The vast majority of land plants transfer part of the organic carbon they produce by photosynthesis to arbuscular mycorrhizal (AM) fungi inside their root cells; the fungi in turn help plants to take up nutrients and water from the soil. This carbon can subsequently be acquired from mycorrhizal fungi by rare nonphotosynthetic 'mycoheterotrophic' plants that tap into the same fungal network. However, recent findings suggest that carbon uptake from AM fungi may exist among some green plants too. If so, this would qualify them as partial mycoheterotrophs (mixotrophs) rather than as pure autotrophs. Here, we discuss the evolutionary, ecophysiological, morphological, genetic, and environmental evidence for the existence and prevalence of this trait. We conclude that there is strong, albeit indirect, evidence for its existence, although its taxonomic distribution remains to be determined. This knowledge gap currently prevents us from inferring the magnitude of AM partial mycoheterotrophy in terrestrial ecosystems, which in turn severely limits our understanding of its role in plant establishment and survival and in ecosystem structure and function.

One of the most intractable problems in bamboo systematics concerns the three-branched bamboos of tribe Arundinarieae (Poaceae: Bambusoideae), which are collectively characterized by having three branches per mid-culm node. Previous phylogenetic studies based on double-digest restriction-site associated DNA sequencing (ddRAD-seq) data confirmed that Oligostachyum, a member of this group, is non-monophyletic, and in particular that Oligostachyum oedogonatum is a problematic species deserving further attention, as it appears to be morphologically and phylogenetically distinct from the other three-branched bamboos. Here we aim to define and confirm the phylogenetic position of O. oedogonatum, by including representatives from across its geographic range. We also provide new insights into the overall phylogeny of the three-branched bamboos and closely related genera, using multiple phylogenomic datasets. While a plastid genome-based tree is very poorly supported, phylogenies inferred using two sets of conserved nuclear genes and single nucleotide polymorphism (SNP) data yield generally well-supported and congruent topologies using coalescent-based approaches. The tree inferred from the largest concatenated gene set is the most dissimilar to other inferences. The nuclear-based data sets all recover a major clade that includes all of the three-branched bamboos and four other genera, which can be distinguished from related taxa due to their possession of three stigmas per floret and leptomorph rhizomes. Notably, eight O. oedogonatum samples form a clade that is distantly related to other members of Oligostachyum, including the type species of the genus (Oligostachyum sulcatum). Population genetic approaches and multi-species coalescent-based analysis of the nuclear data sets imply that seven of these populations can be treated as a single species, O. oedogonatum, but that one population from Jinggangshan is likely an intergeneric hybrid between O. oedogonatum and Pleioblastus. Morphologically, O. oedogonatum differs from all other three-branched bamboos, in having strongly asymmetrically swollen supra-nodal ridges, a laterally compressed spikelet, and rachilla segments that disarticulate readily below fertile florets. Because of its morphological distinctiveness and molecular phylogenetic position, we propose that this taxon should be reinstated as Clavinodum oedogonatum. We provide a new description for this monotypic genus here.

Premise Plastid-based data sets continue to play a major role in our understanding of early flowering-plant relationships, although organellar genomes of major lineages outside the monocots and eudicots remain under-sampled. A tendency of mitochondrial RNA-edit sites to mislead phylogenetic analysis in mixed transcriptomic/genomic data sets needs attention in angiosperm-wide studies, which only rarely consider mitochondrial genomes. Methods We compared mitochondrial- vs. plastid-based phylogenomic inferences, examined the effect of removing putative RNA-edit sites from mitochondrial data, and performed combined organellar analysis (plastid plus filtered mitochondrial genomes). We expanded taxon sampling for multiple angiosperm lineages for phylogenomic analysis using both organellar genomes, representing several poorly sampled lineages (in particular Degeneriaceae, Trimeniaceae) with smaller (few-gene) data sets. Results Plastid-based inferences recover well-supported relationships that align with and build upon previous studies, and recover well-supported internal relationships for two ANA-grade families (Hydatellaceae, Trimeniaceae) sampled for nearly all species. By contrast, unfiltered mitochondrial inferences of angiosperm phylogeny are generally poorly supported, and recover anomalous relationships compared to plastid-based inferences. However, removing putative mitochondrial RNA-edit sites dramatically reduces inter-organellar conflict and improves overall branch support. Conclusions We accounted for phylogenomic discordance between the two organellar genomes regarding overall angiosperm-wide relationships and filled in taxonomic gaps (poorly sampled lineages). Removing RNA edit sites substantially improves congruence in interorganellar inferences by effectively correcting a systematic bias in mitochondrial data. Uncertain relationships persist among five major mesangiosperm lineages in plastid-based inferences, but a clade comprising Chloranthales, Ceratophyllales and eudicots is well supported by filtered mitochondrial data. ### Competing Interest Statement The authors have declared no competing interest. Natural Sciences and Engineering Research Council, https://ror.org/01h531d29 German Academic Exchange Service, https://ror.org/039djdh30 Erasmus+, https://ror.org/01v2m4n16 Agence Nationale de la Recherche, https://ror.org/00rbzpz17, ANR-12-JVS7-0015-01

