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.

Balanophora genomes display massively convergent evolution with other extreme holoparasites and provide novel insights into parasite-host interactions.

Parasitic plants have evolved to be subtly or severely dependent on host plants to complete their life cycle. To provide new insights into the biology of parasitic plants in general, we assembled genomes for members of the sandalwood order Santalales, including a stem hemiparasite (Scurrula) and two highly modified root holoparasites (Balanophora) that possess chimaeric host-parasite tubers. Comprehensive genome comparisons reveal that hemiparasitic Scurrula has experienced a relatively minor degree of gene loss compared with autotrophic plants, consistent with its moderate degree of parasitism. Nonetheless, patterns of gene loss appear to be substantially divergent across distantly related lineages of hemiparasites. In contrast, Balanophora has experienced substantial gene loss for the same sets of genes as an independently evolved holoparasite lineage, the endoparasitic Sapria (Malpighiales), and the two holoparasite lineages experienced convergent contraction of large gene families through loss of paralogues. This unprecedented convergence supports the idea that despite their extreme and strikingly divergent life histories and morphology, the evolution of these and other holoparasitic lineages can be shaped by highly predictable modes of genome reduction. We observe substantial evidence of relaxed selection in retained genes for both hemi- and holoparasitic species. Transcriptome data also document unusual and novel interactions between Balanophora and host plants at the host-parasite tuber interface tissues, with evidence of mRNA exchange, substantial and active hormone exchange and immune responses in parasite and host.

Plastid phylogenomics and molecular evolution of Thismiaceae (Dioscoreales).

PREMISE: Species in Thismiaceae can no longer photosynthesize and instead obtain carbon from soil fungi. Here we infer Thismiaceae phylogeny using plastid genome data and characterize the molecular evolution of this genome. METHODS: We assembled five Thismiaceae plastid genomes from genome skimming data, adding to previously published data for phylogenomic inference. We investigated plastid-genome structural changes, considering locally colinear blocks (LCBs). We also characterized possible shifts in selection pressure in retained genes by considering changes in the ratio of nonsynonymous to synonymous changes (ω). RESULTS: Thismiaceae experienced two major pulses of gene loss around the early diversification of the family, with subsequent scattered gene losses across descendent lineages. In addition to massive size reduction, Thismiaceae plastid genomes experienced occasional inversions, and there were likely two independent losses of the plastid inverted repeat (IR) region. Retained plastid genes remain under generally strong purifying selection (ω << 1), with significant and sporadic weakening or strengthening in several instances. The bifunctional trnE-UUC gene of Thismia huangii may retain a secondary role in heme biosynthesis, despite a probable loss of functionality in protein translation. Several cis-spliced group IIA introns have been retained, despite the loss of the plastid intron maturase, matK. CONCLUSIONS: We infer that most gene losses in Thismiaceae occurred early and rapidly, following the initial loss of photosynthesis in its stem lineage. As a species-rich, fully mycoheterotrophic lineage, Thismiaceae provide a model system for uncovering the unique and divergent ways in which plastid genomes evolve in heterotrophic plants.

Phylotranscriptomic Analyses of Mycoheterotrophic Monocots Show a Continuum of Convergent Evolutionary Changes in Expressed Nuclear Genes From Three Independent Nonphotosynthetic Lineages.

