Nano-Strainer: A workflow for the identification of single-copy nuclear loci for plant systematic studies, using target capture kits and Oxford Nanopore long reads.

In modern plant systematics, target enrichment enables simultaneous analysis of hundreds of genes. However, when dealing with reticulate or polyploidization histories, few markers may suffice, but often are required to be single-copy, a condition that is not necessarily met with commercial capture kits. Also, large genome sizes can render target capture ineffective, so that amplicon sequencing would be preferable; however, knowledge about suitable loci is often missing. Here, we present a comprehensive workflow for the identification of putative single-copy nuclear markers in a genus of interest, by mining a small dataset from target capture using a few representative taxa. The proposed pipeline assesses sequence variability contained in the data from targeted loci and assigns reads to their respective genes, via a combined BLAST/clustering procedure. Cluster consensus sequences are then examined based on four pre-defined criteria presumably indicative for absence of paralogy. This is done by calculating four specialized indices; loci are ranked according to their performance in these indices, and top-scoring loci are considered putatively single- or low copy. The approach can be applied to any probe set. As it relies on long reads, the present contribution also provides template workflows for processing Nanopore-based target capture data. Obtained markers are further tested and then entered into amplicon sequencing. For the detection of possibly remaining paralogy in these data, which might occur in groups with rampant paralogy, we also employ the long-read assembly tool canu. In diploid representatives of the young Compositae genus Leucanthemum, characterized by high levels of polyploidy, our approach resulted in successful amplification of 13 loci. Modifications to remove traces of paralogy were made in seven of these. A species tree from the markers correctly reproduced main relationships in the genus, however, at low resolution. The presented workflow has the potential to valuably support phylogenetic research, for example in polyploid plant groups.

Pedicularis rostratospicata subsp. marsica (P. Sect. Rostratae, Orobanchaceae), a New Subspecies from the Central Apennines (Italy).

The new subspecies Pedicularis rostratospicata subsp. marsica is here described based on morphological and molecular analyses. The new taxon is endemic to few localities of the Central Apennines within the Abruzzo, Lazio and Molise National Park (Central Italy). Pedicularis rostratospicata subsp. marsica can be distinguished from the other two currently accepted subspecies (subsp. rostratospicata and subsp. helvetica) by their taller stems, shorter petioles of basal and middle leaves, narrower blades of basal leaves, longer inflorescences with longer internodes and more flowers, and longer calyx lobes. Molecular analysis confirmed the autonomy of the new taxon. Furthermore, the conservation status assessment of the new subspecies according to IUCN categories and criteria is proposed and discussed, and an analytical key to the three subspecies of P. rostratospicata is presented.

Picks in the Fabric of a Polyploidy Complex: Integrative Species Delimitation in the Tetraploid Leucanthemum Mill. (Compositae, Anthemideae) Representatives.

Based on the results of a preceding species-delimitation analysis for the diploid representatives of the genus Leucanthemum (Compositae, Anthemideae), the present study aims at the elaboration of a specific and subspecific taxonomic treatment of the tetraploid members of the genus. Following an integrative taxonomic approach, species-level decisions on eight predefined morphotaxon hypotheses were based on genetic/genealogical, morphological, ecological, and geographical differentiation patterns. ddRADseq fingerprinting and SNP-based clustering revealed genetic integrity for six of the eight morphotaxa, with no clear differentiation patterns observed between the widespread L. ircutianum subsp. ircutianum and the N Spanish (Cordillera Cantábrica) L. cantabricum and the S French L. delarbrei subsp. delabrei (northern Massif Central) and L. meridionale (western Massif Central). The inclusion of differentiation patterns in morphological (leaf dissection and shape), ecological (climatological and edaphic niches), and geographical respects (pair-wise tests of sympatry vs. allopatry) together with the application of a procedural protocol for species-rank decisions (the 'Wettstein tesseract') led to the proposal of an acknowledgement of the eight predefined morphotaxon hypotheses as six species (two of them with two subspecies). Nomenclatural consequences following from these results are drawn and lead to the following new combinations: Leucanthemum delarbrei subsp. meridionale (Legrand) Oberpr., T.Ott & Vogt, comb. nov. and Leucanthemum ruscinonense (Jeanb. & Timb.-Lagr.) Oberpr., T.Ott & Vogt, comb. et stat. nov.

