Cryptic invasion suggested by a cytogeographic analysis of the halophytic Puccinellia distans complex (Poaceae) in Central Europe.

Introduction: Despite the wealth of studies dealing with the invasions of alien plants, invasions of alien genotypes of native species (cryptic invasions) have been vastly neglected. The impact of cryptic invasions on the biodiversity of plant communities can, however, be significant. Inland saline habitats and halophytes (i.e., salt-tolerant plant species) are especially threatened by this phenomenon as they inhabit fragmented remnants of largely destroyed habitats, but at the same time some of these halophytic species are rapidly spreading along salt-treated roads. To study potential cryptic invasion of halophytes, the patterns of genome size and ploidy variation in the Puccinellia distans complex (Poaceae), the most rapidly spreading roadside halophyte in Central Europe, were investigated. Methods: DNA flow cytometry with confirmatory chromosome counts were employed to assess ploidy levels of 1414 individuals from 133 populations of the P. distans complex. In addition, climatic niche modelling was used to predict the distributions of selected cytotypes. Results: Eight groups differing in ploidy level and/or genome size were discovered, one diploid (2x; 2n = 14), two tetraploid (4xA, 4xB; 2n = 28), one pentaploid (5x; 2n = 35), three hexaploid (6xA, 6xB, 6xC; 2n = 42), and one heptaploid (7x; 2n = 49). The hexaploids (mostly the 6xC cytotype) were widespread through the study area, spreading intensively in both anthropogenic and natural habitats and probably hybridizing with the natural habitat dwelling tetraploids. In contrast, the non-hexaploid cytotypes rarely spread and were predominantly confined to natural habitats. Discussion: The extensive spread of the hexaploid cytotypes along roadsides has most likely facilitated their incursion into natural habitats. The colonization of new natural habitats by the hexaploids may pose a threat to the indigenous Puccinellia populations by compromising their genetic integrity and/or by outcompeting them.

Underestimated diversity in high elevations of a global biodiversity hotspot: two new endemic species of Aethionema (Brassicaceae) from the alpine zone of Iran.

Although the mountains in South-West Asia are a global biodiversity hotspot, our understanding of their biodiversity, especially in the commonly remote alpine and subnival zones, is still limited. This is well exemplified here by Aethionema umbellatum (Brassicaceae), a species considered to have a wide yet disjoint distribution in the Zagros and Yazd-Kerman mountains of western and central Iran. Morphological and molecular phylogenetic data (based on plastid trnL-trnF and nuclear ITS sequences) show that A. umbellatum is restricted to a single mountain range in southwestern Iran (Dena Mts., southern Zagros), whereas populations from central Iran (Yazd-Kerman and central Zagros) and from western Iran (central Zagros) belong to species new to science, A. alpinum and A. zagricum, respectively. Both new species are phylogenetically and morphologically close to A. umbellatum, with which they share unilocular fruits and one-seeded locules. However, they are easily distinguishable by leaf shape, petal size, and fruit characters. This study confirms that the alpine flora of the Irano-Anatolian region is still poorly known. As the proportion of rare and local endemic species in alpine habitats is high, these habitats are of prime interest for conservation efforts.

Contamination of imported kernels by unapproved genome-edited varieties poses a major challenge for monitoring and traceability during transport and handling on a global scale: inferences from a study on feral oilseed rape in Austria.

Novel techniques such as CRISPR/Cas are increasingly being applied for the development of modern crops. However, the regulatory framework for production, labelling and handling of genome-edited organisms varies worldwide. Currently, the European Commission is raising the question whether genome-edited organisms should still be regulated as genetically modified organisms in the future or whether a deregulation should be implemented. In our paper, based on the outcome of a 2-year case study on oilseed rape in Austria, we show that seed spillage during import and subsequent transport and handling activities is a key factor for the unintended dispersal of seeds into the environment, the subsequent emergence of feral oilseed rape populations and their establishment and long-term persistence in natural habitats. These facts must likewise be considered in case of genome-edited oilseed rape contaminants that might be accidentally introduced with conventional kernels. We provide evidence that in Austria a high diversity of oilseed rape genotypes, including some with alleles not known from cultivated oilseed rape in Austria, exists at sites with high seed spillage and low weed management, rendering these sites of primary concern with respect to possible escape of genome-edited oilseed rape varieties into the environment. Since appropriate detection methods for single genome-edited oilseed rape events have only recently started to be successfully developed and the adverse effects of these artificial punctate DNA exchanges remain largely unknown, tracing the transmission and spread of these genetic modifications places high requirements on their monitoring, identification, and traceability.

