Population genomics of the Isoetes appalachiana (Isoetaceae) complex supports a 'diploids-first' approach to conservation.

BACKGROUND AND AIMS: Allopolyploidy is an important driver of diversification and a key contributor to genetic novelty across the tree of life. However, many studies have questioned the importance of extant polyploid lineages, suggesting that the vast majority may constitute evolutionary 'dead ends'. This has important implications for conservation efforts where polyploids and diploid progenitors often compete for wildlife management resources. Isoetes appalachiana is an allotetraploid that is broadly distributed throughout the eastern USA alongside its diploid progenitors, I. valida and I. engelmannii. As such, this species complex provides an excellent opportunity to investigate the processes that underpin the formation and survival of allopolyploid lineages. METHODS: Here we utilized RADseq and whole-chloroplast sequencing to unravel the demographic and evolutionary history of hybridization in this widespread species complex. We developed a modified protocol for phasing RADseq loci from an allopolyploid in order to examine each progenitor's genetic contribution independently in a phylogenetic context. Additionally, we conducted population-level analyses to examine genetic diversity and evidence of gene flow within species. KEY RESULTS: Isoetes appalachiana is the product of multiple phylogenetic origins, suggesting that formation and establishment of allopolyploids are common in this group. Hybridization appears to be unidirectional, with I. engelmannii consistently being the maternal progenitor. Additionally, we find that polyploid lineages are genetically isolated, rarely if ever experiencing gene flow between geographically distinct populations. CONCLUSIONS: Allopolyploid lineages of I. appalachiana appear to form frequently and experience a high degree of genetic isolation following formation. Thus, our results appear to corroborate the hypothesis that the vast majority of recently formed polyploids may represent evolutionary dead ends. However, this does not necessarily lessen the evolutionary importance or ecological impact of polyploidy per se. Accordingly, we propose a conservation strategy that prioritizes diploid taxa, thus preserving downstream processes that recurrently generate allopolyploid diversity.

PURC Provides Improved Sequence Inference for Polyploid Phylogenetics and Other Manifestations of the Multiple-Copy Problem.

Inferring the true biological sequences from amplicon mixtures remains a difficult bioinformatics problem. The traditional approach is to cluster sequencing reads by similarity thresholds and treat the consensus sequence of each cluster as an "operational taxonomic unit" (OTU). Recently, this approach has been improved by model-based methods that correct PCR and sequencing errors in order to infer "amplicon sequence variants" (ASVs). To date, ASV approaches have been used primarily in metagenomics, but they are also useful for determining homeologs in polyploid organisms. To facilitate the usage of ASV methods among polyploidy researchers, we incorporated ASV inference alongside OTU clustering in PURC v2.0, a major update to PURC (Pipeline for Untangling Reticulate Complexes). In addition, PURC v2.0 features faster demultiplexing than the original version and has been updated to be compatible with Python 3. In this chapter we present results indicating that using the ASV approach is more likely to infer the correct biological sequences in comparison to the earlier OTU-based PURC and describe how to prepare sequencing data, run PURC v2.0 under several different modes, and interpret the output.

Genome sequence for the blue-flowered Andean shrub Iochroma cyaneum reveals extensive discordance across the berry clade of Solanaceae.

The tomato (Solanum lycopersicum L.) family, Solanaceae, is a model clade for a wide range of applied and basic research questions. Currently, reference-quality genomes are available for over 30 species from seven genera, and these include numerous crops as well as wild species [e.g., Jaltomata sinuosa (Miers) Mione and Nicotiana attenuata Torr. ex S. Watson]. Here we present the genome of the showy-flowered Andean shrub Iochroma cyaneum (Lindl.) M. L. Green, a woody lineage from the tomatillo (Physalis philadelphica Lam.) subfamily Physalideae. The assembled size of the genome (2.7 Gb) is more similar in size to pepper (Capsicum annuum L.) (2.6 Gb) than to other sequenced diploid members of the berry clade of Solanaceae [e.g., potato (Solanum tuberosum L.), tomato, and Jaltomata]. Our assembly recovers 92% of the conserved orthologous set, suggesting a nearly complete genome for this species. Most of the genomic content is repetitive (69%), with Gypsy elements alone accounting for 52% of the genome. Despite the large amount of repetitive content, most of the 12 I. cyaneum chromosomes are highly syntenic with tomato. Bayesian concordance analysis provides strong support for the berry clade, including I. cyaneum, but reveals extensive discordance along the backbone, with placement of chili pepper and Jaltomata being highly variable across gene trees. The I. cyaneum genome contributes to a growing wealth of genomic resources in Solanaceae and underscores the need for expanded sampling of diverse berry genomes to dissect major morphological transitions.

