Homoploid hybrids, allopolyploids, and high ploidy levels characterize the evolutionary history of a western North American quillwort (Isoëtes) complex.

Polyploidy and hybridization are important processes in seed-free plant evolution. However, a major gap lies in our understanding of how these processes affect the evolutionary history of high-ploidy systems. The heterosporous lycophyte genus Isoëtes is a lineage with many putative hybrids and high-level polyploid taxa (ranging from tetraploid to dodecaploid). Here, we use a complex of western North American Isoëtes, to understand the role of hybridization and high-level polyploidy in generating and maintaining novel diversity. To uncover these processes, we use restriction-site associated DNA sequencing (RADseq), multiple alleles of a single low-copy nuclear marker, whole plastomes, cytology (genome size estimates and chromosome counts), and reproductive status (fertile or sterile). With this dataset, we show that hybridization occurs easily between species in this complex and is bidirectional between identical, but not different, cytotypes. Furthermore, we show that fertile allopolyploids appear to have formed repeatedly from sterile homoploid and interploid hybrids. We propose that low prezygotic reproductive barriers and a high frequency of whole-genome duplication allow for high-level polyploid systems to generate novel lineages, and that these mechanisms may be important in shaping extant Isoëtes diversity.

Low-copy nuclear markers in Isoëtes (Isoëtaceae) identified with transcriptomes.

PREMISE OF THE STUDY: Few genetic markers provide phylogenetic information in closely related species of Isoëtes (Isoëtaceae). We describe the development of primers for several putative low-copy nuclear markers to resolve the phylogeny of Isoëtes, particularly in the southeastern United States. METHODS AND RESULTS: We identified regions of interest in Isoëtes transcriptomes based on low-copy genes in other plants. Primers were designed for these regions and tested with 16 taxa of Isoëtes and one species of Lycopodium. Parts of the pgiC, gapC, and IBR3 gene regions show phylogenetic signal within the North American and Mediterranean clades of Isoëtes. CONCLUSIONS: Transcriptome data prove useful for identification and primer design of low-copy genes. Three new markers show potential for inferring phylogenies in regional clades of Isoëtes, and possibly across the entire genus.

Isoetes mississippiensis: A new quillwort from Mississippi, USA.

Isoetes mississippiensis S.W. Leonard, W.C. Taylor, L.J. Musselman and R.D. Bray (Isoetaceae, Lycopodiophyta) is a new species known from two sites along tributaries of the Pearl River in southern Mississippi. This species is distinguished from other species in the southeastern United States by a combination of character states including a basic diploid (2n=22) chromosome count, laevigate megaspores, and a narrow velum covering less than one-third of the adaxial sporangium wall.

Revealing unknown or extinct lineages within Isoetes (Isoetaceae) using DNA sequencesfrom hybrids.

Isoëtes, a heterosporous lycopod with a fossil record dating back to the Paleozoic, has numerous putative allopolyploids (resulting from hybridization events coupled with doubling of chromosome number). By using the highly variable nucleotide sequences from the second intron of a LFY homologue in Isoëtes, species could be delimited and hybrid origins determined. The data suggest that reticulate evolution is both common and complex within a more derived species complex of Isoëtes. Sequences of identifiable parentage and sequences that are unlike any diploid species known were recovered, leading to the conclusion that one or both of the putative parents have not yet been discovered or are extinct. A range of observations concerning allopolyploid speciation were categorized as follows: (1) verification of previous hypotheses regarding parentage (e.g., I. riparia, I. appalachiana), (2) determination that two morphologically distinct allotetraploid species can share the same parentage (I. azorica and I. acadiensis), (3) recognition of a cryptic allotetraploid species, indicated by the presence of different parental genomes (I. "appalachiana" from Florida), and (4) identification of allotetraploid species with one or two unknown parents (e.g., I. tuckermanii, I. acadiensis, I. azorica, and I. hyemalis). Some sequences from diploid species are remarkably uniform among populations (e.g., I. echinospora from various locations in North America, Iceland, and Wales), while others are variable at the subspecies level (e.g., northern and southern populations within I. engelmannii).