Addressing alpine plant phylogeography using integrative distributional, demographic and coalescent modeling.

Phylogeographic studies of alpine plants have evolved considerably in the last two decades from ad hoc interpretations of genetic data to statistical model-based approaches. In this review we outline the developments in alpine plant phylogeography focusing on the recent approach of integrative distributional, demographic and coalescent (iDDC) modeling. By integrating distributional data with spatially explicit demographic modeling and subsequent coalescent simulations, the history of alpine species can be inferred and long-standing hypotheses, such as species-specific responses to climate change or survival on nunataks during the last glacial maximum, can be efficiently tested as exemplified by available case studies. We also discuss future prospects and improvements of iDDC.

Endemics determine bioregionalization in the alpine zone of the Irano-Anatolian biodiversity hotspot (South-West Asia).

Alpine habitats are characterized by a high rate of range restricted species compared to those of lower elevations. This is also the case for the Irano-Anatolian global biodiversity hotspot in South-West Asia, which is a mountainous area harbouring a high amount of endemic species. Using two quantitative approaches, Endemicity Analysis and Network-Clustering, we want to identify areas of concordant species distribution patterns in the alpine zone of this region as well as to test the hypothesis that, given the high proportion of endemics among alpine species, delimitation of these areas is determined mainly by endemic alpine species, i.e., areas of concordant species distribution patterns are congruent with areas of endemism. Endemicity Analysis identified six areas of concordant species distribution patterns irrespective of dataset (total alpine species versus endemic alpine species), whereas the Network-Clustering approach identified five and four Bioregions from total alpine species and endemic alpine species, respectively. Most of these areas have been previously identified using the endemic flora of different elevational zones. The identified units using both methods and both datasets are strongly congruent, proposing that they reveal meaningful distribution patterns. Bioregionalization in the Irano-Anatolian biodiversity hotspot appears to be strongly influenced by the endemic alpine species, a pattern likely to hold in alpine regions outside the Irano-Anatolian hotspot. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00035-021-00266-7.

The new locally endemic genus Yazdana (Caryophyllaceae) and patterns of endemism highlight the high conservation priority of the poorly studied Shirkuh Mountains (central Iran).

Although mountain ranges are often recognized as global biodiversity hotspots with a high level of endemism, diversity and biogeographic connections of isolated and weakly explored mountains remain poorly understood. This is also the case for Shirkuh Mts. in central Iran. Here, Yazdana shirkuhensis gen. & spec. nov. (Caryophylleae, Caryophyllaceae) is described and illustrated from the high alpine zone of this mountain. Molecular phylogenetic analyses of nuclear and plastid DNA sequence data show that Y. shirkuhensis is related to Cyathophylla and Heterochroa (tribe Caryophylleae). The newly described genus and species accentuate Shirkuh Mts. as a center of endemism, which harbors a high number of narrowly distributed species, mostly in high elevations reaching alpine habitats. As this area is currently not protected, a conservation priority is highlighted for high elevations of Shirkuh Mts.

A multilocus phylogeny of the non-photosynthetic parasitic plant Cistanche (Orobanchaceae) refutes current taxonomy and identifies four major morphologically distinct clades.