Adonis fucensis (A. sect. Adonanthe, Ranunculaceae), a New Species from the Central Apennines (Italy).

Adonis fucensis is herein described as a new species based on morphological and molecular analyses. It is endemic to one locality of the Central Apennines between Amplero and Fucino plains within the NATURA 2000 network in the SAC IT7110205 (Central Italy). The only discovered population is composed of 65 individuals and is at risk of extinction. The conservation status assessment according to IUCN categories and criteria is proposed and discussed. The new species belongs to A. sect. Adonanthe and is morphologically similar to A. volgensis (incl. A. transsilvanica), a species distributed in Hungary, Romania, Bulgaria, and Turkey as well as eastward to SW Siberia and Central Asia. Adonis fucensis can be distinguished from A. volgensis by larger cauline leaves, pentagonal with lobes lanceolate, larger stipules with more lobes and teeth, and larger flowers. Finally, an analytical key to Adonis species belonging to sect. Adonanthe distributed in Europe is presented.

Reticulate Evolution in the Western Mediterranean Mountain Ranges: The Case of the Leucanthemopsis Polyploid Complex.

Polyploidization is one of the most common speciation mechanisms in plants. This is particularly relevant in high mountain environments and/or in areas heavily affected by climatic oscillations. Although the role of polyploidy and the temporal and geographical frameworks of polyploidization have been intensively investigated in the alpine regions of the temperate and arctic biomes, fewer studies are available with a specific focus on the Mediterranean region. Leucanthemopsis (Asteraceae) consists of six to ten species with several infraspecific entities, mainly distributed in the western Mediterranean Basin. It is a polyploid complex including montane, subalpine, and strictly alpine lineages, which are locally distributed in different mountain ranges of Western Europe and North Africa. We used a mixed approach including Sanger sequencing and (Roche-454) high throughput sequencing of amplicons to gather information from single-copy nuclear markers and plastid regions. Nuclear regions were carefully tested for recombinants/PCR artifacts and for paralogy. Coalescent-based methods were used to infer the number of polyploidization events and the age of formation of polyploid lineages, and to reconstruct the reticulate evolution of the genus. Whereas the polyploids within the widespread Leucanthemopsis alpina are autopolyploids, the situation is more complex among the taxa endemic to the western Mediterranean. While the hexaploid, L. longipectinata, confined to the northern Moroccan mountain ranges (north-west Africa), is an autopolyploid, the Iberian polyploids are clearly of allopolyploid origins. At least two different polyploidization events gave rise to L. spathulifolia and to all other tetraploid Iberian taxa, respectively. The formation of the Iberian allopolyploids took place in the early Pleistocene and was probably caused by latitudinal and elevational range shifts that brought into contact previously isolated Leucanthemopsis lineages. Our study thus highlights the importance of the Pleistocene climatic oscillations and connected polyploidization events for the high plant diversity in the Mediterranean Basin.

The Warps and Wefts of a Polyploidy Complex: Integrative Species Delimitation of the Diploid Leucanthemum (Compositae, Anthemideae) Representatives.

Species delimitation-owing to the paramount role of the species rank in evolutionary, ecological, and nature conservation studies-is an essential contribution of taxonomy to biodiversity research. In an 'integrative taxonomy' approach to species delimitation on the diploid level, we searched for evolutionary significant units (the warps and wefts) that gave rise to the polyploid complex of European ox-eye daisies (Leucanthemum; Compositae-Anthemideae). Species discovery and validation methods based on genetic, ecological, geographical, and morphometric datasets were applied to test the currently accepted diploid morpho-species, i.e., morphologically delimited species, in Leucanthemum. Novel approaches were taken in the analyses of RADseq data (consensus clustering), morphometrics of reconstructed leaf silhouettes from digitized herbarium specimens, and quantification of species-distribution overlaps. We show that 17 of the 20 Leucanthemum morpho-species are supported by genetic evidence. The taxonomic rank of the remaining three morpho-species was resolved by combining genealogic, ecologic, geographic, and morphologic data in the framework of von Wettstein's morpho-geographical species concept. We herewith provide a methodological pipeline for the species delimitation in an 'integrative taxonomy' fashion using sources of evidence from genealogical, morphological, ecological, and geographical data in the philosophy of De Queiroz's "Unified Species Concept".

