Genetic factors predict hybrid formation in the British flora.

Natural hybridization can have a profound evolutionary impact, with consequences ranging from the extinction of rare taxa to the origin of new species. Natural hybridization is particularly common in plants; however, our understanding of the general factors that promote or prevent hybridization is hampered by the highly variable outcomes in different lineages. Here, we quantify the influence of different predictors on hybrid formation across species from an entire flora. We combine estimates of hybridization with ecological attributes and a new species-level phylogeny for over 1,100 UK flowering plant species. Our results show that genetic factors, particularly parental genetic distance, as well as phylogenetic position and ploidy, are key determinants of hybrid formation, whereas many other factors such as range overlap and genus size explain much less variation in hybrid formation. Overall, intrinsic genetic factors shape the evolutionary and ecological consequences of natural hybridization across species in a flora.

Comprehensive phylogenomic time tree of bryophytes reveals deep relationships and uncovers gene incongruences in the last 500 million years of diversification.

PREMISE: Bryophytes form a major component of terrestrial plant biomass, structuring ecological communities in all biomes. Our understanding of the evolutionary history of hornworts, liverworts, and mosses has been significantly reshaped by inferences from molecular data, which have highlighted extensive homoplasy in various traits and repeated bursts of diversification. However, the timing of key events in the phylogeny, patterns, and processes of diversification across bryophytes remain unclear. METHODS: Using the GoFlag probe set, we sequenced 405 exons representing 228 nuclear genes for 531 species from 52 of the 54 orders of bryophytes. We inferred the species phylogeny from gene tree analyses using concatenated and coalescence approaches, assessed gene conflict, and estimated the timing of divergences based on 29 fossil calibrations. RESULTS: The phylogeny resolves many relationships across the bryophytes, enabling us to resurrect five liverwort orders and recognize three more and propose 10 new orders of mosses. Most orders originated in the Jurassic and diversified in the Cretaceous or later. The phylogenomic data also highlight topological conflict in parts of the tree, suggesting complex processes of diversification that cannot be adequately captured in a single gene-tree topology. CONCLUSIONS: We sampled hundreds of loci across a broad phylogenetic spectrum spanning at least 450 Ma of evolution; these data resolved many of the critical nodes of the diversification of bryophytes. The data also highlight the need to explore the mechanisms underlying the phylogenetic ambiguity at specific nodes. The phylogenomic data provide an expandable framework toward reconstructing a comprehensive phylogeny of this important group of plants.

Divergence times and the evolution of morphological complexity in an early land plant lineage (Marchantiopsida) with a slow molecular rate.

We present a complete generic-level phylogeny of the complex thalloid liverworts, a lineage that includes the model system Marchantia polymorpha. The complex thalloids are remarkable for their slow rate of molecular evolution and for being the only extant plant lineage to differentiate gas exchange tissues in the gametophyte generation. We estimated the divergence times and analyzed the evolutionary trends of morphological traits, including air chambers, rhizoids and specialized reproductive structures. A multilocus dataset was analyzed using maximum likelihood and Bayesian approaches. Relative rates were estimated using local clocks. Our phylogeny cements the early branching in complex thalloids. Marchantia is supported in one of the earliest divergent lineages. The rate of evolution in organellar loci is slower than for other liverwort lineages, except for two annual lineages. Most genera diverged in the Cretaceous. Marchantia polymorpha diversified in the Late Miocene, giving a minimum age estimate for the evolution of its sex chromosomes. The complex thalloid ancestor, excluding Blasiales, is reconstructed as a plant with a carpocephalum, with filament-less air chambers opening via compound pores, and without pegged rhizoids. Our comprehensive study of the group provides a temporal framework for the analysis of the evolution of critical traits essential for plants during land colonization.

Nuclear DNA content variation and evolution in liverworts.