Phylogenetic relationships of and within non-photosynthetic parasitic lineages are notoriously poorly known, which negatively affects our understanding of parasitic plants. This is also the case for Cistanche (Orobanchaceae), an Old World genus with about two dozen species, whose relationships have not yet been addressed using molecular phylogenetic approaches. Here we infer phylogenetic relationships within the genus, employing a taxonomically and geographically broad sampling covering all previously distinguished infrageneric groups and most of the currently recognized species. A combined matrix of three plastid markers (trnL-trnF, including the trnL intron and the intergenic spacer (IGS), trnS-trnfM IGS and psbA-trnH IGS) and one nuclear marker (ITS) was analyzed using maximum parsimony, maximum likelihood and Bayesian inference. Cistanche falls into four well-supported and geographically differentiated clades: East Asian Clade, Northwest African Clade, Southwest Asian Clade and Widespread Clade. Of those, only the East Asian Clade corresponds to a previously recognized taxonomic section, whereas the others either contain members of two or three sections (Widespread Clade and Southwest Asian Clade, respectively) or have not been taxonomically recognized so far (Northwest African Clade). Whereas the Southwest Asian Clade exhibits strong phylogenetic structure among and partly within species (the East Asian Clade and the Northwest African Clade are monospecific), phylogenetic resolution within the Widespread Clade is often low and hampered by discrepancies between nuclear and plastid markers. Both molecular and morphological data indicate that species diversity in Cistanche is currently underestimated.

An explicit test of Pleistocene survival in peripheral versus nunatak refugia in two high mountain plant species.

Pleistocene climate fluctuations had profound influence on the biogeographical history of many biota. As large areas in high mountain ranges were covered by glaciers, biota were forced either to peripheral refugia (and possibly beyond to lowland refugia) or to interior refugia (nunataks). However, nunatak survival remains controversial as it relies solely on correlative genetic evidence. Here, we test hypotheses of glacial survival using two high alpine plant species (the insect-pollinated Pedicularis asplenifolia and wind-pollinated Carex fuliginosa) in the European Alps. Employing the iDDC (integrative Distributional, Demographic and Coalescent) approach, which couples species distribution modelling, spatial and temporal demographic simulation and Approximate Bayesian Computation, we explicitly test three hypotheses of glacial survival: (a) peripheral survival only, (b) nunatak survival only and (c) peripheral plus nunatak survival. In P. asplenifolia the peripheral plus nunatak survival hypothesis was supported by Bayes factors (BF> 100), whereas in C. fuliginosa the peripheral survival only hypothesis, although best supported, could not be unambiguously distinguished from the peripheral plus nunatak survival hypothesis (BF = 5.58). These results are consistent with current habitat preferences (P. asplenifolia extends to higher elevations) and the potential for genetic swamping (i.e., replacement of local genotypes via hybridization with immigrating genotypes [expected to be higher in the wind-pollinated C. fuliginosa]). Although the persistence of plants on nunataks during glacial periods has been debated and studied over decades, this is one of the first studies to explicitly test the hypothesis instead of solely using correlative evidence.

Endemic diversity and distribution of the Iranian vascular flora across phytogeographical regions, biodiversity hotspots and areas of endemism.

Endemism is one of the most important concepts in biogeography and is of high relevance for conservation biology. Nevertheless, our understanding of patterns of endemism is still limited in many regions of high biodiversity. This is also the case for Iran, which is rich in biodiversity and endemism, but there is no up-to-date account of diversity and distribution of its endemic species. In this study, a comprehensive list of all endemic vascular plant species of Iran, their taxonomic composition and their geographical distribution are presented. To this end, a total of 2,597 (sub)endemic vascular plant species of Iran were documented and their distribution in three phytogeographical regions, two biodiversity hotspots and five areas of endemism were analysed. The Irano-Turanian phytogeographical region harbours 88% of the Iranian endemics, the majority of which are restricted to the Irano-Anatolian biodiversity hotspot (84%). Nearly three quarters of the endemic species are restricted to mountain ranges. The rate of endemism increases along an elevational gradient, causing the alpine zone to harbour a disproportionally high number of endemics. With increasing pastoralism, urbanization, road construction and ongoing climate change, the risk of biodiversity loss in the Iranian mountains is very high, and these habitats need to be more effectively protected.

Phylogenetic Relationships in Orobanchaceae Inferred From Low-Copy Nuclear Genes: Consolidation of Major Clades and Identification of a Novel Position of the Non-photosynthetic Orobanche Clade Sister to All Other Parasitic Orobanchaceae.