Long-read genotyping with SLANG (Simple Long-read loci Assembly of Nanopore data for Genotyping).

Premise: Most phylogenomic library preparation methods and bioinformatic analysis tools in restriction site-associated DNA sequencing (RADseq)/genotyping-by-sequencing (GBS) studies are designed for use with Illumina data. The lack of alternative bioinformatic pipelines hinders the exploration of long-read multi-locus data from other sequencing platforms. The Simple Long-read loci Assembly of Nanopore data for Genotyping (SLANG) pipeline enables locus assembly, orthology estimation, and single-nucleotide polymorphism (SNP) calling using Nanopore-sequenced multi-locus data. Methods and Results: Two test libraries (Leucanthemum spp., Senecio spp.; Compositae) were prepared using an amplified fragment length polymorphism (AFLP)-based method to reduce genome complexity, then Nanopore-sequenced, and analyzed with SLANG. We identified 704 and 448 orthologous loci with 12,368 and 10,048 SNPs, respectively. The constructed phylogenetic networks were identical to a GBS network produced using Leucanthemum Illumina data and were consistent with Senecio species circumscriptions based on morphology. Conclusions: SLANG identifies orthologous loci and extracts SNPs from long-read multi-locus Nanopore data for phylogenetic inference, population genetics, or phylogeographical studies. Combined with an AFLP-based library preparation, SLANG provides an easily scalable, cost-effective, and affordable alternative to Illumina-based RADseq/GBS procedures.

ALLCOPOL: inferring allele co-ancestry in polyploids.

BACKGROUND: Inferring phylogenetic relationships of polyploid species and their diploid ancestors (leading to reticulate phylogenies in the case of an allopolyploid origin) based on multi-locus sequence data is complicated by the unknown assignment of alleles found in polyploids to diploid subgenomes. A parsimony-based approach to this problem has been proposed by Oberprieler et al. (Methods Ecol Evol 8:835-849, 2017), however, its implementation is of limited practical value. In addition to previously identified shortcomings, it has been found that in some cases, the obtained results barely satisfy the applied criterion. To be of better use to other researchers, a reimplementation with methodological refinement appears to be indispensable. RESULTS: We present the AllCoPol package, which provides a heuristic method for assigning alleles from polyploids to diploid subgenomes based on the Minimizing Deep Coalescences (MDC) criterion in multi-locus sequence datasets. An additional consensus approach further allows to assess the confidence of phylogenetic reconstructions. Simulations of tetra- and hexaploids show that under simplifying assumptions such as completely disomic inheritance, the topological errors of reconstructed phylogenies are similar to those of MDC species trees based on the true allele partition. CONCLUSIONS: AllCoPol is a Python package for phylogenetic reconstructions of polyploids offering enhanced functionality as well as improved usability. The included methods are supplied as command line tools without the need for prior programming knowledge.

GinJinn: An object-detection pipeline for automated feature extraction from herbarium specimens.

PREMISE: The generation of morphological data in evolutionary, taxonomic, and ecological studies of plants using herbarium material has traditionally been a labor-intensive task. Recent progress in machine learning using deep artificial neural networks (deep learning) for image classification and object detection has facilitated the establishment of a pipeline for the automatic recognition and extraction of relevant structures in images of herbarium specimens. METHODS AND RESULTS: We implemented an extendable pipeline based on state-of-the-art deep-learning object-detection methods to collect leaf images from herbarium specimens of two species of the genus Leucanthemum. Using 183 specimens as the training data set, our pipeline extracted one or more intact leaves in 95% of the 61 test images. CONCLUSIONS: We establish GinJinn as a deep-learning object-detection tool for the automatic recognition and extraction of individual leaves or other structures from herbarium specimens. Our pipeline offers greater flexibility and a lower entrance barrier than previous image-processing approaches based on hand-crafted features.

Can we use it? On the utility of de novo and reference-based assembly of Nanopore data for plant plastome sequencing.