Across embryophytes there is a significant range in DNA content, both in regards to genome size (total DNA in an unreduced chromosome complement) and degree of endoreduplication (when DNA replication not followed by division resulting in various ploidy levels within the same individual). However, there is little information available on DNA content evolution in liverworts, the likely sister group to all other living plants. This study seeks to detect a phylogenetic structure in the variation in genome size and degree of endopolyploidy within liverworts. Furthermore, we test the hypothesis that shifts in breeding systems and genome size are correlated, as polyploidy is suggested to be a possible mechanism for the evolution of monoecy in liverworts and could therefore be associated with larger genome sizes. Genome size was determined for 67 liverwort species from 33 families using flow cytometry. Estimates for 48 species and 16 families are new to science. A phylogeny was reconstructed using the plastid gene rbcL. Over all taxa analyzed, there was a considerable range in genome size estimates with 1C-values from 0.27 pg (Jungermannia rubra) to 20.46 pg (Phyllothallia fuegiana). Large genome sizes were also found in the Haplomitriopsida. None of the liverwort species showed evidence of endopolyploidy. Although some taxa may be polyploids, a correlation between shifts in genome size and breeding system is lacking. Importantly, genome size variation in liverworts exhibits strong phylogenetic signal (Pagel's λ=0.99955).

Organellar genome, nuclear ribosomal DNA repeat unit, and microsatellites isolated from a small-scale of 454 GS FLX sequencing on two mosses.

Recent innovations in high-throughput DNA sequencing methodology (next generation sequencing technologies [NGS]) allow for the generation of large amounts of high quality data that may be particularly critical for resolving ambiguous relationships such as those resulting from rapid radiations. Application of NGS technology to bryology is limited to assembling entire nuclear or organellar genomes of selected exemplars of major lineages (e.g., classes). Here we outline how organellar genomes and the entire nuclear ribosomal DNA repeat can be obtained from minimal amounts of moss tissue via small-scale 454 GS FLX sequencing. We sampled two Funariaceae species, Funaria hygrometrica and Entosthodon obtusus, and assembled nearly complete organellar genomes and the whole nuclear ribosomal DNA repeat unit (18S-ITS1-5.8S-ITS2-26S-IGS1-5S-IGS2) for both taxa. Sequence data from these species were compared to sequences from another Funariaceae species, Physcomitrella patens, revealing low overall degrees of divergence of the organellar genomes and nrDNA genes with substitutions spread rather evenly across their length, and high divergence within the external spacers of the nrDNA repeat. Furthermore, we detected numerous microsatellites among the 454 assemblies. This study demonstrates that NGS methodology can be applied to mosses to target large genomic regions and identify microsatellites.

Target capture and genome skimming for plant diversity studies.

Recent technological advances in long-read high-throughput sequencing and assembly methods have facilitated the generation of annotated chromosome-scale whole-genome sequence data for evolutionary studies; however, generating such data can still be difficult for many plant species. For example, obtaining high-molecular-weight DNA is typically impossible for samples in historical herbarium collections, which often have degraded DNA. The need to fast-freeze newly collected living samples to conserve high-quality DNA can be complicated when plants are only found in remote areas. Therefore, short-read reduced-genome representations, such as target capture and genome skimming, remain important for evolutionary studies. Here, we review the pros and cons of each technique for non-model plant taxa. We provide guidance related to logistics, budget, the genomic resources previously available for the target clade, and the nature of the study. Furthermore, we assess the available bioinformatic analyses, detailing best practices and pitfalls, and suggest pathways to combine newly generated data with legacy data. Finally, we explore the possible downstream analyses allowed by the type of data generated using each technique. We provide a practical guide to help researchers make the best-informed choice regarding reduced genome representation for evolutionary studies of non-model plants in cases where whole-genome sequencing remains impractical.

A high quality, high molecular weight DNA extraction method for PacBio HiFi genome sequencing of recalcitrant plants.

BACKGROUND: PacBio HiFi sequencing provides highly accurate long-read sequencing datasets which are of great advantage for whole genome sequencing projects. One limitation of the method is the requirement for high quality, high molecular weight input DNA. This can be particularly challenging for plants that frequently contain common and species-specific secondary metabolites, which often interfere with downstream processes. Cape Primroses (genus Streptocarpus), are some of these recalcitrant plants and are selected here as material to develop a high quality, high molecular weight DNA extraction protocol for long read genome sequencing. RESULTS: We developed a DNA extraction method for PacBio HiFi sequencing for Streptocarpus grandis and Streptocarpus kentaniensis. A CTAB lysis buffer was employed to avoid guanidine, and the traditional chloroform and phenol purification steps were replaced with pre-lysis sample washes. Best cells/nucleus lysis was achieved with 4 h at 58 °C. The obtained high quality and high molecular weight DNAs were tested in PacBio SMRTBell™ library preparations, which resulted in circular consensus sequencing (CCS) reads from 17 to 27 Gb per cell, and a read length N50 from 14 to 17 kbp. To evaluate the quality of the reads for whole genome sequencing, they were assembled with HiFiasm into draft genomes, with N50 = 49 Mb and 23 Mb, and L50 = 10 and 11. The longest contigs were 95 Mb and 57 Mb respectively, showing good contiguity as these are longer than the theoretical chromosome length (genome size/chromosome number) of 78 Mb and 55 Mb, for S. grandis and S. kentaniensis respectively. CONCLUSIONS: DNA extraction is a critical step towards obtaining a complete genome assembly. Our DNA extraction method here provided the required high quality, high molecular weight DNA for successful standard-input PacBio HiFi library preparation. The contigs from those reads showed a high contiguity, providing a good starting draft assembly towards obtaining a complete genome. The results obtained here were highly promising, and demonstrated that the DNA extraction method developed here is compatible with PacBio HiFi sequencing and suitable for de novo whole genome sequencing projects of plants.