Mycoheterotrophy is an alternative nutritional strategy whereby plants obtain sugars and other nutrients from soil fungi. Mycoheterotrophy and associated loss of photosynthesis have evolved repeatedly in plants, particularly in monocots. Although reductive evolution of plastomes in mycoheterotrophs is well documented, the dynamics of nuclear genome evolution remains largely unknown. Transcriptome datasets were generated from four mycoheterotrophs in three families (Orchidaceae, Burmanniaceae, Triuridaceae) and related green plants and used for phylogenomic analyses to resolve relationships among the mycoheterotrophs, their relatives, and representatives across the monocots. Phylogenetic trees based on 602 genes were mostly congruent with plastome phylogenies, except for an Asparagales + Liliales clade inferred in the nuclear trees. Reduction and loss of chlorophyll synthesis and photosynthetic gene expression and relaxation of purifying selection on retained genes were progressive, with greater loss in older nonphotosynthetic lineages. One hundred seventy-four of 1375 plant benchmark universally conserved orthologous genes were undetected in any mycoheterotroph transcriptome or the genome of the mycoheterotrophic orchid Gastrodia but were expressed in green relatives, providing evidence for massively convergent gene loss in nonphotosynthetic lineages. We designate this set of deleted or undetected genes Missing in Mycoheterotrophs (MIM). MIM genes encode not only mainly photosynthetic or plastid membrane proteins but also a diverse set of plastid processes, genes of unknown function, mitochondrial, and cellular processes. Transcription of a photosystem II gene (psb29) in all lineages implies a nonphotosynthetic function for this and other genes retained in mycoheterotrophs. Nonphotosynthetic plants enable novel insights into gene function as well as gene expression shifts, gene loss, and convergence in nuclear genomes.

Phylogenomic resolution of order- and family-level monocot relationships using 602 single-copy nuclear genes and 1375 BUSCO genes.

We assess relationships among 192 species in all 12 monocot orders and 72 of 77 families, using 602 conserved single-copy (CSC) genes and 1375 benchmarking single-copy ortholog (BUSCO) genes extracted from genomic and transcriptomic datasets. Phylogenomic inferences based on these data, using both coalescent-based and supermatrix analyses, are largely congruent with the most comprehensive plastome-based analysis, and nuclear-gene phylogenomic analyses with less comprehensive taxon sampling. The strongest discordance between the plastome and nuclear gene analyses is the monophyly of a clade comprising Asparagales and Liliales in our nuclear gene analyses, versus the placement of Asparagales and Liliales as successive sister clades to the commelinids in the plastome tree. Within orders, around six of 72 families shifted positions relative to the recent plastome analysis, but four of these involve poorly supported inferred relationships in the plastome-based tree. In Poales, the nuclear data place a clade comprising Ecdeiocoleaceae+Joinvilleaceae as sister to the grasses (Poaceae); Typhaceae, (rather than Bromeliaceae) are resolved as sister to all other Poales. In Commelinales, nuclear data place Philydraceae sister to all other families rather than to a clade comprising Haemodoraceae+Pontederiaceae as seen in the plastome tree. In Liliales, nuclear data place Liliaceae sister to Smilacaceae, and Melanthiaceae are placed sister to all other Liliales except Campynemataceae. Finally, in Alismatales, nuclear data strongly place Tofieldiaceae, rather than Araceae, as sister to all the other families, providing an alternative resolution of what has been the most problematic node to resolve using plastid data, outside of those involving achlorophyllous mycoheterotrophs. As seen in numerous prior studies, the placement of orders Acorales and Alismatales as successive sister lineages to all other extant monocots. Only 21.2% of BUSCO genes were demonstrably single-copy, yet phylogenomic inferences based on BUSCO and CSC genes did not differ, and overall functional annotations of the two sets were very similar. Our analyses also reveal significant gene tree-species tree discordance despite high support values, as expected given incomplete lineage sorting (ILS) related to rapid diversification. Our study advances understanding of monocot relationships and the robustness of phylogenetic inferences based on large numbers of nuclear single-copy genes that can be obtained from transcriptomes and genomes.

Phylogenomics and plastome evolution of a Brazilian mycoheterotrophic orchid, Pogoniopsis schenckii.

