A. Jonathan Shaw

Phylotranscriptomic analysis of the origin and early diversification of land plants

Significance Early branching events in the diversification of land plants and closely related algal lineages remain fundamental and unresolved questions in plant evolutionary biology. Accurate reconstructions of these relationships are critical for testing hypotheses of character evolution: for example, the origins of the embryo, vascular tissue, seeds, and flowers. We investigated relationships among streptophyte algae and land plants using the largest set of nuclear genes that has been applied to this problem to date. Hypothesized relationships were rigorously tested through a series of analyses to assess systematic errors in phylogenetic inference caused by sampling artifacts and model misspecification. Results support some generally accepted phylogenetic hypotheses, while rejecting others. This work provides a new framework for studies of land plant evolution. Reconstructing the origin and evolution of land plants and their algal relatives is a fundamental problem in plant phylogenetics, and is essential for understanding how critical adaptations arose, including the embryo, vascular tissue, seeds, and flowers. Despite advances in molecular systematics, some hypotheses of relationships remain weakly resolved. Inferring deep phylogenies with bouts of rapid diversification can be problematic; however, genome-scale data should significantly increase the number of informative characters for analyses. Recent phylogenomic reconstructions focused on the major divergences of plants have resulted in promising but inconsistent results. One limitation is sparse taxon sampling, likely resulting from the difficulty and cost of data generation. To address this limitation, transcriptome data for 92 streptophyte taxa were generated and analyzed along with 11 published plant genome sequences. Phylogenetic reconstructions were conducted using up to 852 nuclear genes and 1,701,170 aligned sites. Sixty-nine analyses were performed to test the robustness of phylogenetic inferences to permutations of the data matrix or to phylogenetic method, including supermatrix, supertree, and coalescent-based approaches, maximum-likelihood and Bayesian methods, partitioned and unpartitioned analyses, and amino acid versus DNA alignments. Among other results, we find robust support for a sister-group relationship between land plants and one group of streptophyte green algae, the Zygnematophyceae. Strong and robust support for a clade comprising liverworts and mosses is inconsistent with a widely accepted view of early land plant evolution, and suggests that phylogenetic hypotheses used to understand the evolution of fundamental plant traits should be reevaluated.

Bryophyte Biology: Morphology, anatomy, and classification of the Bryophyta

Introduction With approximately 13 000 species, the Bryophyta compose the second most diverse phylum of land plants. Mosses share with the Marchantiophyta and Anthocerotophyta a haplodiplobiontic life cycle that marks the shift from the haploid-dominated life cycle of the algal ancestors of embryophytes to the sporophyte-dominated life cycle of vascular plants. The gametophyte is free-living, autotrophic, and almost always composed of a leafy stem. Following fertilization a sporophyte develops into an unbranched axis bearing a terminal spore-bearing capsule. The sporophyte remains physically attached to the gametophyte and is at least partially physiologically dependent on the maternal plant. Recent phylogenetic reconstructions suggest that three lineages of early land plants compose an evolutionary grade that spans the transition to land and the origin of plants with branched sporophytes (see Chapter 4). The Bryophyta seem to occupy an intermediate position: their origin predates the divergence of the ancestor to the hornworts and vascular plants but evolved from a common ancestor with liverworts (Qiu et al . 2006). The origin of the earliest land plants can be traced back to the Ordovician and maybe the Cambrian (Strother et al . 2004). Although unambiguous fossils of mosses have only been recovered from sediments dating from younger geological periods (Upper Carboniferous), divergence time estimates based on molecular phylogenies suggest that the origin of mosses dates back to the Ordovician (Newton et al . 2007) and thus that their unique evolutionary history spans at least 400 million years.

