Phylogeography and ecological differentiation of strictly Mediterranean taxa: the case of the Iberian endemic Odontites recordonii.

PREMISE: Ecological drivers for genetic differentiation in Mediterranean climates are still underexplored. We have used the strictly Mediterranean endemic Odontites recordonii as a model species to address this question. This species is one of the three Iberian representatives of the O. vernus group, which are morphologically similar. Thus, it was additionally necessary to clarify their phylogenetic relationships. METHODS: We used amplified fragment length polymorphisms to reveal phylogenetic relationships within O. vernus group, and to reconstruct the phylogeographic patterns within O. recordonii. Additionally, ecological niche models were generated to detect refugia along the Quaternary climatic oscillations. And finally, alleles under natural selection were identified, and correlations between allele presences and environmental variables were calculated in order to shed light on the ecological drivers promoting differentiation. RESULTS: The three species from the O. vernus group were recovered as distinct species. Three genetic groups were found within O. recordonii and a putative refugium was detected for each one. Eighty-one alleles could be under diversifying selection, and 58 alleles showed significant correlations with environmental variables, especially with temperature and precipitation seasonality and summer drought. CONCLUSIONS: The three Iberian species of the O. vernus group are reciprocal monophyletic taxa. The three genetic groups of O. recordonii could have been restricted to narrow refugia during the Quaternary and displayed present distributions in accordance with bioclimatic conditions. Temperature and precipitation seasonality and the intensity of summer drought are definitory climatic parameters of Mediterranean-type climates, and they could have acted as drivers of genetic differentiation on O. recordonii.

Genetic similarities versus morphological resemblance: Unraveling a polyploid complex in a Mediterranean biodiversity hotspot.

The Balkan Peninsula is recognized as one of the hotspots of biodiversity in Europe. This area has shown since the Last Glacial Maximum appropriate conditions for species diversification and hybridization, which has led to the existence of numerous taxonomically unresolved entities. Here, we focus on the Western Balkans and explore the genetic structure and relationships among species belonging to the V. austriaca - V. orbiculata diploid-polyploid complex, including populations showing intermediate morphologies. A combination of nuclear markers (microsatellites), plastid DNA regions (trnH-psbA, ycf6-psbM) and ploidy level estimations using flow cytometry are employed to assess the genetic structure and evolutionary dynamics of this polyploid complex. To reconstruct the evolutionary history, an approximate Bayesian computation approach is combined with projections of the species distribution models onto the climatic scenarios of the Mid-Holocene (6 ka BP) and Last Glacial Maximum (22 ka BP). Four main groups were found: one well-established entity within the diploid level, V. dalmatica, a second diploid-tetraploid group which corresponds to V. orbiculata, a hexaploid cluster harboring V. austriaca subsp. jacquinii individuals, and an enigmatic tetraploid group. According to the molecular data obtained, this latter cluster represents an allopolyploid cryptic lineage −with V. orbiculata and V. dalmatica as putative parents− morphologically similar to V. orbiculata, but genetically more related to V. austriaca subsp. jacquinii. Veronica dalmatica and this "uncertain tetraploid" group are involved in the formation of the hexaploid taxon V. austriaca subsp. jacquinii, with the possibility of recent gene flow among different cytotypes. The present study supports a scenario of diversification from a diploid common ancestor leading to two different but interrelated lineages. The first one would correspond with the diploid V. orbiculata plus tetraploid individuals of this species arising through allo- and autopolyploidization, and the second one would involve all ploidy levels with allopolyploidization being prevalent.

Environmental differences are correlated with the distribution pattern of cytotypes in Veronica subsection Pentasepalae at a broad scale.