PREMISE: Pogoniopsis likely represents an independent photosynthesis loss in orchids. We use phylogenomic data to better identify the phylogenetic placement of this fully mycoheterotrophic taxon, and investigate its molecular evolution. METHODS: We performed likelihood analysis of plastid and mitochondrial phylogenomic data to localize the position of Pogoniopsis schenckii in orchid phylogeny, and investigated the evolution of its plastid genome. RESULTS: All analyses place Pogoniopsis in subfamily Epidendroideae, with strongest support from mitochondrial data, which also place it near tribe Sobralieae with moderately strong support. Extreme rate elevation in Pogoniopsis plastid genes broadly depresses branch support; in contrast, mitochondrial genes are only mildly rate elevated and display very modest and localized reductions in bootstrap support. Despite considerable genome reduction, including loss of photosynthesis genes and multiple translation apparatus genes, gene order in Pogoniopsis plastomes is identical to related autotrophs, apart from moderately shifted inverted repeat (IR) boundaries. All cis-spliced introns have been lost in retained genes. Two plastid genes (accD, rpl2) show significant strengthening of purifying selection. A retained plastid tRNA gene (trnE-UUC) of Pogoniopsis lacks an anticodon; we predict that it no longer functions in translation but retains a secondary role in heme biosynthesis. CONCLUSIONS: Slowly evolving mitochondrial genes clarify the placement of Pogoniopsis in orchid phylogeny, a strong contrast with analysis of rate-elevated plastome data. We documented the effects of the novel loss of photosynthesis: for example, despite massive gene loss, its plastome is fully colinear with other orchids, and it displays only moderate shifts in selective pressure in retained genes.

Mitochondrial genomic data are effective at placing mycoheterotrophic lineages in plant phylogeny.

Fully mycoheterotrophic plants can be difficult to place in plant phylogeny due to elevated substitution rates associated with photosynthesis loss. This potentially limits the effectiveness of downstream analyses of mycoheterotrophy that depend on accurate phylogenetic inference. Although mitochondrial genomic data sets are rarely used in plant phylogenetics, theory predicts that they should be resilient to long-branch artefacts, thanks to their generally slow evolution, coupled with limited rate elevation in heterotrophs. We examined the utility of mitochondrial genomes for resolving contentious higher-order placements of mycoheterotrophic lineages in two test cases: monocots (focusing on Dioscoreales) and Ericaceae. We find Thismiaceae to be distantly related to Burmanniaceae in the monocot order Dioscoreales, conflicting with current classification schemes based on few gene data sets. We confirm that the unusual Afrothismia is related to Taccaceae-Thismiaceae, with a corresponding independent loss of photosynthesis. In Ericaceae we recovered the first well supported relationships among its five major lineages: mycoheterotrophic Ericaceae are not monophyletic, as pyroloids are inferred to be sister to core Ericaceae, and monotropoids to arbutoids. Genes recovered from mitochondrial genomes collectively resolved previously ambiguous mycoheterotroph higher-order relationships. We propose that mitochondrial genomic data should be considered in standardised gene panels for inferring overall plant phylogeny.

Incomplete lineage sorting and local extinction shaped the complex evolutionary history of the Paleogene relict conifer genus, Chamaecyparis (Cupressaceae).

Inferring accurate biogeographic history of plant taxa with an East Asia (EA)-North America (NA) is usually hindered by conflicting phylogenies and a poor fossil record. The current distribution of Chamaecyparis (false cypress; Cupressaceae) with four species in EA, and one each in western and eastern NA, and its relatively rich fossil record, make it an excellent model for studying the EA-NA disjunction. Here we reconstruct phylogenomic relationships within Chamaecyparis using > 1400 homologous nuclear and 61 plastid genes. Our phylogenomic analyses using concatenated and coalescent approaches revealed strong cytonuclear discordance and conflicting topologies between nuclear gene trees. Incomplete lineage sorting (ILS) and hybridization are possible explanations of conflict; however, our coalescent analyses and simulations suggest that ILS is the major contributor to the observed phylogenetic discrepancies. Based on a well-resolved species tree and four fossil calibrations, the crown lineage of Chamaecyparis is estimated to have originated in the upper Cretaceous, followed by diversification events in the early and middle Paleogene. Ancestral area reconstructions suggest that Chamaecyparis had an ancestral range spanning both EA and NA. Fossil records further indicate that this genus is a relict of the "boreotropical" flora, and that local extinctions of European species were caused by global cooling. Overall, our results unravel a complex evolutionary history of a Paleogene relict conifer genus, which may have involved ILS, hybridization and the extinction of local species.