The chloroplast genome harbors plenty of valuable information for phylogenetic research. Illumina short-read data is generally used for de novo assembly of whole plastomes. PacBio or Oxford Nanopore long reads are additionally employed in hybrid approaches to enable assembly across the highly similar inverted repeats of a chloroplast genome. Unlike for PacBio, plastome assemblies based solely on Nanopore reads are rarely found, due to their high error rate and non-random error profile. However, the actual quality decline connected to their use has rarely been quantified. Furthermore, no study has employed reference-based assembly using Nanopore reads, which is common with Illumina data. Using Leucanthemum Mill. as an example, we compared the sequence quality of seven chloroplast genome assemblies of the same species, using combinations of two sequencing platforms and three analysis pipelines. In addition, we assessed the factors which might influence Nanopore assembly quality during sequence generation and bioinformatic processing. The consensus sequence derived from de novo assembly of Nanopore data had a sequence identity of 99.59% compared to Illumina short-read de novo assembly. Most of the errors detected were indels (81.5%), and a large majority of them is part of homopolymer regions. The quality of reference-based assembly is heavily dependent upon the choice of a close-enough reference. When using a reference with 0.83% sequence divergence from the studied species, mapping of Nanopore reads results in a consensus comparable to that from Nanopore de novo assembly, and of only slightly inferior quality compared to a reference-based assembly with Illumina data. For optimal de novo assembly of Nanopore data, appropriate filtering of contaminants and chimeric sequences, as well as employing moderate read coverage, is essential. Based on these results, we conclude that Nanopore long reads are a suitable alternative to Illumina short reads in plastome phylogenomics. Few errors remain in the finalized assembly, which can be easily masked in phylogenetic analyses without loss in analytical accuracy. The easily applicable and cost-effective technology might warrant more attention by researchers dealing with plant chloroplast genomes.

Taming the Red Bastards: Hybridisation and species delimitation in the Rhodanthemum arundanum-group (Compositae, Anthemideae).

Delineating species boundaries in a group of recently diverged lineages is challenging due to minor morphological differences, low genetic differentiation and the occurrence of gene flow among taxa. Here, we employ traditional Sanger sequencing and restriction-site associated DNA (RAD) sequencing, to investigate species delimitation in the close-knit Moroccan daisy group around Rhodanthemum arundanum B.H.Wilcox & al. that diverged recently during the Quaternary. After evaluation of genotyping errors and parameter optimisation in the course of de-novo assembly of RADseq reads in Ipyrad, we assess hybridisation patterns in the study group based on different data assemblies and methods (Neighbor-Net networks, FastStructure and ABBA-BABA tests). RADseq data and Sanger sequences are subsequently used for delimitation of species, using both, multi-species coalescent methods (Stacey and Snapp) and a novel approach based on consensus k-means clustering. In addition to the unveiling of two novel subspecies in the R. arundanum-group, our study provides insights into the performance of different species delimitation methods in the presence of hybridisation and varying quantities of data.

Effects of polyploidy on the coordination of gene expression between organellar and nuclear genomes in Leucanthemum Mill. (Compositae, Anthemideae).

Whole-genome duplications (WGD) through polyploid speciation are associated with disruptions of well-tuned relationships among the three plant cell genomes. Key metabolic processes comprising multi-subunit enzyme complexes, for which partner proteins are both nuclear- and plastid-encoded, are dependent on maintenance of stoichiometric ratios among the subunits to avoid cytonuclear imbalances after WGDs. By using qPCR for gene copy and transcript number quantification, we have studied the relationship of subunit expression in the two gene pairs rbcL/rbcS (the two subunits of RuBisCO) and psbA/psbO (two members of photosystem II) in closely related members of Leucanthemum (Compositae, Anthemideae), comprising a diploid, a tetraploid, and a hexaploid species. While gene copy numbers exhibit the expected pattern of an increase in the nuclear-encoded partner gene relative to the plastid-encoded one, we find that the two partner gene systems behave differently after WGD: While in the psbA/psbO partner gene system, shifts in the gene copy balance caused by polyploidization are not accommodated for through changes in transcription intensities of the two partner genes, the rbcL/rbcS system even shows an unexpected reversed dosage effect with up-regulated transcription intensities on both the nuclear and the plastidal side. We interpret the behavior of the psbA/psbO partner gene system as being due to the stoichiometrically relaxed relationship between the two gene products caused by a fast, damage-provoked combustion of the psbA gene product (the D1 core protein of PSII). Conversely, the finely tuned expression dependencies of the rbcL/rbcS system may be the reason for the observed positive feedback runaway signal as reaction to gene copy imbalances caused by a polyploidization shock.