BACKGROUND AND AIMS: The distribution of cytotypes and its potential correlation with environmental variables represent a cornerstone to understanding the origin and maintenance of polyploid lineages. Although many studies have addressed this question in single species at a regional scale, only a few have attempted to decipher this enigma in groups of closely related species at a broad intercontinental geographical scale. Here, we consider approx. 20 species of a diploid-polyploid complex (Veronica subsect. Pentasepalae) of recent and rapid diversification represented in Europe and North Africa to study the frequency and distribution of cytotypes and their relationship to environmental variables. METHODS: A total of 680 individuals (207 populations) were sampled. Ploidy levels were determined using flow cytometry. Ecological differentiation among cytotypes was tested using climatic and environmental variables related to temperature, precipitation, vegetation and biogeographical region, among others, and by performing univariate and multivariate (constrained principal coordinates analysis) analyses. KEY RESULTS: Four ploidy levels (2x, 4x, 6x and 8x) were found and genome downsizing was observed to occur within the group. Plants of different ploidy level are ecologically differentiated, with hexaploids and octoploids occurring in wetter and colder habitats with a higher seasonality than diploids. A south to north distribution pattern was found, with diploids occupying southern refugial areas and octoploids being more frequent in northern regions of Europe above the permafrost boundary. CONCLUSIONS: The distribution of cytotypes can be explained by ecological differentiation, the geographical position of refuge areas during the Quaternary climatic oscillations as well as by ice and permafrost retreat patterns. The Balkan Peninsula constitutes the most important contact zone between cytotypes. This work provides the first comprehensive ploidy screening within V. subsect. Pentasepalae at a broad scale and indicates that polyploidy and genome downsizing might have contributed to the colonization of new habitats in a recently diverged polyploid complex.

SSR markers for Filago subg. Filago (Gnaphalieae: Asteraceae) and cross-amplification in three other subgenera.

PREMISE OF THE STUDY: Microsatellite primers were developed for the first time in the genus Filago (Gnaphalieae: Asteraceae). These markers will facilitate low-scale phylogenetic, phylogeographic, and population genetic studies within the genus Filago. METHODS AND RESULTS: Ten pairs of polymorphic microsatellite primers (as well as five pairs of monomorphic primers) were identified and optimized on two species of Filago (F. gaditana and F. carpetana) using a microsatellite-enrichment library method and 454 GS-FLX technique. The polymorphic primers amplified tri- to hexanucleotide repeats and showed one to six alleles per locus for both species. Transferability was performed in 29 samples corresponding to nine representative species of Filago. CONCLUSIONS: The results indicate the utility of the newly developed markers, which will be useful to delve into the phylogenetic relationships among the taxa within Filago. These microsatellites will enable studies of phylogeographic, reproductive, and genetic variation.

Phylogeography of an endangered disjunct herb: long-distance dispersal, refugia and colonization routes.

Quaternary glacial cycles appear to have had a consistent role in shaping the genetic diversity and structure of plant species. Despite the unusual combination of the characteristics of the western Mediterranean-Macaronesian area, there are no studies that have specifically examined the effects of palaeoclimatic and palaeogeographic factors on the genetic composition and structure of annual herbs. Astragalus edulis is a disjunct endemic found in the easternmost Canary Islands and the semi-arid areas of north-eastern Africa and south-eastern Iberian Peninsula. This endangered species shows no evident adaptations to long-distance dispersal. Amplified fragment length polymorphism (AFLP) data and plastid DNA sequences were analysed from a total of 360 individuals distributed throughout the range of this species. The modelled potential distribution of A. edulis under current conditions was projected over the climatic conditions of the Last Interglacial (130 ka BP) and Last Glacial Maximum (21 ka BP) to analyse changes in habitat suitability and to look for associations between the modelling and genetic results. Amplified fragment length polymorphism analysis showed clear phylogeographic structure with four distinct genetic clusters. Approximate Bayesian computation (ABC) models based on plastid DNA sequences indicated a Middle Pleistocene long-distance dispersal event as the origin of the populations of the Canary Islands. The models also suggested south-western Morocco as the ancestral area for the species, as well as subsequent colonization of north-eastern Morocco and the Iberian Peninsula. The data compiled indicated the possibility of the presence of refuge areas at favourable locations around the High Atlas and Anti-Atlas mountain ranges. Moreover, palaeodistribution models strongly support the events inferred by ABC modelling and show the potential distribution of the species in the past, suggesting a putative colonization route.

