALA 149043 SALA 155883 SALA 155883 SALA 155883 SALA 149284 SALA 149284 SALA 149284 SALA 149039 SALA 149039 SALA 149287 SALA 149287 SALA 149287 SALA 149369 SALA 149369 SALA 149377 SALA 149377 SALA 149042 SALA 149042 SALA 149387 SALA 149387 SALA 149389 SALA 149389 SALA 149392 SALA 149392 SALA 149041 SALA 149041 Spain; Huesca; Nerín, La Estiba Spain; Huesca; Nerín, La Estiba Spain; Huesca; Nerín, La Estiba Spain; Granada; Sierra de la Sagra Spain; Granada; Sierra de la Sagra Spain; Granada; Sierra de la Sagra Slovakia; Spisšká Nová Ves; Letanovce, Slovensky raj Slovakia; Spisšká Nová Ves; Letanovce, Slovensky raj Slovakia; Spisšká Nová Ves; Letanovce, Slovensky raj Slovkia; Černochov Slovkia; Černochov Slovkia; Černochov Romania; Valea Lunga Romania; Valea Lunga Romania; Valea Lunga Austria; Niederösterreich; Krems, between Weissenkirchen and Dürnstein Austria; Niederösterreich; Krems, between Weissenkirchen and Dürnstein Austria; Niederösterreich; Krems, between Weissenkirchen and Dürnstein Hungary; Gyöngyös, Gyöngyösi Sárhegy Hungary; Gyöngyös, Gyöngyösi Sárhegy Hungary; Gyöngyös, Gyöngyösi Sárhegy Bosnia and Herzegovina; Republika Srpska; between Trebinje and Dubrovnik Bosnia and Herzegovina; Republika Srpska; between Trebinje and Dubrovnik Bosnia and Herzegovina; Republika Srpska; between Trebinje and Dubrovnik Croatia; Dubrovnik, Gromaca Croatia; Dubrovnik, Gromaca Montenegro; Zabljak Montenegro; Zabljak Montenegro; Zabljak Montenegro; Mts. Treskavac, between Borkovici and Boricje Montenegro; Mts. Treskavac, between Borkovici and Boricje Bulgaria; Nova Mahala, near to Nikolaev Bulgaria; Nova Mahala, near to Nikolaev Croatia; Josipdol,between Ostarije and Pribarici Croatia; Josipdol,between Ostarije and Pribarici Greece; Mt. Vermion Greece; Mt. Vermion Bosnia and Herzegovina; Travnik, Vlasic Bosnia and Herzegovina; Travnik, Vlasic Serbia; Devojacki Bunar, Vladimirovac Serbia; Devojacki Bunar, Vladimirovac Bosnia and Herzegovina; Potoci, Porim planina, Rujiste Bosnia and Herzegovina; Potoci, Porim planina, Rujiste 46 17 18 18 18 19 19 19 20 20 21 21 21 22 22 22 23 23 23 24 24 25 25 25 26 26 26 27 27 27 28 28 28 29 29 30 30 31 31 31 32 32 33 34 34 34 35 35 35 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 V. austriaca subsp. jacquinii / V. orbiculata V. austriaca subsp. jacquinii / V. orbiculata V. austriaca subsp. jacquinii / V. orbiculata V. austriaca subsp. jacquinii / V. orbiculata V. austriaca subsp. jacquinii / V. orbiculata V. austriaca subsp. jacquinii / V. orbiculata V. austriaca subsp. jacquinii / V. orbiculata V. affinis jacquinii V. affinis jacquinii V. crinita (= V. thracica) V. crinita (= V. thracica) V. crinita (= V. thracica) V. crinita (= V. thracica) V. crinita (= V. thracica) V. crinita (= V. thracica) V. crinita Kit V. crinita Kit V. crinita Kit V. crinita Kit V. crinita Kit V. crinita (= V. crinita f. bosniaca) Fiala V. crinita (= V. crinita f. bosniaca) Fiala V. crinita (= V. crinita f. bosniaca) Fiala V. kindlii Adamović V. kindlii Adamović V. kindlii Adamović V. kindlii Adamović V. kindlii Adamović V. kindlii Adamović V. kindlii Adamović V. kindlii Adamović V. kindlii Adamović V. kindlii Adamović V. kindlii Adamović V. kindlii Adamović V. kindlii Adamović V. affinis kindlii V. affinis kindlii V. affinis kindlii V. affinis kindlii V. affinis kindlii V. affinis kindlii V. affinis kindlii V. affinis kindlii V. affinis kindlii V. affinis kindlii V. affinis kindlii V. affinis kindlii Unresolved Unresolved Unresolved Unresolved V. dalmatica N. Pad. Gar., Rojas-Andrés, López-González & M.M. Mart. Ort. V. dalmatica N. Pad. Gar., Rojas-Andrés, López-González & M.M. Mart. Ort. V. dalmatica N. Pad. Gar., Rojas-Andrés, López-González & M.M. Mart. Ort. V. dalmatica