PHYLOGEOGRAPHIC STRUCTURE AND CRYPTIC SPECIATION IN THE TRANS-ANTARCTIC MOSS PYRRHOBRYUM MNIOIDES

Abstract Many bryophyte species have distributions that span multiple continents. The hypotheses historically advanced to explain such distributions rely on either long-distance spore dispersal or slow rates of morphological evolution following ancient continental vicariance events. We use phylogenetic analyses of DNA sequence variation at three chloroplast loci (atpB-rbcL spacer, rps4 gene, and trnL intron and 3′ spacer) to examine these two hypotheses in the trans-Antarctic moss Pyrrhobryum mnioides. We find: (1) reciprocal monophyly of Australasian and South American populations, indicating a lack of intercontinental dispersal; (2) shared haplotypes between Australia and New Zealand, suggesting recent or ongoing migration across the Tasman Sea; and (3) reciprocal monophyly among Patagonian and neotropical populations, suggesting no recent migration along the Andes. These results corroborate experimental work suggesting that spore features may be critical determinants of species range. We use the mid-Miocene development of the Atacama Desert, 14 million years ago, to calibrate a molecular clock for the tree. The age of the trans-Antarctic disjunction is estimated to be 80 million years ago, consistent with Gondwanan vicariance, making it among the most ancient documented cases of cryptic speciation. These data are in accord with niche conservatism, but whether the morphological stasis is a product of stabilizing selection or phylogenetic constraint is unknown.

A Linkage Map Reveals a Complex Basis for Segregation Distortion in an Interpopulation Cross in the Moss Ceratodon purpureus

We report the construction of a linkage map for the moss Ceratodon purpureus (n = 13), based on a cross between geographically distant populations, and provide the first experimental confirmation of maternal chloroplast inheritance in bryophytes. From a mapping population of 288 recombinant haploid gametophytes, genotyped at 121 polymorphic AFLP loci, three gene-based nuclear loci, one chloroplast marker, and sex, we resolved 15 linkage groups resulting in a map length of ∼730 cM. We estimate that the map covers more than three-quarters of the C. purpureus genome. Approximately 35% of the loci were sex linked, not including those in recombining pseudoautosomal regions. Nearly 45% of the loci exhibited significant segregation distortion (α = 0.05). Several pairs of unlinked distorted loci showed significant deviations from multiplicative genotypic frequencies, suggesting that distortion arises from genetic interactions among loci. The distorted autosomal loci all exhibited an excess of the maternal allele, suggesting that these interactions may involve nuclear–cytoplasmic factors. The sex ratio of the progeny was significantly male biased, and the pattern of nonrandom associations among loci indicates that this results from interactions between the sex chromosomes. These results suggest that even in interpopulation crosses, multiple mechanisms act to influence segregation ratios.

Bryophyte phylogeny: Advancing the molecular and morphological frontiers

Abstract Revolutionary new concepts of bryophyte relationships have emerged from molecular phylogenetic analyses conducted since the onset of the 21st century. For example, sequence data contradict the historical notion that isophylly in leafy liverworts is plesiomorphic and that simple thalloid liverworts are monophyletic. Also contrary to traditional views are the concepts that Leiosporoceros is genetically distinct from other hornworts and that Oedipodium is sister to the peristomate mosses. Substantial increases in ultrastructural and anatomical data likewise have provided new insights on interrelationships. Because of this recent deluge in evolutionary studies on bryophytes, it is an opportune time to co-examine contemporary morphological knowledge and novel molecular hypotheses. An understanding of bryophyte evolution and biology is essential to identify structural innovations that accompanied early land colonization and to illuminate the evolution of more complicated body plans in tracheophytes. In this review, we examine the progress that has been made since the 1999 International Botanical Congress in clarifying the evolutionary history of the three groups of bryophytes. The state of our knowledge on interrelationships is discussed, with poorly-known, genetically divergent taxa illustrated for each group. Our review of bryophyte evolution includes a reëvaluation of the evolution of sperm cells, sporogenesis, stomata, symbioses, conducting cells and chloroplast ultrastructure in hornworts. We explore the prospects for future discoveries and advances with an emphasis on fundamental evolutionary problems that remain and the challenges that must be met to resolve them.