Macroevolutionary dynamics in the transition of angiosperms to aquatic environments.

Angiosperm lineages in aquatic environments are characterized by high structural and functional diversity, and wide distributions. A long-standing evolutionary riddle is what processes have caused the relatively low diversity of aquatic angiosperms compared to their terrestrial relatives. We use diversification and ancestral reconstruction models with a comprehensive > 10 000 genus angiosperm phylogeny to elucidate the macroevolutionary dynamics associated with transitions of terrestrial plants to water. Our study reveals that net diversification rates are significantly lower in aquatic than in terrestrial angiosperms due to lower speciation and higher extinction. Shifts from land to water started early in angiosperm evolution, but most events were concentrated during the last c. 25 million years. Reversals to a terrestrial habitat started only 40 million years ago, but occurred at much higher rates. Within aquatic angiosperms, the estimated pattern is one of gradual accumulation of lineages, and relatively low and constant diversification rates throughout the Cenozoic. Low diversification rates, together with infrequent water transitions, account for the low diversity of aquatic angiosperms today. The stressful conditions and small global surface of the aquatic habitat available for angiosperms are hypothesized to explain this pattern.

A new carnivorous plant lineage (Triantha) with a unique sticky-inflorescence trap.

Carnivorous plants consume animals for mineral nutrients that enhance growth and reproduction in nutrient-poor environments. Here, we report that Triantha occidentalis (Tofieldiaceae) represents a previously overlooked carnivorous lineage that captures insects on sticky inflorescences. Field experiments, isotopic data, and mixing models demonstrate significant N transfer from prey to Triantha, with an estimated 64% of leaf N obtained from prey capture in previous years, comparable to levels inferred for the cooccurring round-leaved sundew, a recognized carnivore. N obtained via carnivory is exported from the inflorescence and developing fruits and may ultimately be transferred to next year's leaves. Glandular hairs on flowering stems secrete phosphatase, as seen in all carnivorous plants that directly digest prey. Triantha is unique among carnivorous plants in capturing prey solely with sticky traps adjacent to its flowers, contrary to theory. However, its glandular hairs capture only small insects, unlike the large bees and butterflies that act as pollinators, which may minimize the conflict between carnivory and pollination.

Genomic Rearrangements and Sequence Evolution across Brown Algal Organelles.

Organellar genomes serve as useful models for genome evolution and contain some of the most widely used phylogenetic markers, but they are poorly characterized in many lineages. Here, we report 20 novel mitochondrial genomes and 16 novel plastid genomes from the brown algae. We focused our efforts on the orders Chordales and Laminariales but also provide the first plastid genomes (plastomes) from Desmarestiales and Sphacelariales, the first mitochondrial genome (mitome) from Ralfsiales and a nearly complete mitome from Sphacelariales. We then compared gene content, sequence evolution rates, shifts in genome structural arrangements, and intron distributions across lineages. We confirm that gene content is largely conserved in both organellar genomes across the brown algal tree of life, with few cases of gene gain or loss. We further show that substitution rates are generally lower in plastid than mitochondrial genes, but plastomes are more variable in gene arrangement, as mitomes tend to be colinear even among distantly related lineages (with exceptions). Patterns of intron distribution across organellar genomes are complex. In particular, the mitomes of several laminarialean species possess group II introns that have T7-like ORFs, found previously only in mitochondrial genomes of Pylaiella spp. (Ectocarpales). The distribution of these mitochondrial introns is inconsistent with vertical transmission and likely reflects invasion by horizontal gene transfer between lineages. In the most extreme case, the mitome of Hedophyllum nigripes is ∼40% larger than the mitomes of close relatives because of these introns. Our results provide substantial insight into organellar evolution across the brown algae.