Molecular phylogenetic analyses have greatly advanced our understanding of phylogenetic relationships in Orobanchaceae, a model system to study parasitism in angiosperms. As members of this group may lack some genes widely used for phylogenetic analysis and exhibit varying degrees of accelerated base substitution in other genes, relationships among major clades identified previously remain contentious. To improve inferences of phylogenetic relationships in Orobanchaceae, we used two pentatricopeptide repeat (PPR) and three low-copy nuclear (LCN) genes, two of which have been developed for this study. Resolving power and level of support strongly differed among markers. Despite considerable incongruence among newly and previously sequenced markers, monophyly of major clades identified in previous studies was confirmed and, especially in analyses of concatenated data, strongly supported after the exclusion of a small group of East Asian genera (Pterygiella and Phtheirospermum) from the Euphrasia-Rhinanthus clade. The position of the Orobanche clade sister to all other parasitic Orobanchaceae may indicate that the shift to holoparasitism occurred early in the evolution of the family. Although well supported in analyses of concatenated data comprising ten loci (five newly and five previously sequenced), relationships among major clades, most prominently the Striga-Alectra clade, the Euphrasia-Rhinanthus clade, and the Castilleja-Pedicularis clade, were uncertain because of strongly supported incongruence also among well-resolving loci. Despite the limitations of using a few selected loci, congruence among markers with respect to circumscription of major clades of Orobanchaceae renders those frameworks for detailed, species-level, phylogenetic studies.

Ancestral remnants or peripheral segregates? Phylogenetic relationships of two narrowly endemic Euphrasia species (Orobanchaceae) from the eastern European Alps.

Endemism in mountain ranges is considered to be the result of a number of factors, including restriction to refugia during Pleistocene climate fluctuations. However, isolation in glacial refugia cannot explain the origin of narrowly endemic taxa restricted to formerly heavily glaciated areas. Here, we investigate the phylogeny of two narrowly endemic species, Euphrasia inopinata and E. sinuata (Orobanchaceae), found exclusively in formerly heavily glaciated areas of the eastern European Alps. As both species are diploid and very similar to the widespread (allo)polyploid E. minima, we test whether the restricted distributions of E. inopinata and E. sinuata are relictual, i.e. the two species are ancestral diploid remnants of a polyploid complex, or whether they are derived, i.e. the two species are peripheral segregates of a more widespread diploid. Based on internal transcribed spacer (ITS) sequence and amplified fragment length polymorphism (AFLP) fingerprint data it is shown that E. inopinata and E. sinuata, whose diploid ploidy level is confirmed for all analysed individuals via flow cytometry, are phylogenetically closely related to diploid E. alpina s. l. (series Alpinae) instead of E. minima (series Parviflorae). In addition, there is no evidence that these two diploid species participated in the formation of allotetraploid E. minima. Thus, E. inopinata and E. sinuata are interpreted as peripheral segregates of the widespread E. alpina s. l. Shifts in pollination system from allogamy in E. alpina s. l. to autogamy in E. inopinata and E. sinuata, genetic drift in small populations and geographic isolation at the periphery of the range of E. alpina s. str. probably contributed to the morphological and ecological differentiation of E. inopinata and E. sinuata.

Is the incidence of survival in interior Pleistocene refugia (nunataks) underestimated? Phylogeography of the high mountain plant Androsace alpina (Primulaceae) in the European Alps revisited.

Temperate mountain ranges such as the European Alps have been strongly affected by the Pleistocene glaciations. Glacial advances forced biota into refugia, which were situated either at the periphery of mountain ranges or in their interior. Whereas in the Alps peripheral refugia have been repeatedly and congruently identified, support for the latter scenario, termed "nunatak hypothesis," is still limited and no general pattern is recognizable yet. Here, we test the hypothesis of nunatak survival for species growing in the high alpine to subnival zones on siliceous substrate using the cushion plant Androsace alpina (Primulaceae), endemic to the European Alps, as our model species. To this end, we analyzed AFLP and plastid DNA sequence data obtained from a dense and range-wide sampling. Both AFLPs and plastid sequence data identified the southwestern-most population as the most divergent one. AFLP data did not allow for discrimination of interior and peripheral populations, but rather identified two to three longitudinally separated major gene pools. In contrast, in the eastern half of the Alps several plastid haplotypes of regional or local distribution in interior ranges-the Alpine periphery mostly harbored a widespread haplotype-were indicative for the presence of interior refugia. Together with evidence from other Alpine plant species, this study shows that in the eastern Alps silicicolous species of open habitats in the alpine and subnival zone survived, also or exclusively so, in interior refugia. As the corresponding genetic structure may be lost in mostly nuclear-derived, rapidly homogenizing marker systems such as AFLPs or RAD sequencing tags, markers not prone to homogenization, as is the case for plastid sequences (Sanger-sequenced or extracted from an NGS data set) will continue to be important for detecting older, biogeographically relevant patterns.