ENDOPHYTIC XYLARIA (XYLARIACEAE) AMONG LIVERWORTS AND ANGIOSPERMS: PHYLOGENETICS, DISTRIBUTION, AND SYMBIOSIS'

Nuclear ribosomal 18S and internal transcribed spacer (ITS) sequence data were used to identify endophytic fungi cultured from six species of liverworts collected in Jamaica and North Carolina. Comparisons with other published fungal sequences and phylogenetic analyses yielded the following conclusions: (1) the endophytes belong to the ascomycete families Xylariaceae, Hypocreaceae, and Ophiostomataceae, and (2) liverwort endophytes in the genus Xylaria are closely related to each other and to endophytes isolated from angiosperms in China, Puerto Rico, and Europe. Liverwort endophytes are expected to be foragers or endophytic specialists, although little is known about the role of these fungi in symbioses. Features that may indicate a mutualistic role for these endophytes are discussed.

Phylogeny of the Sphagnopsida Based on Chloroplast and Nuclear DNA Sequences

Abstract Most reconstructions of basal land plant relationships derived from morphological or molecular data suggest that the Sphagnopsida form a critical clade at or near the base of the mosses (Bryophyta s.s.). The Sphagnopsida include two orders: Sphagnales and Ambuchananiales, each with one family. The Ambuchananiaceae is monotypic, with A. leucobryoides of Tasmania. Nucleotide sequences from five genomic regions, two from the nuclear genome (ITS and 26S nuclear ribosomal DNA) and three from the chloroplast genome (psbT, rpl16, trnL) were subjected to cladistic analyses in order to assess 1) the relationship between Ambuchanania and Sphagnum, 2) the polarity of evolutionary change in Sphagnum (i.e., infer a root for the infrageneric phylogeny), 3) monophyly of the four large sections of Sphagnum (Acutifolia, Cuspidata, Sphagnum, and Subsecunda) and 4) phylogenetic relationships of the smaller or monotypic sections. Ambuchanania is resolved as the sister group to Sphagnum and is not nested within the latter as a highly derived species. Polarity of evolutionary change in Sphagnum is ambiguous; alternative hypotheses suggested by molecular data place either the sect. Subsecunda or the sect. Sphagnum as sister to all other species. The four large sections of Sphagnum are each monophyletic if circumscribed to include species traditionally placed in monotypic sections. Sphagnum macrophyllum (sect. Isocladus), is nested within the Subsecunda. Sphagnum pylaesii (sect. Hemitheca), is nested within the Cuspidata and is closely related to S. tenellum (sect. Mollusca). Sphagnum wulfianum (sect. Polyclada), is nested within the Acutifolia, closely related to S. fimbriatum and S. girgensohnii. Sphagnum aongstroemii (sect. Insulosa) is either nested within the Acutifolia, or is sister to other species of Acutifolia. Molecular evidence supports a sister group relationship between the sections Rigida and Sphagnum, and between the sections Squarrosa and Acutifolia. Molecular data suggest that phylogenetic structure in Sphagnum can be accommodated by four large sections without segregating morphologically distinctive taxa into smaller sections, as is traditionally done. A revised classification is proposed in which the genus is divided into four sections: Acutifolia, Cuspidata, Sphagnum, and Subsecunda.

Polarity of peatmoss (Sphagnum) evolution: who says bryophytes have no roots?

The class Sphagnopsida (Bryophyta) includes two genera: Ambuchanania and Sphagnum. Ambuchanania contains just one rare species known from two Tasmanian localities, but Sphagnum comprises a speciose clade of mosses that dominates many wetland ecosystems, especially in the boreal zone of the Northern Hemisphere. Recent phylogenetic analyses have resolved well-supported clades within Sphagnum, but polarizing Sphagnum evolution has been problematic because the genus is so isolated that it is difficult to determine homologies between morphological and/or molecular traits within Sphagnum with those of any potential outgroup. DNA sequences from 16 genomic regions representing the mitochondrial, chloroplast, and nuclear genomes (ca. 16 kilobases) were obtained from 24 species of Sphagnum plus one species each from Takakia and Andreaea in order to resolve a rooted phylogeny. Two tropical species, S. sericeum and S. lapazense, were resolved as sister to the rest of the genus and are extremely divergent from all other sphagna. The main Sphagnum lineage consists of two clades; one includes the sections Sphagnum, Rigida, and Cuspidata, and the other includes Subsecunda, Acutifolia, and Squarrosa. The placement of section Subsecunda is weakly supported, but other nodes are strongly supported by maximum parsimony, maximum likelihood, and Bayesian analyses. In addition to homogeneous Bayesian analyses, heterogeneous models were employed to account for different patterns of nucleotide substitution among genomic regions.