Discordant Phylogenomic Placement of Hydnoraceae and Lactoridaceae Within Piperales Using Data From All Three Genomes.

Phylogenetic relationships within the magnoliid order Piperales have been studied extensively, yet the relationships of the monotypic family Lactoridaceae and the holoparasitic Hydnoraceae to the remainder of the order remain a matter of debate. Since the first confident molecular phylogenetic placement of Hydnoraceae among Piperales, different studies have recovered various contradictory topologies. Most phylogenetic hypotheses were inferred using only a few loci and have had incomplete taxon sampling at the genus level. Based on these results and an online survey of taxonomic opinion, the Angiosperm Phylogeny Group lumped both Hydnoraceae and Lactoridaceae in Aristolochiaceae; however, the latter family continues to have unclear relationships to the aforementioned taxa. Here we present extensive phylogenomic tree reconstructions based on up to 137 loci from all three subcellular genomes for all genera of Piperales. We infer relationships based on a variety of phylogenetic methods, explore instances of phylogenomic discordance between the subcellular genomes, and test alternative topologies. Consistent with these phylogenomic results and a consideration of the principles of phylogenetic classification, we propose to exclude Hydnoraceae and Lactoridaceae from the broad circumscription of Aristolochiaceae, and instead favor recognition of four monophyletic and morphologically well circumscribed families in the perianth-bearing Piperales: Aristolochiaceae, Asaraceae, Hydnoraceae, and Lactoridaceae, with a total of six families in the order.

A bi-organellar phylogenomic study of Pandanales: inference of higher-order relationships and unusual rate-variation patterns.

We used a bi-organellar phylogenomic approach to address higher-order relationships in Pandanales, including the first molecular phylogenetic study of the panama-hat family, Cyclanthaceae. Our genus-level study of plastid and mitochondrial gene sets includes a comprehensive sampling of photosynthetic lineages across the order, and provides a framework for investigating clade ages, biogeographic hypotheses and organellar molecular evolution. Using multiple inference methods and both organellar genomes, we recovered mostly congruent and strongly supported relationships within and between families, including the placement of fully mycoheterotrophic Triuridaceae. Cyclanthaceae and Pandanaceae plastomes have slow substitution rates, contributing to weakly supported plastid-based relationships in Cyclanthaceae. While generally slowly evolving, mitochondrial genomes exhibit sporadic rate elevation across the order. However, we infer well-supported relationships even for slower evolving mitochondrial lineages in Cyclanthaceae. Clade age estimates across photosynthetic lineages are largely consistent with previous studies, are well correlated between the two organellar genomes (with slightly younger inferences from mitochondrial data), and support several biogeographic hypotheses. We show that rapidly evolving non-photosynthetic lineages may bias age estimates upwards at neighbouring photosynthetic nodes, even using a relaxed clock model. Finally, we uncovered new genome structural variants in photosynthetic taxa at plastid inverted repeat boundaries that show promise as interfamilial phylogenetic markers.

Organellomic data sets confirm a cryptic consensus on (unrooted) land-plant relationships and provide new insights into bryophyte molecular evolution.