Correction: Phylogeny, character evolution and spatiotemporal diversification of the species-rich and world-wide distributed tribe Rubieae (Rubiaceae).

[This corrects the article DOI: 10.1371/journal.pone.0207615.].

Phylogeny, character evolution and spatiotemporal diversification of the species-rich and world-wide distributed tribe Rubieae (Rubiaceae).

The Rubiaceae tribe Rubieae has a world-wide distribution with up to 1,000 species. These collectively exhibit an enormous ecological and morphological diversity, making Rubieae an excellent group for macro- and microevolutionary studies. Previous molecular phylogenetic analyses used only a limited sampling within the tribe or missed lineages crucial for understanding character evolution in this group. Here, we analyze sequences from two plastid spacer regions as well as morphological and biogeographic data from an extensive and evenly distributed sampling to establish a sound phylogenetic framework. This framework serves as a basis for our investigation of the evolution of important morphological characters and the biogeographic history of the Rubieae. The tribe includes three major clades, the Kelloggiinae Clade (Kelloggia), the Rubiinae Clade (Didymaea, Rubia) and the most species-rich Galiinae Clade (Asperula, Callipeltis, Crucianella, Cruciata, Galium, Mericarpaea, Phuopsis, Sherardia, Valantia). Within the Galiinae Clade, the largest genera Galium and Asperula are para- and polyphyletic, respectively. Smaller clades, however, usually correspond to currently recognized taxa (small genera or sections within genera), which may be used as starting points for a refined classification in this clade. Life-form (perennial versus annual), flower shape (long versus short corolla tube) and fruit characters (dry versus fleshy, with or without uncinate hairs) are highly homoplasious and have changed multiple times independently. Inference on the evolution of leaf whorls, a characteristic feature of the tribe, is sensitive to model choice. Multi-parted leaf whorls appear to have originated from opposite leaves with two small interpetiolar stipules that are subsequently enlarged and increased in number. Early diversification of Rubieae probably started during the Miocene in western Eurasia. Disjunctions between the Old and the New World possibly are due to connections via a North Atlantic land bridge. Diversification of the Galiineae Clade started later in the Miocene, probably in the Mediterranean, from where lineages reached, often multiple times, Africa, eastern Asia and further on the Americas and Australia.

Hotspots within a global biodiversity hotspot - areas of endemism are associated with high mountain ranges.

Conservation biology aims at identifying areas of rich biodiversity. Currently recognized global biodiversity hotspots are spatially too coarse for conservation management and identification of hotspots at a finer scale is needed. This might be achieved by identification of areas of endemism. Here, we identify areas of endemism in Iran, a major component of the Irano-Anatolian biodiversity hotspot, and address their ecological correlates. Using the extremely diverse sunflower family (Asteraceae) as our model system, five consensus areas of endemism were identified using the approach of endemicity analysis. Both endemic richness and degree of endemicity were positively related to topographic complexity and elevational range. The proportion of endemic taxa at a certain elevation (percent endemism) was not congruent with the proportion of total surface area at this elevation, but was higher in mountain ranges. While the distribution of endemic richness (i.e., number of endemic taxa) along an elevational gradient was hump-shaped peaking at mid-elevations, the percentage of endemism gradually increased with elevation. Patterns of endemic richness as well as areas of endemism identify mountain ranges as main centres of endemism, which is likely due to high environmental heterogeneity and strong geographic isolation among and within mountain ranges. The herein identified areas can form the basis for defining areas with conservation priority in this global biodiversity hotspot.

Dating the Species Network: Allopolyploidy and Repetitive DNA Evolution in American Daisies (Melampodium sect. Melampodium, Asteraceae).