N. Pad. Gar., Rojas-Andrés, López-González & M.M. Mart. Ort. V. dalmatica N. Pad. Gar., Rojas-Andrés, López-González & M.M. Mart. Ort. V. thracica Velen. V. thracica Velen. V. thracica Velen. V. thracica Velen. V. thracica Velen. V. thracica Velen. Unresolved Unresolved Unresolved Unresolved Unresolved Unresolved Unresolved Unresolved Unresolved Unresolved Unresolved Unresolved V. teucrioides Boiss. & Heldr. V. teucrioides Boiss. & Heldr. V. teucrioides Boiss. & Heldr. BR102-5 SA377-1 SA377-5 SA377-8 SA386-1 SA386-2 SA386-3 MO5552-2 MO5552-3 MS1227-3 MS1227-1 MS1227-4 MS1244-1 MS1244-3 MS1244-4 BR137-13 BR137-2 BR137-7 BR271-3 BR271-5 Frajman-12562-1 Frajman-12562-2 Frajman-12562-3 BR258-18 BR258-8 BR258-9 MO5558-10 MO5558-5 MO5558-8 MO5569-16 MO5569-17 MO5569-18 MO6090-12 MO6090-4 MO6092-34 MO6092-6 MS1259-12 MS1259-8 MS1259-9 SA394-1 SA394-6 SA394-BIS SA393*-1 SA393*-2 SA393*-5 NLG119bis-1 NLG119bis-2 NLG119bis-3 4x 4x 4x 4x 2x 2x 2x 2x 2x 2x * 2x 2x 2x 2x 2x 2x 2x 2x * * * * * * 2x 2x 2x 2x 2x 2x 2x 2x 2x 2x 2x 4x 4x 4x 2x 2x 2x * 4x 4x 2x 2x 2x SALA 149041 SALA 149355 SALA 149355 SALA 149355 SALA 149292 SALA 149292 SALA 149292 SALA 149274 SALA 149274 SALA 149038 SALA 149038 SALA 149038 SALA 149386 SALA 149386 SALA 149386 SALA 149288 SALA 149288 SALA 149288 SALA 157045 SALA 157045 SALA 149244 SALA 149244 SALA 149244 SALA 157013 SALA 157013 SALA 157013 SALA 149277 SALA 149277 SALA 149277 SALA 149278 SALA 149278 SALA 149278 SALA 157011 SALA 157011 SALA 157012 SALA 157012 SALA 149346 SALA 149346 SALA 149346 SALA 149280 SALA 149280 SALA 149281 SALA 149347 SALA 149347 SALA 149347 SALA 155864 SALA 155864 SALA 155864 Bosnia and Herzegovina; Potoci, Porim planina, Rujiste Bosnia and Herzegovina; Sarajevo, Trebevik Bosnia and Herzegovina; Sarajevo, Trebevik Bosnia and Herzegovina; Sarajevo, Trebevik Montenegro; Kotor, Lovćen Montenegro; Kotor, Lovćen Montenegro; Kotor, Lovćen Bosnia and Herzegovina; betweenTjentište and Gacko Bosnia and Herzegovina; betweenTjentište and Gacko Bulgaria, Plovdiv, Popovitsa Bulgaria, Plovdiv, Popovitsa Bulgaria, Plovdiv, Popovitsa Bulgaria; Varna, between Vinitsa and aladza's Monastery Bulgaria; Varna, between Vinitsa and aladza's Monastery Bulgaria; Varna, between Vinitsa and aladza's Monastery Serbia; between Zlot and Brestovac Serbia; between Zlot and Brestovac Serbia; between Zlot and Brestovac Romania; Deva, Cetate Romania; Deva, Cetate Bosnia and Herzegovina; Ravan planina, Summit of Mt. Tajan Bosnia and Herzegovina; Ravan planina, Summit of Mt. Tajan Bosnia and Herzegovina; Ravan planina, Summit of Mt. Tajan Greece; Boras Sky Station, summit of Kaimaktsalan Greece; Boras Sky Station, summit of Kaimaktsalan Greece; Boras Sky Station, summit of Kaimaktsalan Montenegro; Cakor, near to Kosovo border Montenegro; Cakor, near to Kosovo border Montenegro; Cakor, near to Kosovo border FYROM; Gevgelija, near to Kozuf Sky Station FYROM; Gevgelija, near to Kozuf Sky Station FYROM; Gevgelija, near to Kozuf Sky Station FYROM; Pelister, near to Pelister summit FYROM; Pelister, near to Pelister summit FYROM; Pelister, near to Pelister summit FYROM; Pelister, near to Pelister summit Greece; Mt. Vermion Greece; Mt. Vermion Greece; Mt. Vermion Montenegro; between Pljevlja and Bobovo Montenegro; between Pljevlja and Bobovo Montenegro; between Pljevlja and Bobovo Montenegro; Zabljak Montenegro; Zabljak Montenegro; Zabljak FYROM; Galichica, between Trpejca and Oteševo FYROM; Galichica, between Trpejca and Oteševo FYROM; Galichica, between Trpejca and Oteševo 47 36 37 38 39 39 39 40 40 40 41 41 41 42 42 42 43 43 43 43 44 44 44 45 45 45 45 45 45 46 46 46 47 47 47 48 48 48 49 49 49 50 50 50 51 52 53 54 54 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 V. krylovii Schischk. V. krylovii