PREMISE: Phylogenetic trees of bryophytes provide important evolutionary context for land plants. However, published inferences of overall embryophyte relationships vary considerably. We performed phylogenomic analyses of bryophytes and relatives using both mitochondrial and plastid gene sets, and investigated bryophyte plastome evolution. METHODS: We employed diverse likelihood-based analyses to infer large-scale bryophyte phylogeny for mitochondrial and plastid data sets. We tested for changes in purifying selection in plastid genes of a mycoheterotrophic liverwort (Aneura mirabilis) and a putatively mycoheterotrophic moss (Buxbaumia), and compared 15 bryophyte plastomes for major structural rearrangements. RESULTS: Overall land-plant relationships conflict across analyses, generally weakly. However, an underlying (unrooted) four-taxon tree is consistent across most analyses and published studies. Despite gene coverage patchiness, relationships within mosses, liverworts, and hornworts are largely congruent with previous studies, with plastid results generally better supported. Exclusion of RNA edit sites restores cases of unexpected non-monophyly to monophyly for Takakia and two hornwort genera. Relaxed purifying selection affects multiple plastid genes in mycoheterotrophic Aneura but not Buxbaumia. Plastid genome structure is nearly invariant across bryophytes, but the tufA locus, presumed lost in embryophytes, is unexpectedly retained in several mosses. CONCLUSIONS: A common unrooted tree underlies embryophyte phylogeny, [(liverworts, mosses), (hornworts, vascular plants)]; rooting inconsistency across studies likely reflects substantial distance to algal outgroups. Analyses combining genomic and transcriptomic data may be misled locally for heavily RNA-edited taxa. The Buxbaumia plastome lacks hallmarks of relaxed selection found in mycoheterotrophic Aneura. Autotrophic bryophyte plastomes, including Buxbaumia, hardly vary in overall structure.

Sequencing and Analyzing the Transcriptomes of a Thousand Species Across the Tree of Life for Green Plants.

The 1,000 Plants (1KP) initiative was the first large-scale effort to collect next-generation sequencing (NGS) data across a phylogenetically representative sampling of species for a major clade of life, in this case theViridiplantae, or green plants. As an international multidisciplinary consortium, we focused on plant evolution and its practical implications. Among the major outcomes were the inference of a reference species tree for green plants by phylotranscriptomic analysis of low-copy genes, a survey of paleopolyploidy (whole-genome duplications) across the Viridiplantae, the inferred evolutionary histories for many gene families and biological processes, the discovery of novel light-sensitive proteins for optogenetic studies in mammalian neuroscience, and elucidation of the genetic network for a complex trait (C4 photosynthesis). Altogether, 1KP demonstrated how value can be extracted from a phylodiverse sequencing data set, providing a template for future projects that aim to generate even more data, including complete de novo genomes, across the tree of life.

A Target Capture-Based Method to Estimate Ploidy From Herbarium Specimens.

Whole genome duplication (WGD) events are common in many plant lineages, but the ploidy status and possible occurrence of intraspecific ploidy variation are unknown for most species. Standard methods for ploidy determination are chromosome counting and flow cytometry approaches. While flow cytometry approaches typically use fresh tissue, an increasing number of studies have shown that recently dried specimens can be used to yield ploidy data. Recent studies have started to explore whether high-throughput sequencing (HTS) data can be used to assess ploidy levels by analyzing allelic frequencies from single copy nuclear genes. Here, we compare different approaches using a range of yam (Dioscorea) tissues of varying ages, drying methods and quality, including herbarium tissue. Our aims were to: (1) explore the limits of flow cytometry in estimating ploidy level from dried samples, including herbarium vouchers collected between 1831 and 2011, and (2) optimize a HTS-based method to estimate ploidy by considering allelic frequencies from nuclear genes obtained using a target-capture method. We show that, although flow cytometry can be used to estimate ploidy levels from herbarium specimens collected up to fifteen years ago, success rate is low (5.9%). We validated our HTS-based estimates of ploidy using 260 genes by benchmarking with dried samples of species of known ploidy (Dioscorea alata, D. communis, and D. sylvatica). Subsequently, we successfully applied the method to the 85 herbarium samples analyzed with flow cytometry, and successfully provided results for 91.7% of them, comprising species across the phylogenetic tree of Dioscorea. We also explored the limits of using this HTS-based approach for identifying high ploidy levels in herbarium material and the effects of heterozygosity and sequence coverage. Overall, we demonstrated that ploidy diversity within and between species may be ascertained from historical collections, allowing the determination of polyploidization events from samples collected up to two centuries ago. This approach has the potential to provide insights into the drivers and dynamics of ploidy level changes during plant evolution and crop domestication.