Chloroplast, mitochondrial, and nuclear microsatellites from the southern Appalachian hornwort, Nothoceros aenigmaticus (Dendrocerotaceae).

PREMISE OF THE STUDY: New microsatellite primers were developed for testing genetic differentiation within Nothoceros aenigmaticus and their potential use in other Nothoceros species. The microsatellites are designed to investigate partitioning of genetic variation in a taxon with a peculiar sex allopatry in the southern Appalachian Mountains and relationships with conspecific sexual populations from Mexico. METHODS AND RESULTS: We used two methods for microsatellite development: an enriched library and second-generation shotgun sequence reads. From these two methods, a total of nine primer pairs were selected and tested on 89 southern Appalachian N. aenigmaticus accessions, nine Mexican accessions, and 16 N. vincentianus accessions. Three mitochondrial loci were recovered from the enriched library method and six loci from 454 shotgun sequencing: three were from the chloroplast and three from the nucleus. The primers amplified repeats with two to 20 alleles per locus. CONCLUSIONS: New microsatellite primers were developed for testing genetic differentiation within N. aenigmaticus and potentially for use in other Nothoceros species. We present one of the first reports of highly polymorphic mitochondrial microsatellites in plants.

Frequent pseudogenization and loss of the plastid-encoded sulfate-transport gene cysA throughout the evolution of liverworts.

PREMISE OF THE STUDY: The presence or absence of a functional copy of a plastid gene may reflect relaxed selection, and may be phylogenetically significant, reflecting shared ancestry. In some liverworts, the plastid gene cysA is a pseudogene (inferred to be nonfunctional). We surveyed 63 liverworts from all major clades to determine whether the loss of cysA is phylogenetically significant, whether intact copies of cysA are under selective constraints, and whether rates of nucleotide substitution differ in other plastid genes from taxa with and without a functional copy of cysA. METHODS: Primers annealing to flanking and internal regions were used to amplify and sequence cysA from 61 liverworts. Two additional cysA sequences were downloaded from NCBI. The ancestral states of cysA were reconstructed on a phylogenetic hypothesis inferred from seven markers. Rates of nucleotide substitution were estimated for three plastid loci to reflect the intrinsic mutation rate in the plastid genome. The ratio of nonsynonymous to synonymous substitutions was estimated for intact copies of cysA to infer selective constraints. KEY RESULTS: Throughout liverworts, cysA has been lost up to 29 times, yet intact copies of cysA are evolving under selective constraints. Gene loss is more frequent in groups with an increased substitution rate in the plastid genome. CONCLUSIONS: The number of inferred losses of cysA in liverworts exceeds any other reported plastid gene. Despite frequent losses, cysA is evolving under purifying selection in liverworts that retain the gene. It appears that cysA is lost more frequently in lineages characterized by a higher rate of nucleotide substitutions in the plastid.

Barcode UK: A complete DNA barcoding resource for the flowering plants and conifers of the United Kingdom.

