Testing genome skimming for species discrimination in the large and taxonomically difficult genus Rhododendron

Abstract

Standard plant DNA barcodes based on 2–3 plastid regions, and nrDNA ITS show variable levels of resolution, and fail to discriminate among species in many plant groups. Genome skimming to recover complete plastid genome sequences and nrDNA arrays has been proposed as a solution to address these resolution limitations. However, few studies have empirically tested what gains are achieved in practice. Of particular interest is whether adding substantially more plastid and nrDNA characters will lead to an increase in discriminatory power, or whether the resolution limitations of standard plant barcodes are fundamentally due to plastid genomes and nrDNA not tracking species boundaries. To address this, we used genome skimming to recover near‐complete plastid genomes and nuclear ribosomal DNA from Rhododendron species and compared discrimination success with standard plant barcodes. We sampled 218 individuals representing 145 species of this species‐rich and taxonomically difficult genus, focusing on the global biodiversity hotspots of the Himalaya‐Hengduan Mountains. Only 33% of species were distinguished using ITS+matK+rbcL+trnH‐psbA. In contrast, 55% of species were distinguished using plastid genome and nrDNA sequences. The vast majority of this increase is due to the additional plastid characters. Thus, despite previous studies showing an asymptote in discrimination success beyond 3–4 plastid regions, these results show that a demonstrable increase in discriminatory power is possible with extensive plastid genome data. However, despite these gains, many species remain unresolved, and these results also reinforce the need to access multiple unlinked nuclear loci to obtain transformative gains in species discrimination in plants.

Testing the Complete Plastome for Species Discrimination, Cryptic Species Discovery and Phylogenetic Resolution in Cephalotaxus (Cephalotaxaceae)

“…Our species discrimination results indicated that the complete plastome can provide more variation to discriminate Cephalotaxus species compared to the standard barcodes, which has also been demonstrated in other studies (e.g., Ji et al, 2020 ; Song et al, 2020 ; Fu et al, 2022 ). In addition, the complete plastome approach based on genome skimming can circumvent issues of locus choice, and low PCR and sequencing recovery rate, which are sometimes encountered in standard DNA barcoding studies ( Ruhsam et al, 2015 ; Curci et al, 2016 ).…”

“…The inclusion of multiple individuals from a single species sampled is highly important to confirm species delimitation successfully and accurately ( Liu et al, 2012 ; Fu et al, 2022 ). Therefore, in this study, all species/varieties of Cephalotaxus had more than one individual included (two to five individuals per taxon).…”

“…The benefits of the complete plastome have been argued for its utilization in plant species delimitation, although this may not solve delimitation failures resulting from rare biparental plastid inheritance, introgression and hybridization, such as presumed for Salix ( Percy et al, 2014 ), and Calligonum ( Song et al, 2020 ). However, plastome barcoding has shown a great promise for reliably distinguishing closely related species ( Kane et al, 2012 ; Ruhsam et al, 2015 ; Zhang et al, 2017 ; Zhu et al, 2018 ; Fu et al, 2022 ). In addition, the plastid genome has a large number of evolutionarily informative variation, which has been widely used for phylogenetic reconstruction at deep to shallow levels in land plants ( Moore et al, 2010 ; Yu et al, 2018 ; Ji et al, 2019 ; Li et al, 2019 , 2021 ), including conifers (e.g., Fu et al, 2019 ; Zhang et al, 2019 ; Ji et al, 2021 ).…”

Providing Scale to a Known Taxonomic Unknown—At Least a 70-Fold Increase in Species Diversity in a Cosmopolitan Nominal Taxon of Lichen-Forming Fungi

“…Although genome skimming approaches, such as those implemented here, can provide crucial genome-scale data [ 48 , 75 , 76 ], more cost- and time-efficient methods, such as target enrichment sequencing [ 77 , 78 ] or restriction-site associated sequencing (RADseq) [ 26 , 47 , 79 ] might be more appropriate for this clade given the high number of samples that will likely need to be included. Ultimately, genome-scale data can also be used to reconstruct a robust phylogeny for this group to explore additional evolutionary, phylogeographic, and taxonomic questions.…”

Genome Skimming Contributes to Clarifying Species Limits in Paris Section Axiparis (Melanthiaceae)

“…With next-generation DNA sequencing (NGS) technologies, genome-wide sequence variation has begun to replace one or a few sequence loci for the identification and delimitation of plant species ( Li et al, 2015 ; Coissac et al, 2016 ; Hollingsworth et al, 2016 ). The genome skimming approach, which uses NGS technologies to generate multi-copy and highly repetitive genome components, such as whole plastid genomes (plastomes) and nuclear ribosomal DNA (nrDNA) clusters via relatively low coverage genome sequencing ( Straub et al, 2012 ), has been increasingly used for species identification and delimitation in recent years ( Nock et al, 2011 ; Kane et al, 2012 ; Dodsworth, 2015 ; Li et al, 2015 ; Ruhsam et al, 2015 ; Firetti et al, 2017 ; Fu et al, 2019 , 2021 ; Ji et al, 2019a , 2020 , 2021 ; Knope et al, 2020 ; Ślipiko et al, 2020 ; Su et al, 2021 ). Compared with restriction-site associated DNA sequencing (RAD-seq; Miller et al, 2007 ; Baird et al, 2008 ), another NSG-based technique that is extensively used to generate genomic data for plant species identification and delimitation (e.g., Wu et al, 2018 ; Donkpegan et al, 2020 ; Ma et al, 2020 ; Zhou et al, 2020 ; Li et al, 2021 ), the promising advantage of using genome skimming for species identification and delimitation is the avoidance of problems encountered with RAD-seq ( Kane et al, 2012 ; Hollingsworth et al, 2016 ), such as only applying to diploids and generating asymmetric data between distinctly related taxa.…”