Allopolyploidy has played an important role in the evolution of the flowering plants. Genome mergers are often accompanied by significant and rapid alterations of genome size and structure via chromosomal rearrangements and altered dynamics of tandem and dispersed repetitive DNA families. Recent developments in sequencing technologies and bioinformatic methods allow for a comprehensive investigation of the repetitive component of plant genomes. Interpretation of evolutionary dynamics following allopolyploidization requires both the knowledge of parentage and the age of origin of an allopolyploid. Whereas parentage is typically inferred from cytogenetic and phylogenetic data, age inference is hampered by the reticulate nature of the phylogenetic relationships. Treating subgenomes of allopolyploids as if they belonged to different species (i.e., no recombination among subgenomes) and applying cross-bracing (i.e., putting a constraint on the age difference of nodes pertaining to the same event), we can infer the age of allopolyploids within the framework of the multispecies coalescent within BEAST2. Together with a comprehensive characterization of the repetitive DNA fraction using the RepeatExplorer pipeline, we apply the dating approach in a group of closely related allopolyploids and their progenitor species in the plant genus Melampodium (Asteraceae). We dated the origin of both the allotetraploid, Melampodium strigosum, and its two allohexaploid derivatives, Melampodium pringlei and Melampodium sericeum, which share both parentage and the direction of the cross, to the Pleistocene ($<$1.4 Ma). Thus, Pleistocene climatic fluctuations may have triggered formation of allopolyploids possibly in short intervals, contributing to difficulties in inferring the precise temporal order of allopolyploid species divergence of M. sericeum and M. pringlei. The relatively recent origin of the allopolyploids likely played a role in the near-absence of major changes in the repetitive fraction of the polyploids' genomes. The repetitive elements most affected by the postpolyploidization changes represented retrotransposons of the Ty1-copia lineage Maximus and, to a lesser extent, also Athila elements of Ty3-gypsy family.

Marker Development for Phylogenomics: The Case of Orobanchaceae, a Plant Family with Contrasting Nutritional Modes.

Phylogenomic approaches, employing next-generation sequencing (NGS) techniques, have revolutionized systematic and evolutionary biology. Target enrichment is an efficient and cost-effective method in phylogenomics and is becoming increasingly popular. Depending on availability and quality of reference data as well as on biological features of the study system, (semi-)automated identification of suitable markers will require specific bioinformatic pipelines. Here, we established a highly flexible bioinformatic pipeline, BaitsFinder, to identify putative orthologous single copy genes (SCGs) and to construct bait sequences in a single workflow. Additionally, this pipeline has been constructed to be able to cope with challenging data sets, such as the nutritionally heterogeneous plant family Orobanchaceae. To this end, we used transcriptome data of differing quality available for four Orobanchaceae species and, as reference, SCG data from monkeyflower (Erythranthe guttata, syn. Mimulus g.; 1,915 genes) and tomato (Solanum lycopersicum; 391 genes). Depending on whether gaps were permitted in initial blast searches of the four Orobanchaceae species against the reference, our pipeline identified 1,307 and 981 SCGs with average length of 994 bp and 775 bp, respectively. Automated bait sequence construction (using 2× tiling) resulted in 38,170 and 21,856 bait sequences, respectively. In comparison to the recently published MarkerMiner 1.0 pipeline BaitsFinder identified about 1.6 times as many SCGs (of at least 900 bp length). Skipping steps specific to analyses of Orobanchaceae, BaitsFinder was successfully used in a group of non-parasitic plants (three Asteraceae species and, as reference, SCG data from Arabidopsis thaliana based on previously compiled SCGs). Thus, BaitsFinder is expected to be broadly applicable in groups, where only transcriptomes or partial genome data of differing quality are available.

A novel method to infer the origin of polyploids from Amplified Fragment Length Polymorphism data reveals that the alpine polyploid complex of Senecio carniolicus (Asteraceae) evolved mainly via autopolyploidy.