Microsatellite marker development for the tetraploid Veronica aragonensis (Plantaginaceae) using next-generation sequencing and high-resolution melting analyses.

PREMISE OF THE STUDY: The tetraploid Veronica aragonensis (Plantaginaceae) is a narrow endemic to the Iberian Peninsula. Specific microsatellite markers were developed to investigate genetic structure and diversity. METHODS AND RESULTS: A total of 15 polymorphic markers were characterized on three populations of V. aragonensis, using a microsatellite-enriched library on an Ion Torrent sequencer and high-resolution melting (HRM) analyses to rapidly discard nonreliable, multicopy, and/or monomorphic loci. Allele number per locus ranged from one to five, and levels of observed heterozygosity per population varied from 0.142 ± 0.301 to 0.281 ± 0.369. Most primers also amplified in the closely related species V. rosea and in three subspecies of V. tenuifolia. CONCLUSIONS: The species-specific microsatellite markers developed here represent an essential tool to provide genetic information on the population level for V. aragonensis. The low levels of variation detected highlight the importance of continued efforts to improve conservation of the species.

Divide and conquer! Data-mining tools and sequential multivariate analysis to search for diagnostic morphological characters within a plant polyploid complex (Veronica subsect. Pentasepalae, Plantaginaceae).

This study exhaustively explores leaf features seeking diagnostic characters to aid the classification (assigning cases to groups, i.e. populations to taxa) in a polyploid plant-species complex. A challenging case study was selected: Veronica subsection Pentasepalae, a taxonomically intricate group. The "divide and conquer" approach was implemented-that is, a difficult primary dataset was split into more manageable subsets. Three techniques were explored: two data-mining tools (artificial neural networks and decision trees) and one unsupervised discriminant analysis. However, only the decision trees and discriminant analysis were finally used to select diagnostic traits. A previously established classification hypothesis based on other data sources was used as a starting point. A guided discriminant analysis (i.e. involving manual character selection) was used to produce a grouping scheme fitting this hypothesis so that it could be taken as a reference. Sequential unsupervised multivariate analysis enabled the recognition of all species and infraspecific taxa; however, a suboptimal classification rate was achieved. Decision trees resulted in better classification rates than unsupervised multivariate analysis, but three complete taxa were misidentified (not present in terminal nodes). The variable selection led to a different grouping scheme in the case of decision trees. The resulting groups displayed low misclassification rates when analyzed using artificial neural networks. The decision trees as well as the discriminant analysis are recommended in the search of diagnostic characters. Due to the high sensitivity that artificial neural networks have to the combination of input/output layers, they are proposed as evaluation tools for morphometric studies. The "divide and conquer" principle is a promising strategy, providing success in the present case study.

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

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 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.

Development of 14 microsatellite markers in Odontites vernus s.l. (Orobanchaceae) and cross-amplification in related taxa.

PREMISE OF THE STUDY: Microsatellite primers were developed for the first time in the root hemiparasite herb Odontites vernus (Orobanchaceae). These markers will be useful to investigate the role of polyploidization in the evolution of this diploid-tetraploid complex, as well as the extent of gene flow between different ploidy levels. METHODS AND RESULTS: Fourteen polymorphic and reproducible loci were identified and optimized from O. vernus using a microsatellite-enriched library and 454 Junior sequencing. The set of primers amplified di- to pentanucleotide repeats and showed two to 13 alleles per locus. Transferability was tested in 30 taxa (19 belonging to Odontites and 11 from eight other genera of Orobanchaceae tribe Rhinantheae). CONCLUSIONS: The results indicate the utility of the newly developed microsatellites in O. vernus and several other species, which will be useful for taxon delimitation and conservation genetics studies.

