DiasMorph: a dataset of morphological traits and images of Central European diaspores.

We present DiasMorph, a dataset of images and traits of diaspores from 1,442 taxa in 519 genera, and 96 families from Central Europe, totalling 94,214 records. The dataset was constructed following a standardised and reproducible image analysis method. The image dataset consists of diaspores against a high-contrast background, enabling a simple and efficient segmentation process. The quantitative traits records go beyond traditional morphometric measurements, and include colour and contour features, which are made available for the first time in a large dataset. These measurements correspond to individual diaspores, an input currently unavailable in traits databases, and allow for several approaches to explore the morphological traits of these species. Additionally, information regarding the presence and absence of appendages and structures both in the images and diaspores of the assessed taxa is also included. By making these data available, we aim to encourage initiatives to advance on new tools for diaspore identification, further our understanding of morphological traits functions, and provide means for the continuous development of image analyses applications.

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.

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.

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.

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.

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.

'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.