Despite its evolutionary and ecological relevance, the mode of polyploid origin has been notoriously difficult to be reconstructed from molecular data. Here, we present a method to identify the putative parents of polyploids and thus to infer the mode of their origin (auto- vs. allopolyploidy) from Amplified Fragment Length Polymorphism (AFLP) data. To this end, we use Cohen's d of distances between in silico polyploids, generated within a priori defined scenarios of origin from a priori delimited putative parental entities (e.g. taxa, genetic lineages), and natural polyploids. Simulations show that the discriminatory power of the proposed method increases mainly with increasing divergence between the lower-ploid putative ancestors and less so with increasing delay of polyploidization relative to the time of divergence. We apply the new method to the Senecio carniolicus aggregate, distributed in the European Alps and comprising two diploid, one tetraploid and one hexaploid species. In the eastern part of its distribution, the S. carniolicus aggregate was inferred to comprise an autopolyploid series, whereas for western populations of the tetraploid species, an allopolyploid origin involving the two diploid species was the most likely scenario. Although this suggests that the tetraploid species has two independent origins, other evidence (ribotype distribution, morphology) is consistent with the hypothesis of an autopolyploid origin with subsequent introgression by the second diploid species. Altogether, identifying the best among alternative scenarios using Cohen's d can be straightforward, but particular scenarios, such as allopolyploid origin vs. autopolyploid origin with subsequent introgression, remain difficult to be distinguished.

Molecular and karyological data confirm that the enigmatic genus Platypholis from Bonin-Islands (SE Japan) is phylogenetically nested within Orobanche (Orobanchaceae).

Molecular phylogenetic studies have greatly improved our understanding of phylogenetic relationships of non-photosynthetic parasitic broomrapes (Orobanche and related genera, Orobanchaceae), but a few genera have remained unstudied. One of those is Platypholis, whose sole species, Platypholis boninsimae, is restricted to the Bonin-Islands (Ogasawara Islands) about 1000 km southeast of Japan. Based on overall morphological similarity, Platypholis has been merged with Orobanche, but this hypothesis has never been tested with molecular data. Employing maximum likelihood and Bayesian analyses on a family-wide data set (two plastid markers, matK and rps2, and three nuclear markers, ITS, phyA and phyB) as well as on an ITS data set focusing on Orobanche s. str., it is shown that P. boninsimae Maxim. is phylogenetically closely linked to or even nested within Orobanche s. str. This position is supported both by morphological evidence and by the newly obtained chromosome number of 2n = 38, which is characteristic for the genus Orobanche s. str.

rDNA Loci Evolution in the Genus Glechoma (Lamiaceae).

Glechoma L. (Lamiaceae) is distributed in eastern Asia and Europe. Understanding chromosome evolution in Glechoma has been strongly hampered by its small chromosomes, constant karyotype and polyploidy. Here phylogenetic patterns and chromosomal variation in Glechoma species are considered, using genome sizes, chromosome mapping of 5S and 35S rDNAs by fluorescence in situ hybridisation (FISH), and phylogenetic analyses of internal transcribed spacers (nrITS) of 35S rDNA and 5S rDNA NTS sequences. Species and populations of Glechoma are tetraploid (2n = 36) with base chromosome number of x = 9. Four chromosomes carry pericentric 5S rDNA sites in their short arms in all the species. Two to four of these chromosomes also carry 35S rDNA in subterminal regions of the same arms. Two to four other chromosomes have 35S rDNA sites, all located subterminally within short arms; one individual possessed additional weak pericentric 35S rDNA signals on three other chromosomes. Five types of rDNA locus distribution have been defined on the basis of 35S rDNA variation, but none is species-specific, and most species have more than one type. Glechoma hederacea has four types. Genome size in Glechoma ranges from 0.80 to 0.94 pg (1C), with low levels of intrapopulational variation in all species. Phylogenetic analyses of ITS and NTS sequences distinguish three main clades coinciding with geographical distribution: European (G. hederacea-G. hirsuta), Chinese and Korean (G. longituba), and Japanese (G. grandis). The paper presents the first comparative cytogenetic analyses of Glechoma species including karyotype structure, rDNA location and number, and genome size interpreted in a phylogenetic context. The observed variation suggests that the genus is still in genomic flux. Genome size, but not rDNA loci number and distribution, provides a character for species delimitation which allows better inferences of interspecific relationships to be made, in the absence of well-defined morphological differentiation.