'At the crossroads towards polyploidy': genomic divergence and extent of homoploid hybridization are drivers for the formation of the ox-eye daisy polyploid complex (Leucanthemum, Compositae-Anthemideae).

Polyploidy plays a paramount role in phytodiversity, but the causes of this evolutionary pathway require further study. Here, we use phylogenetic methods to examine possible polyploidy-promoting factors by comparing diploid representatives of the comprehensive European polyploid complex Leucanthemum with members of its strictly diploid North African counterpart Rhodanthemum. We investigate genetic divergence and gene flow among all diploid lineages of both genera to evaluate the role of genomic differentiation and hybridization for polyploid speciation. To test whether hybridization in Leucanthemum has been triggered by the geological conditions during its diversification, we additionally generate a time-calibrated phylogeny of 46 species of the subtribe Leucantheminae. Leucanthemum shows a significantly higher genetic divergence and hybridization signal among diploid lineages compared with Rhodanthemum, in spite of a similar crown age and diversification pattern during the Quaternary. Our study demonstrates the importance of genetic differentiation among diploid progenitors and their concurrent affinity for natural hybridization for the formation of a polyploid complex. Furthermore, the role of climate-induced range overlaps on hybridization and polyploid speciation during the Quaternary is discussed.

Ragweeds and relatives: Molecular phylogenetics of Ambrosiinae (Asteraceae).

Ambrosiinae are one of the most distinct subtribes in the Heliantheae alliance (Asteraceae), mainly due to specialization toward wind pollination. Taxa of the subtribe are principally native to the Americas, although some species have attained a cosmopolitan distribution. Members of subtribe Engelmanniinae are considered close to Ambrosiinae, due to shared morphological traits. However, the placement of Ambrosiinae within the Heliantheae alliance has not yet been corroborated by phylogenetic analyses. In the present study, we test the circumscription of subtribe Ambrosiinae and examine relationships among its genera. We used sequence information from three plastid (psbA-trnH, trnQ-rps16 and trnL-F) and two nuclear (ITS and D35) marker regions. Phylogenetic inference analyses were conducted, applying Bayesian Inference (BI) and Maximum Likelihood (ML). Subtribe Ambrosiinae is found monophyletic or nearly so in all analyses. The genera Dugesia and Rojasianthe (previously considered part of subtribe Engelmanniinae) in some cases cluster together with Ambrosiinae; these genera are clearly not part of Engelmanniinae. Within Ambrosiinae, the genera Parthenium and Parthenice occupy basal positions, whereas members of the genus Ambrosia are the most derived representatives of the subtribe. Previous subdivision of Ambrosiinae into "Iveae" (members having androgynous capitula and free achenes) and "Ambrosieae" (genera with unisexual heads and achenes enclosed in burs) is not corroborated. Results also allow consideration of relationships among species and subgeneric groups within Parthenium, Iva, and Ambrosia.

Are there morphological and life-history traits under climate-dependent differential selection in S Tunesian Diplotaxis harra (Forssk.) Boiss. (Brassicaceae) populations?

Adaptation of morphological, physiological, or life-history traits of a plant species to heterogeneous habitats through the process of natural selection is a paramount process in evolutionary biology. We have used a population genomic approach to disentangle selection-based and demography-based variation in morphological and life-history traits in the crucifer Diplotaxis harra (Forssk.) Boiss. (Brassicaceae) encountered in populations along aridity gradients in S Tunisia. We have genotyped 182 individuals from 12 populations of the species ranging from coastal to semidesert habitats using amplified fragment length polymorphism (AFLP) fingerprinting and assessed a range of morphological and life-history traits from their progeny cultivated under common-garden conditions. Application of three different statistical approaches for searching AFLP loci under selection allowed us to characterize candidate loci, for which their association with the traits assessed was tested for statistical significance and correlation with climate data. As a key result of this study, we find that only the shape of cauline leaves seems to be under differential selection along the aridity gradient in S Tunisian populations of Diplotaxis harra, while for all other traits studied neutral biogeographical and/or random factors could not be excluded as explanation for the variation observed. The counter-intuitive finding that plants from populations with more arid habitats produce broader leaves under optimal conditions of cultivation than those from more mesic habitats is interpreted as being ascribable to selection for a higher plasticity in this trait under more unpredictable semidesert conditions compared to the more predictable ones in coastal habitats.

