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Abstract Heterotrophic plants are among the most recalcitrant from a systematics perspective because of reduced morphological and genomic features, and often extreme substitution rate heterogeneity. The orchid subtribe Calypsoinae exemplifies this, containing several lineages that have lost leaves and photosynthesis. In particular, relationships of the leafy Asian Oreorchis and the leafless American Corallorhiza have been contentious. Here we used nuclear sequence capture to resolve relationships within Calypsoinae and addressed the monophyly of Corallorhiza and Oreorchis, for which previous studies have highlighted conflicting patterns of monophyly or paraphyly, depending on the data analysed. Nuclear analyses provided strong support for a monophyletic Corallorhiza and paraphyletic Oreorchis, the latter with two strongly supported clades. As in previous studies, plastid analyses recovered strongly supported paraphyletic assemblages for both genera. Topology tests using plastid and nuclear relationships and data rejected the constrained topologies, further revealing strong cytonuclear conflict. Network-based analyses revealed a lack of evidence for hybridization, suggesting incomplete lineage sorting associated with biological and historical factors have driven intergenomic conflict. Additionally, we found that loci identified as putatively lost in holomycotrophic Corallorhiza species are functionally enriched for organellar functions. The study provides a strong case for the resurrection of Kitigorchis as the sister of Corallorhiza, with two species, Kitigorchis erythrochrysea and Kitigorchis indica, and highlights the challenges associated with phylogenetics of lineages containing mycoheterotrophs. 

Abstract The capability to generate densely sampled single nucleotide polymorphism (SNP) data is essential in diverse subdisciplines of biology, including crop breeding, pathology, forensics, forestry, ecology, evolution and conservation. However, the wet‐laboratory expertise and bioinformatics training required to conduct genome‐scale variant discovery remain limiting factors for investigators with limited resources.Here we present ISSRseq, a PCR‐based method for reduced representation of genomic variation using simple sequence repeats as priming sites to sequence inter simple sequence repeat (ISSR) regions. Briefly, ISSR regions are amplified with single primers, pooled, used to construct sequencing libraries with a commercially available kit, and sequenced on the Illumina platform. We also present a flexible bioinformatic pipeline that assembles ISSR loci, calls and hard filters variants, outputs data matrices in common formats, and conducts population analyses using R.Using three angiosperm species as case studies, we demonstrate that ISSRseq is highly repeatable, necessitates only simple wet‐laboratory skills and commonplace instrumentation, is flexible in terms of the number of single primers used, and can generate genomic‐scale variant discovery on par with existing RRS methods which require more complex wet‐laboratory procedures.ISSRseq represents a straightforward approach to SNP genotyping in any organism, and we predict that this method will be particularly useful for those studying population genomics and phylogeography of non‐model organisms. Furthermore, the ease of ISSRseq relative to other RRS methods should prove useful to those lacking advanced expertise in wet‐laboratory methods or bioinformatics. 

Lineage-based species definitions applying coalescent approaches to species delimitation have become increasingly popular. Yet, the application of these methods and the recognition of lineage-only definitions have recently been questioned. Species delimitation criteria that explicitly consider both lineages and evidence for ecological role shifts provide an opportunity to incorporate ecologically meaningful data from multiple sources in studies of species boundaries. Here, such criteria were applied to a problematic group of mycoheterotrophic orchids, the Corallorhiza striata complex, analysing genomic, morphological, phenological, reproductive-mode, niche, and fungal host data. A recently developed method for generating genomic polymorphism data-ISSRseq-demonstrates evidence for four distinct lineages, including a previously unidentified lineage in the Coast Ranges and Cascades of California and Oregon, USA. There is divergence in morphology, phenology, reproductive mode, and fungal associates among the four lineages. Integrative analyses, conducted in population assignment and redundancy analysis frameworks, provide evidence of distinct genomic lineages and a similar pattern of divergence in the extended data, albeit with weaker signal. However, none of the extended data sets fully satisfy the condition of a significant role shift, which requires evidence of fixed differences. The four lineages identified in the current study are recognized at the level of variety, short of comprising different species. This study represents the most comprehensive application of lineage + role to date and illustrates the advantages of such an approach.