A step-by-step protocol for meiotic chromosome counts in flowering plants: A powerful and economical technique revisited.

PREMISE: Counting chromosomes is a fundamental botanical technique, yet it is often intimidating and increasingly sidestepped. Once mastered, the basic protocol can be applied to a broad range of taxa and research questions. It also reveals an aspect of the plant genome that is accessible with only the most basic of resources-access to a microscope with 1000× magnification is the most limiting factor. METHODS AND RESULTS: Here we provide a detailed protocol for choosing, staining, and squashing angiosperm pollen mother cells. The protocol is supplemented by figures and two demonstration videos. CONCLUSIONS: The protocol we provide will hopefully demystify and reinvigorate a powerful and once commonplace botanical technique that is available to researchers regardless of their location and resources.

Organelle Genome Inheritance in Deparia Ferns (Athyriaceae, Aspleniineae, Polypodiales).

Organelle genomes of land plants are predominately inherited maternally but in some cases can also be transmitted paternally or biparentally. Compared to seed plants (>83% genera of angiosperms and >12% genera of gymnosperms), plastid genome (plastome) inheritance has only been investigated in fewer than 2% of fern genera, and mitochondrial genome (mitogenome) from only one fern genus. We developed a new and efficient method to examine plastome and mitogenome inheritance in a fern species-Deparia lancea (Athyriaceae, Aspleniineae, Polypodiales), and found that plastid and mitochondrial DNAs were transmitted from only the maternal parentage to a next generation. To further examine whether both organelle genomes have the same manner of inheritance in other Deparia ferns, we sequenced both plastid and mitochondrial DNA regions of inter-species hybrids, and performed phylogenetic analyses to identify the origins of organellar DNA. Evidence from our experiments and phylogenetic analyses support that both organelle genomes in Deparia are uniparentally and maternally inherited. Most importantly, our study provides the first report of mitogenome inheritance in eupolypod ferns, and the second one among all ferns.

Next-generation polyploid phylogenetics: rapid resolution of hybrid polyploid complexes using PacBio single-molecule sequencing.

Difficulties in generating nuclear data for polyploids have impeded phylogenetic study of these groups. We describe a high-throughput protocol and an associated bioinformatics pipeline (Pipeline for Untangling Reticulate Complexes (Purc)) that is able to generate these data quickly and conveniently, and demonstrate its efficacy on accessions from the fern family Cystopteridaceae. We conclude with a demonstration of the downstream utility of these data by inferring a multi-labeled species tree for a subset of our accessions. We amplified four c. 1-kb-long nuclear loci and sequenced them in a parallel-tagged amplicon sequencing approach using the PacBio platform. Purc infers the final sequences from the raw reads via an iterative approach that corrects PCR and sequencing errors and removes PCR-mediated recombinant sequences (chimeras). We generated data for all gene copies (homeologs, paralogs, and segregating alleles) present in each of three sets of 50 mostly polyploid accessions, for four loci, in three PacBio runs (one run per set). From the raw sequencing reads, Purc was able to accurately infer the underlying sequences. This approach makes it easy and economical to study the phylogenetics of polyploids, and, in conjunction with recent analytical advances, facilitates investigation of broad patterns of polyploid evolution.

An Exploration into Fern Genome Space.