Schischk. V. krylovii Schischk. V. linearis (Bornm.) Rojas-Andrés & M.M. Mart. Ort. V. linearis (Bornm.) Rojas-Andrés & M.M. Mart. Ort. V. linearis (Bornm.) Rojas-Andrés & M.M. Mart. Ort. V. linearis (Bornm.) Rojas-Andrés & M.M. Mart. Ort. V. linearis (Bornm.) Rojas-Andrés & M.M. Mart. Ort. V. linearis (Bornm.) Rojas-Andrés & M.M. Mart. Ort. V. linearis (Bornm.) Rojas-Andrés & M.M. Mart. Ort. V. linearis (Bornm.) Rojas-Andrés & M.M. Mart. Ort. V. linearis (Bornm.) Rojas-Andrés & M.M. Mart. Ort. V. affinis linearis V. affinis linearis V. affinis linearis V. orbiculata A. Kern V. orbiculata A. Kern V. orbiculata A. Kern V. orbiculata A. Kern V. orbiculata A. Kern V. orbiculata A. Kern V. orbiculata A. Kern V. orbiculata A. Kern V. orbiculata A. Kern V. orbiculata A. Kern V. orbiculata A. Kern V. orbiculata A. Kern V. orbiculata A. Kern V. orsiniana Ten. V. orsiniana Ten. V. orsiniana Ten. V. orsiniana Ten. V. orsiniana Ten. V. orsiniana Ten. V. orsiniana Ten. V. orsiniana Ten. V. orsiniana Ten. V. orsiniana Ten. V. orsiniana Ten. V. orsiniana Ten. V. orsiniana Ten. V. orsiniana Ten. V. orsiniana Ten. V. affinis orsiniana V. affinis orsiniana V. affinis orsiniana V. affinis orsiniana V. affinis orsiniana V. linearis (Bornm.) Rojas-Andrés & M.M. Mart. Ort. V. linearis (Bornm.) Rojas-Andrés & M.M. Mart. Ort. V. linearis (Bornm.) Rojas-Andrés & M.M. Mart. Ort. V. kindlii Adamović V. kindlii Adamović V. kindlii Adamović V. kindlii Adamović V. kindlii Adamović Albach 1275 s.n s.n MO6094-19 MO6094-1 MO6094-3 NLG151-10 NLG151-11 NLG151-8 NPG51-1 NPG51-2 NPG51-3 NLG125-26 NLG125-29 NLG125-32 BR100-12 BR100-13 BR100-1 BR100-3 BR110-1 BR110-3 BR110-4 MO5537-12 MO5537-13 MO5537-1 MO5537-4 MO5537-5 MO5537-7 BR201-11 BR201-1 BR201-2 BR205-2 BR205-4 BR205-5 BR213-17 BR213-3 BR213-5 BR239-10 BR239-1 BR239-5 MO6056-1 MO6056-3 MO6056-9 MO6078-1 MO6079-6 MO6079*-3 NLG108-2 NLG108-3 2x * * * 2x 2x 2x 2x 2x 2x 2x 2x 2x 2x 2x 4x 4x 2x 2x 2x 2x 2x 2x 2x 2x 4x 4x 4x 2x 2x 2x 2x 2x 2x 2x 2x 2x 2x 2x 2x 2x 2x 2x 2x 2x 2x 2x 2x OLD ALTB ALTB SALA 153001 SALA 153001 SALA 153001 SALA 157040 SALA 157040 SALA 157040 SALA 157038 SALA 157038 SALA 157038 SALA 153000 SALA 153000 SALA 153000 SALA 149336 SALA 149336 SALA 149336 SALA 149336 SALA 149294 SALA 149294 SALA 149294 SALA 149337 SALA 149337 SALA 149337 SALA 149337 SALA 149337 SALA 149337 SALA 149301 SALA 149301 SALA 149301 SALA 149304 SALA 149304 SALA 149304 SALA 149309 SALA 149309 SALA 149309 SALA 155118 SALA 155118 SALA 155118 SALA 155070 SALA 155070 SALA 155070 SALA 155877 SALA 155878 SALA 155876 SALA 155881 SALA 155881 Russia; Republic of Altai, Chike-Taman-Pass Russia; Altay region, Charyshsky distr. Russia; Altay region, Petropavlovsky distr. FYROM; between Modrište and Zdunje, near to Jezero Kozjak FYROM; between Modrište and Zdunje, near to Jezero Kozjak FYROM; between Modrište and Zdunje, near to Jezero Kozjak FYROM; Skopje, near to Nova Breznica FYROM; Skopje, near to Nova Breznica FYROM; Skopje, near to Nova Breznica FYROM; Prilep, Sivec, marble mountain FYROM; Prilep, Sivec, marble mountain FYROM; Prilep, Sivec, marble mountain FYROM; Brod, near to Barbaros port FYROM; Brod, near to Barbaros port FYROM; Brod, near to Barbaros port Bosnia and Herzegovina; Mostar, Hum mountain Bosnia and Herzegovina; Mostar, Hum mountain Bosnia and Herzegovina; Mostar, Hum mountain Bosnia and Herzegovina; Mostar, Hum mountain Croatia; Peljesak Peninsule, between Trstenik and Pijavicino Croatia; Peljesak Peninsule, between Trstenik and Pijavicino Croatia; Peljesak Peninsule, between Trstenik and Pijavicino Croatia; Makarska, between Omis and Makarska, Gornja Brela Croatia; Makarska, between Omis and Makarska, Gornja Brela Croatia; Makarska, between Omis and