DNA barcoding and metabarcoding provide new avenues for investigating biological systems. These techniques require well-curated reference libraries with extensive coverage. Generating an exhaustive national DNA barcode reference library can open up new avenues of research in ecology, evolution and conservation, yet few studies to date have created such a resource. In plant DNA barcoding, herbarium collections provide taxonomically robust material but also pose challenges in lab processing. Here, we present a national DNA barcoding resource covering all of the native flowering plants and conifers of the United Kingdom. This represents 1,482 plant species, with the majority of specimens (81%) sourced from herbaria. Using Sanger sequencing of the plant DNA barcode markers, rbcL, matK, and ITS2, at least one DNA barcode was retrieved from 98% of the UK flora. We sampled from multiple individuals, resulting in a species coverage for rbcL of 96% (4,477 sequences), 90% for matK (3,259 sequences) and 75% for ITS2 (2,585 sequences). Sequence recovery was lower for herbarium material compared to fresh collections, with the age of the specimen having a significant effect on the success of sequence recovery. Species level discrimination was highest with ITS2, however, the ability to successfully retrieve a sequence was lowest for this region. Analyses of the genetic distinctiveness of species across a complete flora showed DNA barcoding to be informative for all but the most taxonomically complex groups. The UK flora DNA barcode reference library provides an important resource for many applications that require plant identification from DNA.

High rates of gene flow by pollen and seed in oak populations across Europe.

Gene flow is a key factor in the evolution of species, influencing effective population size, hybridisation and local adaptation. We analysed local gene flow in eight stands of white oak (mostly Quercus petraea and Q. robur, but also Q. pubescens and Q. faginea) distributed across Europe. Adult trees within a given area in each stand were exhaustively sampled (range [239, 754], mean 423), mapped, and acorns were collected ([17,147], 51) from several mother trees ([3], [47], 23). Seedlings ([65,387], 178) were harvested and geo-referenced in six of the eight stands. Genetic information was obtained from screening distinct molecular markers spread across the genome, genotyping each tree, acorn or seedling. All samples were thus genotyped at 5-8 nuclear microsatellite loci. Fathers/parents were assigned to acorns and seedlings using likelihood methods. Mating success of male and female parents, pollen and seed dispersal curves, and also hybridisation rates were estimated in each stand and compared on a continental scale. On average, the percentage of the wind-borne pollen from outside the stand was 60%, with large variation among stands (21-88%). Mean seed immigration into the stand was 40%, a high value for oaks that are generally considered to have limited seed dispersal. However, this estimate varied greatly among stands (20-66%). Gene flow was mostly intraspecific, with large variation, as some trees and stands showed particularly high rates of hybridisation. Our results show that mating success was unevenly distributed among trees. The high levels of gene flow suggest that geographically remote oak stands are unlikely to be genetically isolated, questioning the static definition of gene reserves and seed stands.

Plant DNA sequencing for phylogenetic analyses: from plants to sequences.

DNA sequences are important sources of data for phylogenetic analysis. Nowadays, DNA sequencing is a routine technique in molecular biology laboratories. However, there are specific questions associated with project design and sequencing of plant samples for phylogenetic analysis, which may not be familiar to researchers starting in the field. This chapter gives an overview of methods and protocols involved in the sequencing of plant samples, including general recommendations on the selection of species/taxa and DNA regions to be sequenced, and field collection of plant samples. Protocols of plant sample preparation, DNA extraction, PCR and cloning, which are critical to the success of molecular phylogenetic projects, are described in detail. Common problems of sequencing (using the Sanger method) are also addressed. Possible applications of second-generation sequencing techniques in plant phylogenetics are briefly discussed. Finally, orientation on the preparation of sequence data for phylogenetic analyses and submission to public databases is also given.

Microsatellite primers in the Peltigera dolichorhiza complex (lichenized ascomycete, Peltigerales).

PREMISE OF THE STUDY: Microsatellite primers were developed for the lichen-forming fungus Peltigera dolichorhiza to investigate partitioning of genetic variation in a widespread, morphologically and chemically variable taxon likely to represent a complex of cryptic lineages, including P. neopolydactyla. • METHODS AND RESULTS: Using next generation shotgun sequence reads, 331 primer pairs were designed to amplify microsatellite sequences from an African accession of P. dolichorhiza. Eleven primer pairs representing the longest repeat units identified were tested on 15 P. dolichorhiza accessions from Africa (incl. Réunion), South America, Papua New Guinea, and on two accessions of P. neopolydactyla from North America. The primers amplified di-, tri, tetra-, and pentanucelotide repeats with 3-8 alleles per locus. All individuals represent distinct multiloci genotypes. • CONCLUSIONS: These results indicate the utility of the new microsatellite primers for testing genetic differentiation within the widespread complex of P. dolichorhiza.