"Fix Me Another Marguerite!": Species delimitation in a group of intensively hybridizing lineages of ox-eye daisies (Leucanthemum Mill., Compositae-Anthemideae).

Delineating species boundaries in the framework of the multi-species coalescent (MSC) proves to be a reliable, objective, and reproducible method in an increasing number of studies. However, the underlying model assumes the lack of gene flow after speciation; an assumption which may be frequently violated in plant evolution. This study evaluates the robustness of currently available species delimitation methods implemented in beast (BFD, BFD*, and dissect) in the closely-knit ox-eye daisy group around Leucanthemum ageratifolium Pau. Comprising five taxa being allopatrically distributed between northern Spain and southern Italy this study group shows signs of hybridization with the widespread and codistributed species Leucanthemum vulgare (Vaill.) Lam. to various extent. As expected, our empirical analyses based on both AFLP fingerprinting and sequence data demonstrate that the robustness of species delimitation results is considerably influenced by the intensity of hybridization among species and the number of hybrid individuals included. Therefore, we set up a methodological pipeline with a first step of identification and subsequent removal of individuals showing admixed genetic patterns caused by actual interbreeding using AFLP-fingerprint and morphometric data, followed by application of different Bayesian MSC species delimitation methods based on the remnant individuals using both AFLP-fingerprint and sequence data (four nuclear markers, five concatenated intergenic spacer regions of the plastid genome). The results argue for acknowledgement of Leucanthemum laciniatum, L. legraeanum, and L. ligusticum as independent species, show the close relationship of L. ageratifolium, L. monspeliense, and L. vulgare, and give rise to the description of three nothospecies new to science.

Detecting reticulate relationships among diploid Leucanthemum Mill. (Compositae, Anthemideae) taxa using multilocus species tree reconstruction methods and AFLP fingerprinting.

We examined the evolutionary history of the diploid representatives of the genus Leucanthemum Mill. (Compositae, Anthemideae), which constitutes an extensive polyploid complex comprising around 41 species with ploidy levels ranging from 2x to 22x. The inference of phylogenetic relationships even on the diploid level is complicated in this genus due to the overlay of hybridisation and incomplete lineage sorting processes leading to incongruence among gene trees based on nuclear and plastid sequence information. Species tree and network reconstructions were based on gene trees from nine low-copy nuclear markers and the concatenated sequence information for five intergenic spacer regions of the chloroplast genome, either sequenced by Roche 454 pyrosequencing techniques or traditional Sanger sequencing techniques. Additional phylogenetic information came from multi-locus AFLP-fingerprinting of representative individuals of all diploid taxa under study and the subsequent analysis of AFLP patterns with Bayesian clustering and network reconstruction methods. To distinguish between hybridisation and incomplete lineage sorting, we developed and utilized a new 'hybrid index' calculation for individual taxa of the data set, which was compared to a simulated null-distribution assuming the occurrence of incomplete lineage sorting alone for pinpointing taxa with a significant hybrid signal. As a result, two species groups with contrasting patterns of gene flow and/or hybrid speciation signals could be identified in the diploids of Leucanthemum: (a) an early-diverging stock of allopatrically distributed diploid species with a lack of evidence for recent hybridisation events among its members and (b) a more recently radiated taxon assemblage with morphologically less clearly circumscribed taxa and a pronounced signal of gene flow among lineages and several candidate taxa, for which a homoploid hybrid origin may be considered.

Is the extremely rare Iberian endemic plant species Castrilanthemum debeauxii (Compositae, Anthemideae) a 'living fossil'? Evidence from a multi-locus species tree reconstruction.