Makarska, Gornja Brela Croatia; Makarska, between Omis and Makarska, Gornja Brela Croatia; Makarska, between Omis and Makarska, Gornja Brela Croatia; Makarska, between Omis and Makarska, Gornja Brela France; Department of Hérault, Saint-Chinian, between Malibert and Pardailhan France; Department of Hérault, Saint-Chinian, between Malibert and Pardailhan France; Department of Hérault, Saint-Chinian, between Malibert and Pardailhan France; Department of Lozère, Cévennes, Aven Armand France; Department of Lozère, Cévennes, Aven Armand France; Department of Lozère, Cévennes, Aven Armand France; Department of Alpes-Maritimes, between Tende and Col de Tende's tunnel France; Department of Alpes-Maritimes, between Tende and Col de Tende's tunnel France; Department of Alpes-Maritimes, between Tende and Col de Tende's tunnel Spain; Huesca; 7km from Benabarre, road A1606 to Laguarrés Spain; Huesca; 7km from Benabarre, road A1606 to Laguarrés Spain; Huesca; 7km from Benabarre, road A1606 to Laguarrés Spain; Castellón, Rambla de las truchas Spain; Castellón, Rambla de las truchas Spain; Castellón, Rambla de las truchas Italy; Calabria, Monte Pollino, Coll di Dragone to Voscari Italy; Calabria, Monte Pollino, Coll di Dragone to Voscari Italy; Calabria, Monte Pollino, Coll di Dragone to Voscari Italy; Calabria, between Ceci and Lorica, ca. 7 km to the west from Lorica Italy; Calabria, between Ceci and Lorica, ca. 7 km to the west from Lorica 48 55 56 56 56 57 57 57 58 58 58 59 59 59 60 60 60 60 61 61 61 62 62 62 63 63 63 64 64 64 65 65 65 66 66 67 67 68 68 69 69 70 70 71 71 71 72 72 72 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 V. affinis orsiniana V. prostrata L. V. prostrata L. V. prostrata L. V. prostrata L. V. prostrata L. V. prostrata L. V. prostrata L. V. prostrata L. V. prostrata L. V. prostrata L. V. prostrata L. V. prostrata L. V. rhodopea (Velen.) Degen. ex Stoj. & Stef. V. rhodopea (Velen.) Degen. ex Stoj. & Stef. V. rhodopea (Velen.) Degen. ex Stoj. & Stef. V. rhodopea (Velen.) Degen. ex Stoj. & Stef. V. rosea Desf. V. rosea Desf. V. rosea Desf. V. rosea Desf. V. rosea Desf. V. rosea Desf. V. rosea Desf. V. rosea Desf. V. rosea Desf. V. rosea Desf. V. rosea Desf. V. rosea Desf. V. rosea Desf. V. rosea Desf. V. rosea Desf. V. satureiifolia Poit. & Turpin V. satureiifolia Poit. & Turpin V. satureiifolia Poit. & Turpin V. satureiifolia Poit. & Turpin V. satureiifolia Poit. & Turpin V. satureiifolia Poit. & Turpin V. satureiifolia Poit. & Turpin V. satureiifolia Poit. & Turpin V. satureiifolia Poit. & Turpin V. satureiifolia Poit. & Turpin V. sennenii (Pau) M.M. Mart. Ort. & E. Rico V. sennenii (Pau) M.M. Mart. Ort. & E. Rico V. sennenii (Pau) M.M. Mart. Ort. & E. Rico V. sennenii (Pau) M.M. Mart. Ort. & E. Rico V. sennenii (Pau) M.M. Mart. Ort. & E. Rico V. sennenii (Pau) M.M. Mart. Ort. & E. Rico V. kindlii Adamović NLG109-1 BR182-12 BR182-14 BR182-9 MO6037-2 MO6037-3 MO6037-7 MS1239-1 MS1239-2 MS1239-3 NPG6-10 NPG6-13 NPG6-9 BR12-10 BR12-1 BR12-3 BR12-6 DP783-14 DP783-19 DP783-2 MO5502-10 MO5502-14 MO5502-5 MO5510-3 MO5510-4 MO5510-6 NLG88-12 NLG88-25 NLG88-27 VL87-13 VL87-4 VL87-7 BR204-11 BR204-3 BR244-21 BR244-24 DA603-1 DA603-5 MO6070-4 MO6070-5 NPG2-10 NPG2-12 BR223-1 BR223-2 BR223-5 BR224-1 BR224-2 BR224-5 2x 2x 2x 2x 2x 2x 2x 2x 2x 2x 2x 2x 2x 2x 2x 2x 2x 2x 2x 2x 2x 2x 2x 4x 2x 4x 2x 2x 2x 2x 2x 2x 4x 4x 4x 4x * * 4x 4x 4x 4x 8x 8x 8x 8x 8x 8x SALA 155879 SALA 149040 SALA 149040 SALA 149040 SALA 149315 SALA 149315 SALA 149315 SALA 149317 SALA 149317 SALA 149317 SALA 155856 SALA 155856 SALA 155856 SALA 149321 SALA 149321 SALA 149321 SALA 149321 SALA 149323 SALA 149323 SALA 149323 SALA 149324 SALA 149324 SALA 149324 SALA 149338 SALA 149338 SALA 149338 SALA 155071 SALA 155071 