Revealing the evolutionary history of a reticulate polyploid complex in the genusIsoëtes

Abstract

Polyploidy and hybridization are important processes in the evolution of spore-dispersed plants. Few studies, however, focus these dynamics in heterosporous lycophytes, such as Isoetes, where polyploid hybrids are common and thought to be important in the generation of their extant diversity. We investigate reticulate evolution in a complex of western North American quillworts (Isoetes) and provide insights into the evolutionary history of hybrids, and the role of polyploidy in maintaining novel diversity. We utilize low copy nuclear markers, whole plastomes, restriction site-associated DNA sequencing, cytology, and reproductive status (fertile or sterile) to investigate the reticulate evolutionary history of western North American Isoetes. We reconstruct the reticulate evolutionary history and directionality of hybridization events in this complex. The presence of high level polyploids, plus frequent homoploid and interploid hybridization suggests that there are low prezygotic reproductive barriers in this complex, hybridization is common and bidirectional between similar (but not divergent) cytotypes, and that allopolyploidization is important to restore fertility in some hybrid taxa. Our data provide five lines of evidence suggesting that hybridization and polyploidy can occur with frequency in the genus, and these evolutionary processes may be important in shaping extant Isoetes diversity.

PURC v2.0: A Program for Improved Sequence Inference for Polyploid Phylogenetics and Other Manifestations of the Multiple-Copy Problem

“…The output from PURC is one alignment for each locus, which includes all the copies present in each of the accessions, labelled by their accession ID and coverage (the number of reads that constitute that sequence). Since its release, PURC has been used to investigate allopolyploidy in genus-level datasets in diverse plant lineages (Morales-Briones and Tank, 2019;Dauphin et al, 2018;Suissa et al, 2020;Blischak et al, 2020), in studies of cytotype variation within species (Kao et al, 2020), and for applications where polyploidy itself was incidental to the primary research questions (Kao et al, 2019;Chery et al, 2019;Wolfe et al, 2021;Frost et al, 2020). Most notably, Blischak et al (2018) created a program to adapt PURC to data generated by microfluidic PCR, reducing one of the main limitations of amplicon-based approaches (the time and expense associated with PCR itself).…”

“…PURC v2.0 was run on four replicates of the five-taxon mock community, performing OTU clustering with default parameter values (clustering thresholds by round: 0.997, 0.995, 0.990, 0.997, final size threshold = 4) and inferring ASVs (DADA2) with a maximum of five expected errors allowed per read and length outliers removed based on Tukey's outlier test (Tukey, 1977). We also tested data from species of Isoetes from Suissa et al (2020) using the same parameters.…”

“…Most replicates contained OTUs identical to ASVs, but OTUs with small sequence deviations were common and a few spurious OTUs with 75%-92% identity to ASVs were also generated. Because the Isoetes data from Suissa et al (2020) are empirical and the true sequences are unknown, we evaluated ASV-versus OTU-based inferences of these sequences using two independent PCR amplifications of each of three individuals, thought to represent one diploid and two allotetraploids (I. echinospora Taylor #6989-1, I. maritima Taylor #6983, and I. aff. "new hybrid A" Taylor #6988-2, respectively).…”

“…PURC v2.0 also includes PacBio's lima tool (https://lima.how) as a new method for demultiplexing reads on Linux operating systems. Using lima, we could recover an average of 74% more reads from the Suissa et al (2020) data than with the original BLAST-based method in PURC, including recovering one individual for which no sequences were identified using BLAST; the persample increase ranged from 24-640% (Figure 1 C). The number of both OTUs and ASVs increased in the lima-demultiplexed dataset but ASVs fluctuated less, with 74% of samples having the same number of ASVs as in the BLAST-demultiplexed data versus only 26% for OTUs.…”