The present study provides results of multi-species coalescent species tree analyses of DNA sequences sampled from multiple nuclear and plastid regions to infer the phylogenetic relationships among the members of the subtribe Leucanthemopsidinae (Compositae, Anthemideae), to which besides the annual Castrilanthemum debeauxii (Degen, Hervier & É.Rev.) Vogt & Oberp., one of the rarest flowering plant species of the Iberian Peninsula, two other unispecific genera (Hymenostemma, Prolongoa), and the polyploidy complex of the genus Leucanthemopsis belong. Based on sequence information from two single- to low-copy nuclear regions (C16, D35, characterised by Chapman et al. (2007)), the multi-copy region of the nrDNA internal transcribed spacer regions ITS1 and ITS2, and two intergenic spacer regions of the cpDNA gene trees were reconstructed using Bayesian inference methods. For the reconstruction of a multi-locus species tree we applied three different methods: (a) analysis of concatenated sequences using Bayesian inference (MrBayes), (b) a tree reconciliation approach by minimizing the number of deep coalescences (PhyloNet), and (c) a coalescent-based species-tree method in a Bayesian framework ((∗)BEAST). All three species tree reconstruction methods unequivocally support the close relationship of the subtribe with the hitherto unclassified genus Phalacrocarpum, the sister-group relationship of Castrilanthemum with the three remaining genera of the subtribe, and the further sister-group relationship of the clade of Hymenostemma+Prolongoa with a monophyletic genus Leucanthemopsis. Dating of the (∗)BEAST phylogeny supports the long-lasting (Early Miocene, 15-22Ma) taxonomical independence and the switch from the plesiomorphic perennial to the apomorphic annual life-form assumed for the Castrilanthemum lineage that may have occurred not earlier than in the Pliocene (3Ma) when the establishment of a Mediterranean climate with summer droughts triggered evolution towards annuality.

The reticulate evolutionary history of the polyploid NW Iberian Leucanthemum pluriflorum clan (Compositae, Anthemideae) as inferred from nrDNA ETS sequence diversity and eco-climatological niche-modelling.

The genus Leucanthemum Mill. is a species-rich polyploid complex of southern and central Europe, comprising 41 species with ploidy levels ranging from 2x to 22x. The Leucanthemum pluriflorum clan, a geographically isolated species group of the NW Iberian Peninsula, comprises the diploid L. pluriflorum, the tetraploids Leucanthemumircutianum subsp. pseudosylvaticum and Leucanthemum×corunnense (being a putative hybrid taxon based on a cross between L. pluriflorum and Leucanthemummerinoi), and the two hexaploids Leucanthemumsylvaticum and L. merinoi. In order to reconstruct the evolutionary history of this species group, we analysed sequence variation at the external transcribed spacer region of the nuclear ribosomal repeat (nrDNA ETS) for its members and for a number of other diploid species of Leucanthemum. Our results indicate that there are two major ETS ribotypes present in Leucanthemum, with some of the diploid species fixed for either of the two types and several species (among them L. pluriflorum) exhibiting both types. This polymorphism at the nrDNA ETS locus suggests either gene flow among some of the diploid species (possibly via polyploids) or a homoploid hybrid origin of some of those diploids. Additionally, patterns of ETS ribotype sharing among populations of the four species of the L. pluriflorum clan suggest that the tetraploid L. ircutianum subsp. pseudosylvaticum and the hexaploids L. sylvaticum and L. merinoi have an allopolyploid origin with L. pluriflorum as the maternal parent. Eco-climatological modelling of present and past (last glacial maximum, LGM) distribution areas of the members of the L. pluriflorum clan indicates that the diploid L. pluriflorum may have undergone geographical differentiation into northern (Galician) and southern (central Portuguese) coastal lineages that could account for the two chloroplast haplotype groups observable in the tetra- and hexaploids. Later climatic changes in the Holocene could then have led to the extinction of southern diploid lineages. A distinct overlap of present and past (LGM) potential distribution ranges of L. pluriflorum with those of the N Iberian endemics Leucanthemumgallaecicum and Leucanthemumgaudinii subsp. cantabricum may indicate that one of the latter species may have acted as the paternal parent in the formation of the polyploids of the clan.