SALA 155071 SALA 149326 SALA 149326 SALA 149326 SALA 149356 SALA 149356 SALA 155108 SALA 155108 WU WU SALA 155113 SALA 155113 SALA 154213 SALA 154213 SALA 149394 SALA 149394 SALA 149394 SALA 149395 SALA 149395 SALA 149395 Italy; Calabria, between Ceci and Lorica, ca. 7 km to the west from Lorica Austria; Niederösterreich; Rohrendorf bei Krems, Saubühel Austria; Niederösterreich; Rohrendorf bei Krems, Saubühel Austria; Niederösterreich; Rohrendorf bei Krems, Saubühel France; Department of Hautes-Alpes, Gap, Col de Bayard France; Department of Hautes-Alpes, Gap, Col de Bayard France; Department of Hautes-Alpes, Gap, Col de Bayard Bulgaria; Madara, near to the village Bulgaria; Madara, near to the village Bulgaria; Madara, near to the village Italy; Abruzzo, Gioia dei Marsi, from Sperone to Monte Serrato Italy; Abruzzo, Gioia dei Marsi, from Sperone to Monte Serrato Italy; Abruzzo, Gioia dei Marsi, from Sperone to Monte Serrato Bulgaria; Belmeken Bulgaria; Belmeken Bulgaria; Belmeken Bulgaria; Belmeken Morocco; prov. Ifrane, Azrou, near to Djebel Hebri, western slope Morocco; prov. Ifrane, Azrou, near to Djebel Hebri, western slope Morocco; prov. Ifrane, Azrou, near to Djebel Hebri, western slope Algeria; Col de Krorchef Algeria; Col de Krorchef Algeria; Col de Krorchef Algeria; Batna, summit of Djebel Ichali Algeria; Batna, summit of Djebel Ichali Algeria; Batna, summit of Djebel Ichali Marocco; Souss-Massa-Drâa, Jebel Siroua, northern route Marocco; Souss-Massa-Drâa, Jebel Siroua, northern route Marocco; Souss-Massa-Drâa, Jebel Siroua, northern route Morocco; Bab Taza, to the north from Jbel L'akraa Morocco; Bab Taza, to the north from Jbel L'akraa Morocco; Bab Taza, to the north from Jbel L'akraa France; Department of Lozère, Cévennes France; Department of Lozère, Cévennes Spain; Huesca, Aragüés del Puerto, Llanos de Lizara Spain; Huesca, Aragüés del Puerto, Llanos de Lizara Germany; Rheinland-Pfalz, Nature protection area Uhlerborn Germany; Rheinland-Pfalz, Nature protection area Uhlerborn Spain; Huesca, Borau, Las Blancas Spain; Huesca, Borau, Las Blancas France; Department of Seine and Marne, Fontainebleau forest France; Department of Seine and Marne, Fontainebleau forest Spain; Álava, Salinas de Añana, from Sobrón to collado de la Rastrilla Spain; Álava, Salinas de Añana, from Sobrón to collado de la Rastrilla Spain; Álava, Salinas de Añana, from Sobrón to collado de la Rastrilla Spain; Cantabria, Sonabia Spain; Cantabria, Sonabia Spain; Cantabria, Sonabia 49 73 74 74 74 75 75 75 76 76 76 77 77 77 78 78 78 79 79 79 80 80 80 81 81 81 82 82 82 83 83 83 84 84 85 85 86 86 87 87 88 88 89 89 90 90 91 91 91 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 V. affinis sennenii V. tenuifolia subsp. fontqueri (Pau) M.M. Mart. Ort. & E. Rico V. tenuifolia subsp. fontqueri (Pau) M.M. Mart. Ort. & E. Rico V. tenuifolia subsp. fontqueri (Pau) M.M. Mart. Ort. & E. Rico V. tenuifolia subsp. fontqueri (Pau) M.M. Mart. Ort. & E. Rico V. tenuifolia subsp. fontqueri (Pau) M.M. Mart. Ort. & E. Rico V. tenuifolia subsp. fontqueri (Pau) M.M. Mart. Ort. & E. Rico V. tenuifolia subsp. fontqueri (Pau) M.M. Mart. Ort. & E. Rico V. tenuifolia subsp. fontqueri (Pau) M.M. Mart. Ort. & E. Rico V. tenuifolia subsp. fontqueri (Pau) M.M. Mart. Ort. & E. Rico V. tenuifolia subsp. javalambrensis (Pau) Molero & J. Pujadas V. tenuifolia subsp. javalambrensis (Pau) Molero & J. Pujadas V. tenuifolia subsp. javalambrensis (Pau) Molero & J. Pujadas V. tenuifolia subsp. javalambrensis (Pau) Molero & J. Pujadas V. tenuifolia subsp. javalambrensis (Pau) Molero & J. Pujadas V. tenuifolia subsp. javalambrensis (Pau) Molero & J. Pujadas V. tenuifolia subsp. javalambrensis (Pau) Molero & J. Pujadas V. tenuifolia subsp. javalambrensis (Pau) Molero & J. Pujadas V. tenuifolia subsp. javalambrensis (Pau) Molero & J. Pujadas V. tenuifolia subsp. tenuifolia Asso V. tenuifolia subsp. tenuifolia Asso V. tenuifolia subsp. tenuifolia Asso V. tenuifolia subsp. tenuifolia Asso V. tenuifolia subsp. tenuifolia Asso V. tenuifolia subsp. tenuifolia Asso V. teucrioides Boiss. & Heldr. V. teucrioides Boiss. & Heldr. V. teucrioides Boiss. & Heldr. V. teucrioides Boiss. & Heldr. V. teucrioides Boiss. & Heldr. V. teucrioides Boiss. & Heldr. V. teucrium L. var. angustifolia V. teucrium L. var. angustifolia V. teucrium L. var. angustifolia V. teucrium L. var. angustifolia V. teucrium L. var. angustifolia V. teucrium L. var. angustifolia V. teucrium L. var. angustifolia V. teucrium L. var. angustifolia V. teucrium L. var. teucrium V. teucrium L. var. teucrium V. teucrium L. var. teucrium V. teucrium L. var. teucrium V. teucrium L. var. teucrium V. teucrium L. var. teucrium V. turrilliana Stoj. & Stef. V. turrilliana Stoj. & Stef. V. turrilliana Stoj. & Stef. V. sennenii (Pau) M.M. Mart. Ort. & E. Rico V. angustifolia (Vahl) Bernh. V. angustifolia (Vahl) Bernh. V. angustifolia (Vahl) Bernh. V. angustifolia (Vahl) Bernh. V. angustifolia (Vahl) Bernh. V. angustifolia (Vahl) Bernh. V. angustifolia (Vahl) Bernh. V. angustifolia (Vahl) Bernh. V. teucrium L. V. teucrium L. V. teucrium L. V. teucrium L. V. teucrium L. V. teucrium L. MO6070BIS s.n. s.n. s.n. MO1519-3-3 MO1519-3-4 MO1519-3-6 NPG27-1 NPG27-3 NPG27-5 BR222-3 BR222-4 BR222-7 NLG23-1 NLG23-24 NLG23-6 NLG05-2 NLG05-5 NLG05-7 BR241-12 BR241-1 BR241-3 MO6058-15 MO6058-5 MO6058-8 BR130-15 BR130-1 BR130-8 BR48-10 BR48-11 BR48-1 BR168-11 BR168-7 BR211-18 BR211-2 MO6022-14 MO6022-7 NPG3-1 NPG3-3 MO4574-10 MO4574-2 MO6025-3 MO6025-6 MO6042-17B MO6042-8A BR41-10 BR41-1 BR41-2 8x * * 2x 2x 2x 2x 2x 2x 2x 2x 2x 2x 2x 2x 2x 2x 2x 2x 2x 2x 2x 2x 2x 2x 2x 2x 2x 2x 2x 2x 8x 8x 8x 8x 8x 8x 8x 8x 8x 8x 8x 8x 8x 8x 2x 2x 2x SALA 155112 GDA 57010 GDA 57010 GDA 57010 SALA 120855 SALA 120855 SALA 120855 MGC 46659 MGC 46659 MGC 46659 SALA 149328 SALA 149328 SALA 149328 SALA 155079 SALA 155079 SALA 155079 SALA 155105 SALA 155105 SALA 155105 SALA 155117 SALA 155117 SALA 155117 SALA 155096 SALA 155096 SALA 155096 SALA 149329 SALA 149329 SALA 149329 SALA 149330 SALA 149330 SALA 149330 SALA 149399 SALA 149399 SALA 149409 SALA 149409 SALA 149413 SALA 149413 SALA 154237 SALA 154237 SALA 149044 SALA 149044 SALA 149414 SALA 149414 SALA 149419 SALA 149418 SALA 149331 SALA 149331 SALA 149331 Spain; Huesca, Borau, Las Blancas Spain; Granada, Calar de los Tejoletos Spain; Granada, Calar de los Tejoletos Spain; Granada, Calar de los Tejoletos Spain; Almería, Dalías, Sierra de Gádor, Cerro de La Atalaya and Dos Hermanas Spain; Almería, Dalías, Sierra de Gádor, Cerro de La Atalaya and Dos Hermanas Spain; Almería, Dalías, Sierra de Gádor, Cerro de La Atalaya and Dos Hermanas Spain; Ronda, Sierra de Las Nieves, Los Quejigales, Puerto de Los Pilones Spain; Ronda, Sierra de Las Nieves, Los Quejigales, Puerto de Los Pilones Spain; Ronda, Sierra de Las Nieves, Los Quejigales, Puerto de Los Pilones Spain; Salamanca, La Mata de la Armuña Spain; Salamanca, La Mata de la Armuña Spain; Salamanca, La Mata de la Armuña Spain; León, Montes de Valdueza, La Guiana, Los Apóstoles Spain; León, Montes de Valdueza, La Guiana, Los Apóstoles Spain; León, Montes de Valdueza, La Guiana, Los Apóstoles Spain; Guadalajara, Atienza, Naharrós Spain; Guadalajara, Atienza, Naharrós Spain; Guadalajara, Atienza, Naharrós Spain; Castellón, Morella. San Cristobal's chapel Spain; Castellón, Morella. San Cristobal's chapel Spain; Castellón, Morella. San Cristobal's chapel Spain; Huesca, Arro, road to Los Molinos and S. Vitorián Monastery Spain; Huesca, Arro, road