Designing conservation strategies to preserve the genetic diversity of Astragalus edulis Bunge, an endangered species from western Mediterranean region.

Astragalus edulis (Fabaceae) is an endangered annual species from the western Mediterranean region that colonized the SE Iberian Peninsula, NE and SW Morocco, and the easternmost Macaronesian islands (Lanzarote and Fuerteventura). Although in Spain some conservation measures have been adopted, it is still necessary to develop an appropriate management plan to preserve genetic diversity across the entire distribution area of the species. Our main objective was to use population genetics as well as ecological and phylogeographic data to select Relevant Genetic Units for Conservation (RGUCs) as the first step in designing conservation plans for A. edulis. We identified six RGUCs for in situ conservation, based on estimations of population genetic structure and probabilities of loss of rare alleles. Additionally, further population parameters, i.e. occupation area, population size, vulnerability, legal status of the population areas, and the historical haplotype distribution, were considered in order to establish which populations deserve conservation priority. Three populations from the Iberian Peninsula, two from Morocco, and one from the Canary Islands represent the total genetic diversity of the species and the rarest allelic variation. Ex situ conservation is recommended to complement the preservation of A. edulis, given that effective in situ population protection is not feasible in all cases. The consideration of complementary phylogeographic and ecological data is useful for management efforts to preserve the evolutionary potential of the species.

Characterization of 12 polymorphic SSR markers in Veronica subsect. Pentasepalae (Plantaginaceae) and cross-amplification in 10 other subgenera.

PREMISE OF THE STUDY: Microsatellite primers were developed in the perennial herbs of the diploid-polyploid complex Veronica subsect. Pentasepalae (Plantaginaceae) to investigate the role that hybridization has played in the evolution of the group, which includes several endangered species. METHODS AND RESULTS: Twelve pairs of primers leading to polymorphic and readable markers were identified and optimized from V. jacquinii and V. orbiculata using a microsatellite-enriched library method and 454 GS-FLX technique. The set of primers amplified dinucleotide to pentanucleotide repeats, and the number of alleles per locus ranged from one to six, one to 11, and one to nine for V. orsiniana, V. javalambrensis, and V. rosea, respectively. Transferability analyses were performed in 20 species corresponding to 10 different subgenera. CONCLUSIONS: These results indicate the utility of the newly developed microsatellites across Veronica subsect. Pentasepalae, which will help in the study of gene flow patterns and genetic structure.

Systematic significance of seed morphology in veronica (plantaginaceae): a phylogenetic perspective.

BACKGROUND AND AIMS: A new infrageneric rearrangement for Veronica has been proposed based on the most recent evidence from DNA sequence data, morphological evidence, and biogeographical considerations. Looking for morphological synapomorphies for each monophyletic subgenus has been problematic, due to difficulties arising from widespread homoplasy (mainly parallel evolution). In an attempt to overcome these difficulties, previously underexplored morphological characters are starting to be studied in more depth. METHODS: A molecular phylogenetic hypothesis was used based on sequences of ITS (nuclear ribosomal DNA) and plastid trnL-F regions, as a framework to test the use of seed coat ultrastructure (studied under scanning electron microscope) in the systematics of the genus. A sample of 132 taxa representing ten of the 13 subgenera in Veronica, excluding the species of the southern hemisphere Hebe complex and the exclusively North American subgenus Synthyris, was studied. KEY RESULTS AND CONCLUSIONS: The results demonstrate that, in many cases, the ultrastucture of the testa can be employed to assess relationships of taxa within the genus, and the character provides additional support for molecular trees. Further characters relevant for the classification of Veronica, i.e. base chromosome number, iridoid chemical data, life cycle, inflorescence position, have been taken into consideration in a discussion where an attempt is made to highlight the best traits to characterize each subgenus investigated.