Laura L. Forrest

A DNA barcode for land plants

DNA barcoding involves sequencing a standard region of DNA as a tool for species identification. However, there has been no agreement on which region(s) should be used for barcoding land plants. To provide a community recommendation on a standard plant barcode, we have compared the performance of 7 leading candidate plastid DNA regions (atpF–atpH spacer, matK gene, rbcL gene, rpoB gene, rpoC1 gene, psbK–psbI spacer, and trnH–psbA spacer). Based on assessments of recoverability, sequence quality, and levels of species discrimination, we recommend the 2-locus combination of rbcL+matK as the plant barcode. This core 2-locus barcode will provide a universal framework for the routine use of DNA sequence data to identify specimens and contribute toward the discovery of overlooked species of land plants.

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).

Unraveling the evolutionary history of the liverworts (Marchantiophyta): multiple taxa, genomes and analyses

Abstract Nucleotide sequence data from three chloroplast genes (rbcL, rps4 and psbA), one nuclear gene (the ribosomal LSU) and one mitochondrial gene (nad5) were assembled for 173 species in 117 genera of liverworts, making this the largest molecular phylogeny of the group to date. Analyses of these data provide support for the monophyly of the liverworts, and for previously resolved backbone relationships within the Marchantiophyta. The earliest divergence involves the “simple thalloid” taxa of the Haplomitriaceae and Treubiaceae. A Blasiaceae/complex thalloid clade is resolved as sister to all remaining liverworts. The leafy liverworts do not resolve as monophyletic. The separation of the Aneuraceae/Metzgeriaceae from all other simple thalloids and their placement within the “leafy” clade as sister to the enigmatic leafy genus Pleurozia, as suggested in earlier molecular phylogenies, is also supported by this far larger data set.

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.

Extant diversity of bryophytes emerged from successive post-Mesozoic diversification bursts

Unraveling the macroevolutionary history of bryophytes, which arose soon after the origin of land plants but exhibit substantially lower species richness than the more recently derived angiosperms, has been challenged by the scarce fossil record. Here we demonstrate that overall estimates of net species diversification are approximately half those reported in ferns and ∼30% those described for angiosperms. Nevertheless, statistical rate analyses on time-calibrated large-scale phylogenies reveal that mosses and liverworts underwent bursts of diversification since the mid-Mesozoic. The diversification rates further increase in specific lineages towards the Cenozoic to reach, in the most recently derived lineages, values that are comparable to those reported in angiosperms. This suggests that low diversification rates do not fully account for current patterns of bryophyte species richness, and we hypothesize that, as in gymnosperms, the low extant bryophyte species richness also results from massive extinctions.

A recircumscription of Begonia based on nuclear ribosomal sequences

Abstract.With c. 1400 known species, Begonia is one of the largest plant genera. In order to address the evolution of Begonia we have produced maximum parsimony and maximum likelihood cladograms for 26S and ITS sequence data. Sequences were obtained from a total of 35 species of Begonia, one species of Symbegonia and two species of Datisca. The resulting phylogenetic hypotheses suggest that the most basal members of Begonia are from Africa, with American and Asian clades nested within the paraphyletic African Begonia. Despite marked morphological heterogeneity the endemic Begonia of Madagascar and the south Indian ocean islands form a monophyletic group. As currently circumscribed, Begonia is paraphyletic with the New Guinean endemic Symbegonia nested deeply within it and most closely related to species from the Asian section Petermannia. Analysis of a smaller ITS dataset, including three accessions of Symbegonia and nine accessions of Begonia section Petermannia, further suggests that Symbegonia is nested within section Petermannia, resolving within a monophyletic clade of New Guinean species. Morphological synapomorphies of Symbegonia are reviewed and this taxon is sunk into the genus Begonia, where it is given sectional status.

A Phylogeny of Begonia Using Nuclear Ribosomal Sequence Data and Morphological Characters

Abstract With ca. 1400 species, Begonia is one of the largest plant genera. To address the evolution of Begonia we have produced maximum parsimony cladograms for nuclear large subunit and internal transcribed spacer sequence data in combination with 34 informative morphological characters. Data were obtained from 64 species of Begonia, the monotypic genus Hillebrandia, and both species of Datisca. The resulting phylogenetic hypotheses supported three main clades within Begonia, two internally resolved clades of African plants, and one unresolved transcontinental clade containing species from southern Africa, America, Asia, and the Socotran archipelago. Morphological characters often support well-resolved molecular clades. Tepal number in the staminate and carpellate flowers and fruit characters are discussed. None of the morphological characters sampled, including traditionally emphasized characters in sectional delimitations such as locule number and number of placental branches, provides a basis for subdividing Begonia into easily circumscribed monophyletic groups. Implications for future sectional classifications of Begonia are discussed.

Lichen-symbiotic cyanobacteria associated with Peltigera have an alternative vanadium-dependent nitrogen fixation system

In past decades, environmental nitrogen fixation has been attributed almost exclusively to the action of enzymes in the well-studied molybdenum-dependent nitrogen fixation system. However, recent evidence has shown that nitrogen fixation by alternative pathways may be more frequent than previously suspected. In this study, the nitrogen fixation systems employed by lichen-symbiotic cyanobacteria were examined to determine whether their diazotrophy can be attributed, in part, to an alternative pathway. The mining of metagenomic data (generated through pyrosequencing) and PCR assays were used to determine which nitrogen-fixation systems are present in cyanobacteria from the genus Nostoc associated with four samples from different geographical regions, representing different lichen-forming fungal species in the genus Peltigera. A metatranscriptomic sequence library from an additional specimen was examined to determine which genes associated with N2 fixation are transcriptionally expressed. Results indicated that both the standard molybdenum-dependent system and an alternative vanadium-dependent system are present and actively transcribed in the lichen symbiosis. This study shows for the first time that an alternative system is utilized by cyanobacteria associated with fungi. The ability of lichen-associated cyanobacteria to switch between pathways could allow them to colonize a wider array of environments, including habitats characterized by low temperature and trace metal (e.g. molybdenum) availability. We discuss the implications of these findings for environmental studies that incorporate acetylene-reduction assay data.

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

UNLABELLED 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.

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.