to Los Molinos and S. Vitorián Monastery Spain; Huesca, Arro, road to Los Molinos and S. Vitorián Monastery FYROM; Mavrovo, Bistra planina, between Mavrovo and Galicnik FYROM; Mavrovo, Bistra planina, between Mavrovo and Galicnik FYROM; Mavrovo, Bistra planina, between Mavrovo and Galicnik Greece; Mt. Olimpo, Sparmos Greece; Mt. Olimpo, Sparmos Greece; Mt. Olimpo, Sparmos France; Department of Haute-Savoie, Monte Salève near to Ginebra France; Department of Haute-Savoie, Monte Salève near to Ginebra France; Department of Alpes-Maritimes, Grasse; near to Caussols, col de l'Êcre France; Department of Alpes-Maritimes, Grasse; near to Caussols, col de l'Êcre France; Department of Eure-et-Loir, Châteaudun, Thiville France; Department of Eure-et-Loir, Châteaudun, Thiville France; Department of Essonne, Valpuiseaux. La Lieu France; Department of Essonne, Valpuiseaux. La Lieu Bulgaria; near to Tran, between Pernik and Tran Bulgaria; near to Tran, between Pernik and Tran Germany; Nordrhein-Westfalen, Euskirchen, between Iversheim and Arloff Germany; Nordrhein-Westfalen, Euskirchen, between Iversheim and Arloff Austria; Kärnten, Villach, Oberschütt to Unterschütt Austria; Kärnten, Villach, Oberschütt to Unterschütt Turkey; Kirklareli, from Dereköy to Armağan Turkey; Kirklareli, from Dereköy to Armağan Turkey; Kirklareli, from Dereköy to Armağan 50 92 92 92 93 93 93 94 95 236 237 238 239 240 241 242 243 V. turrilliana Stoj. & Stef. V. turrilliana Stoj. & Stef. V. turrilliana Stoj. & Stef. V. turrilliana Stoj. & Stef. V. turrilliana Stoj. & Stef. V. turrilliana Stoj. & Stef. ** V. orientalis Mill. ** V. orientalis Mill. BR45-2 BR45-3 BR45-5 MS1247-1 MS1247-2 MS1247-9 MA3463 MA3437 2x 2x 2x 2x 2x 2x 6x 4x SALA 149333 SALA 149333 SALA 149333 SALA 149334 SALA 149334 SALA 149334 OLD & VANF OLD & VANF Turkey; 6 Km. from Vize, to Kömürköy-Alkpinars Turkey; 6 Km. from Vize, to Kömürköy-Alkpinars Turkey; 6 Km. from Vize, to Kömürköy-Alkpinars Bulgaria; 15 Km to the north of Malko Turnovo Bulgaria; 15 Km to the north of Malko Turnovo Bulgaria; 15 Km to the north of Malko Turnovo Turkey; Prov. Van, Güzeldere Pass Turkey; Prov. Van, between Gürpinar and Güzelsu * Ploidy level not available ** Outgroup Table S1. Individuals of the diploid-polyploid complex Veronica subsection Pentasepalae included in the present study. Population (Pop.) and individual (Ind.) codes, collector number, DNA ploidy level, voucher code and location are indicated for each individual. The initial and final taxonomic assignments according to the results presented here are shown. 51 No. of fragments AFLP primer combinations Color dye tinyFLP Holland's Scripts MseI-CAT EcoRI-ACT 6-FAM 309 222 MseI-CTG EcoRI-AAG VIC 249 217 MseI-CTT EcoRI-ACC NED 310 466 MseI-CAA EcoRI-ACC PET 259 256 1127 1161 Total no. of fragments Table S2. Primer combinations and fluorescent dye labels used in AFLP genotyping. Number of fragments generated by each automated AFLP scoring methodology is shown. 52 Optimized Parameter Variable/Fixed Begin End Step 6FAM VIC NED PET Minimum peak height (rfu) Variable 50 200 50 100 150 200 200 Maximum peak width (bp) Fixed 1.0 * * 1.0 1.0 1.0 1.0 Minimum peak size (bp) Variable 50 150 50 50 100 50 50 Maximum peak size (bp) Variable 350 500 50 500 450 450 350 Size tolerance range (bp) Variable 0.4 1.5 0.1 0.5 0.7 0.5 0.6 Minimum peak-peak distance (bp) Fixed 0.0 * * 0.0 0.0 0.0 0.0 Peak Height difference (%) Fixed 0.0 * * 0 0 0 0 Minimum allele frequency (%) Fixed 1.0 * * 1.0 1.0 1.0 1.0 Maximum allele frequency (%) Fixed 99.0 * * 99.0 99.0 99.0 99.0 Global R * * * * 0.992410 0.988047 0.984625 0.985349 Significance level of global R * * * * 0.001 0.001 0.001 0.001 Table S3. Parameter space used to find the optimal