Selecting barcoding loci for plants: evaluation of seven candidate loci with species-level sampling in three divergent groups of land plants.

There has been considerable debate, but little consensus regarding locus choice for DNA barcoding land plants. This is partly attributable to a shortage of comparable data from all proposed candidate loci on a common set of samples. In this study, we evaluated the seven main candidate plastid regions (rpoC1, rpoB, rbcL, matK, trnH-psbA, atpF-atpH, psbK-psbI) in three divergent groups of land plants [Inga (angiosperm); Araucaria (gymnosperm); Asterella s.l. (liverwort)]. Across these groups, no single locus showed high levels of universality and resolvability. Interspecific sharing of sequences from individual loci was common. However, when multiple loci were combined, fewer barcodes were shared among species. Evaluation of the performance of previously published suggestions of particular multilocus barcode combinations showed broadly equivalent performance. Minor improvements on these were obtained by various new three-locus combinations involving rpoC1, rbcL, matK and trnH-psbA, but no single combination clearly outperformed all others. In terms of absolute discriminatory power, promising results occurred in liverworts (e.g. c. 90% species discrimination based on rbcL alone). However, Inga (rapid radiation) and Araucaria (slow rates of substitution) represent challenging groups for DNA barcoding, and their corresponding levels of species discrimination reflect this (upper estimate of species discrimination = 69% in Inga and only 32% in Araucaria; mean = 60% averaging all three groups).

Pleistocene and pre-Pleistocene Begonia speciation in Africa.

This paper presents a historical biogeographic analysis of African Begonia based on combined internal transcribed spacer (ITS) and trnL intron sequences. Age range estimates for Begonia in Africa ranged from only 1.5 Ma for some terminal nodes to 27 Ma for basal nodes when the ages of Réunion (2 Ma) andMayotte (5.4 Ma) were used to date the split between Begonia salaziensis and Begonia comorensis. Assuming a more recent origin age for Begonia salaziensis (2 Ma) provided age estimates in other parts of the phylogeny which agreed with patterns observed in other African organisms. A large proportion of the Begonia diversity seen today in Africa is of pre-Pleistocene origin. Species of Pleistocene origin are concentrated in species-rich groups such as sections Loasibegonia, Scutobegonia, and Tetraphila, which have their centre of diversity in western Central Africa. Phylogenetically isolated taxa such as Begonia longipetiolata, Begonia iucunda, and Begonia thomeana date to the late Miocene, a period of extended aridification on the African continent that had severe effects on African rain forest species. A general pattern is identified where phylogenetically isolated species occur outside the main identified rain forest refuges. Endemic species on the island of São Tomé such as Begonia baccata, Begonia molleri, and Begonia subalpestris appear to be palaeoendemics. Of these species, the most recent age estimate is for B. baccata, which is dated at ca. 3 Ma. Therefore, São Tomé appears to have functioned as an important (if previously unrecognised) pre-Pleistocene refuge. On the mainland, areas such as the Massif of Chaillu in Gabon, southern Congo (Brazzaville), and far western areas of Congo (Kinshasa) have played similar roles to São Tomé.

Phylogenetic position and biogeography of Hillebrandia sandwicensis (Begoniaceae): a rare Hawaiian relict.

The Begoniaceae consist of two genera, Begonia, with approximately 1400 species that are widely distributed in the tropics, and Hillebrandia, with one species that is endemic to the Hawaiian Islands and the only member of the family native to those islands. To help explain the history of Hillebrandia on the Hawaiian Archipelago, phylogenetic relationships of the Begoniaceae and the Cucurbitales were inferred using sequence data from 18S, rbcL, and ITS, and the minimal age of both Begonia and the Begoniaceae were indirectly estimated. The analyses strongly support the placement of Hillebrandia as the sister group to the rest of the Begoniaceae and indicate that the Hillebrandia lineage is at least 51-65 million years old, an age that predates the current Hawaiian Islands by about 20 million years. Evidence that Hillebrandia sandwicensis has survived on the Hawaiian Archipelago by island hopping from older, now denuded islands to younger, more mountainous islands is presented. Various scenarios for the origin of ancestor to Hillebrandia are considered. The geographic origin of source populations unfortunately remains obscure; however, we suggest a boreotropic or a Malesian-Pacific origin is most likely. Hillebrandia represents the first example in the well-studied Hawaiian flora of a relict genus.