Phylogenetic Relationships among the Mosses Based on Heterogeneous Bayesian Analysis of Multiple Genes from Multiple Genomic Compartments

Abstract Nucleotide sequences from eight nuclear, chloroplast, and mitochondrial genes were obtained from 30 mosses (plus four outgroup liverworts) in order to resolve phylogenetic relationships among the major clades of division Bryophyta. Phylogenetic analyses were conducted using maximum parsimony, maximum likelihood (ML), and Bayesian inference. Inferences were compared from Bayesian analyses using homogeneous and several heterogeneous models. Estimates of clade confidence were based on bootstrap analyses, posterior probabilities (in Bayesian analyses) and novel combined approaches. Most ingroup relationships were congruent among analyses, but support for individual clades depended on the analytical approach. Increasingly parameterized models of nucleotide substitution in the likelihood analyses provided significantly higher goodness-of-fit to the data. The results suggest that 1) the Bryophyta, including Sphagnum and Takakia, are monophyletic, 2) Andreaea and Andreaeobryum form a monophyletic group, 3) Oedipodium griffithianum is sister to all other operculate taxa, 4) mosses with nematodontous peristomes are paraphyletic and basal to arthrodontous mosses, 5) Diphyscium is sister to all other arthrodontous mosses, 6) Encalypta is sister to the Funariaceae, and 6) mosses with diplolepideous-alternate peristomes form a monophyletic group. Implications of the phylogenetic hypothesis for morphological evolution in mosses include 1) a pseudopodium has arisen independently in Sphagnum and Andreaea, 2) the mucilage hairs of Andreaeobryum and Takakia are non-homologous, 3) the stomata found in Sphagnum are not homologous to those of other mosses, and 4) that stomata were absent in the ancestor of all mosses.

Horizontal transfer of an adaptive chimeric photoreceptor from bryophytes to ferns

Significance Despite being one of the oldest groups of land plants, the majority of living ferns resulted from a relatively recent diversification following the rise of angiosperms. To exploit fully the new habitats created by angiosperm-dominated ecosystems, ferns had to evolve novel adaptive strategies to cope with the low-light conditions exerted by the angiosperm canopy. Neochrome, an unconventional photoreceptor that allows ferns to “see the light” better, was likely part of the solution. Surprisingly, we discovered that fern neochrome was derived from a bryophyte lineage via horizontal gene transfer (HGT). This finding not only provides the first evidence that a plant-to-plant HGT can have a profound evolutionary impact but also has implications for the evolution of photosensory systems in plants. Ferns are well known for their shade-dwelling habits. Their ability to thrive under low-light conditions has been linked to the evolution of a novel chimeric photoreceptor—neochrome—that fuses red-sensing phytochrome and blue-sensing phototropin modules into a single gene, thereby optimizing phototropic responses. Despite being implicated in facilitating the diversification of modern ferns, the origin of neochrome has remained a mystery. We present evidence for neochrome in hornworts (a bryophyte lineage) and demonstrate that ferns acquired neochrome from hornworts via horizontal gene transfer (HGT). Fern neochromes are nested within hornwort neochromes in our large-scale phylogenetic reconstructions of phototropin and phytochrome gene families. Divergence date estimates further support the HGT hypothesis, with fern and hornwort neochromes diverging 179 Mya, long after the split between the two plant lineages (at least 400 Mya). By analyzing the draft genome of the hornwort Anthoceros punctatus, we also discovered a previously unidentified phototropin gene that likely represents the ancestral lineage of the neochrome phototropin module. Thus, a neochrome originating in hornworts was transferred horizontally to ferns, where it may have played a significant role in the diversification of modern ferns.