scoring parameters using optiFLP, and optimized parameters detected for each fluorescent-labelled primer combination 53 Figure Captions Fig. 1. Maps of sampling sites. Population codes follow Table S1, symbol shapes represent ploidy level (○ 2x; □ 4x; Δ 6x; ◊ 8x; ☆ mixed-ploidy populations), and colors indicate cluster affiliation. An asterisk (*) indicates missing data for DNA-ploidy level. A, B, Locations of the 93 populations of Veronica subsect. Pentasepalae analyzed in this study. C, Detailed distribution map of studied populations from the Balkan Peninsula. Fig. 2. A, Neighbor-Net network based on 1127 AFLP scored fragments of 241 individuals of Veronica subsect. Pentasepalae using Jaccard’s genetic distances. Individual codes are shown following Table S1. Arcs delimits taxa whose initial taxonomic determination and ploidy are shown. Range of colors of the arcs differentiates the four clusters identified by the Structure analyses. Bootstrap values (BS) > 50% are shown. B, Bayesian clustering analyses based on the entire AFLP dataset. Four main clusters from a STRUCTURE analyses with K = 4 are represented by different colors. Black lines separate different populations which are indicated below the graph (population codes follow Table S1). Fig. 3. Principal Coordinate Analysis (PCoA) of the AFLP dataset of 241 individuals based on Jaccard’s distances and DCENTER module. Axis 1 and Axis 2 explain 8.36 and 6.20% of the variation, respectively. A, The two clusters supported by genetic structure analyses using Kmeans clustering algorithm are represented by colors. B, Colors indicate the four genetic clusters from K = 4 estimated by Bayesian clustering analyses using Structure. 54 Fig. 4. Genetic structure analysis based on AFLP data of 58 individuals from 26 populations (corresponding with group IV/cluster D, which comprises most polyploid taxa from central Europe and north of Spain). A, Bayesian model-based clustering at K = 2 using Structure. Colors represent different clusters (dark blue vs. light blue) and black lines separate individuals of different populations, which are indicated below the graph (codes follow Table S1). Taxonomic names from each population are shown above the graph. B, Part of the NeighborNet (presented in Fig. 2) representing the 58 analyzed individuals. C, Distribution map of populations included in genetic structure analysis of cluster D. Supplementary data. Fig. S1. Neighbor-joining tree based on the AFLP complete dataset (1127 fragments; 243 individuals from 95 populations including V. orientalis as outgroup) inferred from Nei-Li distances. Individual labels follow Table S1. Initial taxonomic assignment and ploidy level are given. Arcs delimit each taxa and range of colors indicate the four clusters identified by the Structure analyses with K = 4. Bootstrap values (BS) > 50% are shown and asterisks in branches indicate absence of BS values. Supplementary data. Fig. S2. Genetic Structure analyses based on the entire AFLP dataset at K = 4 (A), partial data subsets (B), and whole dataset at K = 20 (C). Each individual is represented by one horizontal column. Colors represent different clusters and black lines separate different populations which are indicated in the left side of the graph (codes follow Table S1). Taxonomic names from each population are shown on the right side of the figure. 55 56 57 58 Highlights  Multiple autopolyploidization events occurred in the tetraploid V. satureiifolia.  A new diploid species, V. dalmatica, is identified and fully described.  Cryptic speciation is observed within the subsection.  An amphi-Adriatic distribution of V. kindlli is demonstrated. Graphical abstract 59

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