Ferns are one of the few remaining major clades of land plants for which a complete genome sequence is lacking. Knowledge of genome space in ferns will enable broad-scale comparative analyses of land plant genes and genomes, provide insights into genome evolution across green plants, and shed light on genetic and genomic features that characterize ferns, such as their high chromosome numbers and large genome sizes. As part of an initial exploration into fern genome space, we used a whole genome shotgun sequencing approach to obtain low-density coverage (∼0.4X to 2X) for six fern species from the Polypodiales (Ceratopteris, Pteridium, Polypodium, Cystopteris), Cyatheales (Plagiogyria), and Gleicheniales (Dipteris). We explore these data to characterize the proportion of the nuclear genome represented by repetitive sequences (including DNA transposons, retrotransposons, ribosomal DNA, and simple repeats) and protein-coding genes, and to extract chloroplast and mitochondrial genome sequences. Such initial sweeps of fern genomes can provide information useful for selecting a promising candidate fern species for whole genome sequencing. We also describe variation of genomic traits across our sample and highlight some differences and similarities in repeat structure between ferns and seed plants.

Transcriptome-mining for single-copy nuclear markers in ferns.

BACKGROUND: Molecular phylogenetic investigations have revolutionized our understanding of the evolutionary history of ferns-the second-most species-rich major group of vascular plants, and the sister clade to seed plants. The general absence of genomic resources available for this important group of plants, however, has resulted in the strong dependence of these studies on plastid data; nuclear or mitochondrial data have been rarely used. In this study, we utilize transcriptome data to design primers for nuclear markers for use in studies of fern evolutionary biology, and demonstrate the utility of these markers across the largest order of ferns, the Polypodiales. PRINCIPAL FINDINGS: We present 20 novel single-copy nuclear regions, across 10 distinct protein-coding genes: ApPEFP_C, cryptochrome 2, cryptochrome 4, DET1, gapCpSh, IBR3, pgiC, SQD1, TPLATE, and transducin. These loci, individually and in combination, show strong resolving power across the Polypodiales phylogeny, and are readily amplified and sequenced from our genomic DNA test set (from 15 diploid Polypodiales species). For each region, we also present transcriptome alignments of the focal locus and related paralogs-curated broadly across ferns-that will allow researchers to develop their own primer sets for fern taxa outside of the Polypodiales. Analyses of sequence data generated from our genomic DNA test set reveal strong effects of partitioning schemes on support levels and, to a much lesser extent, on topology. A model partitioned by codon position is strongly favored, and analyses of the combined data yield a Polypodiales phylogeny that is well-supported and consistent with earlier studies of this group. CONCLUSIONS: The 20 single-copy regions presented here more than triple the single-copy nuclear regions available for use in ferns. They provide a much-needed opportunity to assess plastid-derived hypotheses of relationships within the ferns, and increase our capacity to explore aspects of fern evolution previously unavailable to scientific investigation.

rbcL and matK earn two thumbs up as the core DNA barcode for ferns.

BACKGROUND: DNA barcoding will revolutionize our understanding of fern ecology, most especially because the accurate identification of the independent but cryptic gametophyte phase of the fern's life history--an endeavor previously impossible--will finally be feasible. In this study, we assess the discriminatory power of the core plant DNA barcode (rbcL and matK), as well as alternatively proposed fern barcodes (trnH-psbA and trnL-F), across all major fern lineages. We also present plastid barcode data for two genera in the hyperdiverse polypod clade--Deparia (Woodsiaceae) and the Cheilanthes marginata group (currently being segregated as a new genus of Pteridaceae)--to further evaluate the resolving power of these loci. PRINCIPAL FINDINGS: Our results clearly demonstrate the value of matK data, previously unavailable in ferns because of difficulties in amplification due to a major rearrangement of the plastid genome. With its high sequence variation, matK complements rbcL to provide a two-locus barcode with strong resolving power. With sequence variation comparable to matK, trnL-F appears to be a suitable alternative barcode region in ferns, and perhaps should be added to the core barcode region if universal primer development for matK fails. In contrast, trnH-psbA shows dramatically reduced sequence variation for the majority of ferns. This is likely due to the translocation of this segment of the plastid genome into the inverted repeat regions, which are known to have a highly constrained substitution rate. CONCLUSIONS: Our study provides the first endorsement of the two-locus barcode (rbcL+matK) in ferns, and favors trnL-F over trnH-psbA as a potential back-up locus. Future work should focus on gathering more fern matK sequence data to facilitate universal primer development.