Rapidly radiated groups are usually accompanied by unclear lineage and taxa delineation, which complicates their better understanding in terms of biodiversity, evolutionary processes, and taxonomic treatment. The most species-rich orchid subtribe, Pleurothallidinae, exemplifies an extremely diverse group with a complex evolutionary history associated with Andean orography. Here we combined multi-loci phylogeny reconstruction (HybSeq), genome-wide traits (inferred by flow cytometry), spatial analyses, and biogeography to investigate the evolutionary intricacy of one clade of Pleurothallidinae orchids. To achieve deep insights, we performed multiple species tree reconstruction approaches with the implementation of custom scripts to reveal sources of topological discrepancies and alternative evolutionary scenarios. The phylogeny clearly resolves the delimitation of the main evolutionary lineages corresponding to the accepted genera, with the exception of the genus Specklinia, which is divided into three distinct monophyletic lineages whose taxonomic treatment is proposed. Genome-wide characters (especially genome size) show an association with precipitation seasonality in a geographical context, and partial endoreplication, a unique character of orchids, is geographically restricted to the Andes, Central America, and the Caribbean. Specifically, the Andean region exemplifies the prevalence of bigger genome size and higher GC content, resulting from a higher proportion of species with partial endoreplication. The Andean origin of the clade was also revealed by biogeographic analysis. Our comprehensive approach has provided deep insights into the evolution of this clade and may be a useful tool for unraveling the intricate evolutionary history of similarly complex lineages.

• Keywords
• Andes, GC content, HybSeq, Pleurothallidinae, endoreplication, evolution, genome size, spatial analysis,
• MeSH
• Biological Evolution * MeSH
• Genome Size MeSH
• Phylogeny * MeSH
• Genome, Plant * genetics   MeSH
• Evolution, Molecular MeSH
• Orchidaceae * genetics   classification   MeSH
• Publication type
• Journal Article MeSH

The estimation of nuclear DNA content has been by far the most popular application of flow cytometry in plants. Because flow cytometry measures relative fluorescence intensities of nuclei stained by a DNA fluorochrome, ploidy determination, and estimation of the nuclear DNA content in absolute units both require comparison to a reference standard of known DNA content. This implies that the quality of the results obtained depends on the standard selection and use. Internal standardization, when the nuclei of an unknown sample and the reference standard are isolated, stained, and measured simultaneously, is mandatory for precise measurements. As DNA peaks representing G1 /G0 nuclei of the sample and standard appear on the same histogram of fluorescence intensity, the quotient of their position on the fluorescence intensity axis provides the quotient of DNA amounts. For the estimation of DNA amounts in absolute units, a number of well-established standards are now available to cover the range of known plant genome sizes. Since there are different standards in use, the standard and the genome size assigned to it has always to be reported. When none of the established standards fits, the introduction of a new standard species is needed. For this purpose, the regression line approach or simultaneous analysis of the candidate standard with several established standards should be prioritized. Moreover, the newly selected standard organism has to fulfill a number of requirements: it should be easy to identify and maintain, taxonomically unambiguous, globally available, with known genome size stability, lacking problematic metabolites, suitable for isolation of sufficient amounts of nuclei, and enabling measurements with low coefficients of variation of DNA peaks, hence suitable for the preparation of high quality samples.

• Keywords
• C-value, GC content, best practices, flow cytometry, genome size, plant sciences, plant standard species, standardization,
• MeSH
• DNA, Plant genetics   MeSH
• Genome, Plant * MeSH
• Ploidies * MeSH
• Flow Cytometry methods   MeSH
• Reference Standards MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• DNA, Plant MeSH

Knowledge of population variation across species' ranges is a prerequisite for correctly assessing the overall variability of any group of organisms and provides an invaluable basis for unraveling evolutionary history, optimizing taxonomy and devising effective conservation strategies. Here, we examine the genus Neotinea, which represents a relatively recently delimited monophyletic genus of orchids, for which a detailed study of its overall variability was lacking. We applied a suite of biosystematic methods, consisting of flow cytometry, multivariate and geometric morphometrics, and analysis of genomic SNP data, to identify phylogenetic lineages within the genus, to delineate phenotypic variation relevant to these lineages, and to identify potential cryptic taxa within lineages. We found clear differentiation into four major lineages corresponding to the groups usually recognized within the genus: Neotinea maculata as a distinct and separate taxon, the Neotinea lactea group comprising two Mediterranean taxa N. lactea and Neotinea conica, the Neotinea ustulata group comprising two phenologically distinct varieties, and the rather complex Neotinea tridentata group comprising two major lineages and various minor lineages of unclear taxonomic value. N. conica constitutes both a monophyletic group within N. lactea and a distinct phenotype within the genus and merits its proposed subspecies-level recognition. By contrast, the spring and summer flowering forms of N. ustulata (var. ustulata and var. aestivalis) were confirmed to be distinct only morphologically, not phylogenetically. The most complex pattern emerged in the N. tridentata group, which splits into two main clades, one containing lineages from the Balkans and eastern Mediterranean and the other consisting of plants from Central Europe and the central Mediterranean. These individual lineages differ in genome size and show moderate degrees of morphological divergence. The tetraploid Neotinea commutata is closely related to the N. tridentata group, but our evidence points to an auto- rather than an allopolyploid origin. Our broad methodological approach proved effective in recognizing cryptic lineages among the orchids, and we propose the joint analysis of flow cytometric data on genome size and endopolyploidy as a useful and beneficial marker for delineating orchid species with partial endoreplication.

• Keywords
• RADseq, cryptic diversity, genome size, geometric morphometric, multivariate morphometric, orchids (Orchidaceae), partial endoreplication,
• Publication type
• Journal Article MeSH