SummaryAfrothismia is a genus of non-photosynthetic mycoheterotrophs from the forests of continental tropical Africa. Multiple phylogenetic inferences using molecular data recover the genus as sister to a clade comprising mycoheterotrophic Thismiaceae and the photosynthetic family Taccaceae, contrary to earlier placements of Afrothismia and Thismiaceae within Burmanniaceae. Morphological support for separating Afrothismia from the rest of Thismiaceae has depended on the zygomorphic flowers of Afrothismia (although some species of Thismia are also zygomorphic), and their clusters of root tubers, each with a terminal rootlet. The number of described species of Afrothismia has recently increased from four to 16, with seven more species as yet undescribed; these discoveries have added morphological characters that support its distinction from Thismiaceae. Most notably, the ovary in Afrothismia has a single stalked placenta, and circumscissile fruits from which seeds are exserted by placental elevation (vs in Thismiaceae, three placentas, a deliquescing fruit lid, and seeds not exserted). Afrothismia stamens are inserted in the lower part of the floral tube, where they are attached to the stigma, and individual flowers are subtended by a single large dorsal bract. In contrast, in Thismiaceae, stamens are inserted at the mouth of the tube, free of and distant from the stigma, and each flower is subtended by a loose whorl of (2 –) 3 (– 4) bracts. Here we formally characterise Afrothismiaceae and review what is known of its development, seed germination, interactions with mycorrhizal Glomeromycota, biogeography, phylogeny and pollination biology. All but one (Afrothismia insignis; Vulnerable) of the 13 species assessed on the IUCN Red List of Threatened Species are either Endangered or Critically Endangered; one species (A. pachyantha) is considered extinct.

Angiosperms are the cornerstone of most terrestrial ecosystems and human livelihoods(1,2). A robust understanding of angiosperm evolution is required to explain their rise to ecological dominance. So far, the angiosperm tree of life has been determined primarily by means of analyses of the plastid genome(3,4). Many studies have drawn on this foundational work, such as classification and first insights into angiosperm diversification since their Mesozoic origins(5-7). However, the limited and biased sampling of both taxa and genomes undermines confidence in the tree and its implications. Here, we build the tree of life for almost 8,000 (about 60%) angiosperm genera using a standardized set of 353 nuclear genes(8). This 15-fold increase in genus-level sampling relative to comparable nuclear studies(9) provides a critical test of earlier results and brings notable change to key groups, especially in rosids, while substantiating many previously predicted relationships. Scaling this tree to time using 200 fossils, we discovered that early angiosperm evolution was characterized by high gene tree conflict and explosive diversification, giving rise to more than 80% of extant angiosperm orders. Steady diversification ensued through the remaining Mesozoic Era until rates resurged in the Cenozoic Era, concurrent with decreasing global temperatures and tightly linked with gene tree conflict. Taken together, our extensive sampling combined with advanced phylogenomic methods shows the deep history and full complexity in the evolution of a megadiverse clade.

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.

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.

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.

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.

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.

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.

EDITORIAL article Front. Plant Sci., 10 November 2022Sec. Plant Development and EvoDevo https://doi.org/10.3389/fpls.2022.1076169

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.

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.

Abstract 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.

Target enrichment represents a useful, cost-effective method for researchers working on the phylogenomics of non-model organisms (e.g., Cronn et al., 2012; Hale et al., 2020). The ability to sequence a customizable predefined genomic subset for several dozens or even hundreds of taxa allows in-depth analyses and the testing of phylogenetic hypotheses in ways that were not previously possible (reviewed in McKain et al., 2018). The most popular methods for targeted sequencing of genomic loci in phylogenomics include (long-)amplicon sequencing (Rothfels et al., 2017) and hybridization capture (Mandel et al., 2014; Weitemier et al., 2014). Targeted amplicon sequencing is based on single-fragment PCR amplification or by using multiplexing methods such as a microfluidic PCR-based amplification of multiple pre-selected genomic regions (e.g., Zhang and Ozdemir, 2009; Ho et al., 2014), which can then be pooled and sequenced. Massively parallel amplicon sequencing was first used in medical diagnostics (Turner et al., 2009) and was later applied to metazoan phylogenetics (Bybee et al., 2011; O'Neill et al., 2013). Microfluidic PCR and long-amplicon sequencing were subsequently applied in plant systematics (Uribe-Convers et al., 2014, 2016; Gostel et al., 2015). Amplicon-based methods can be time consuming as they require careful optimization and validation of primers. These methods are also susceptible to many of the common problems in PCR (such as nonspecific products, inability to amplify large loci in their entirety, or simply no products). Recently, amplicon approaches have been largely supplanted by hybridization-based targeted enrichment, which allows for relatively rapid probe design with reference to a few related transcriptomes or genomes, and allows simultaneous and efficient recovery of many hundreds of genes. Target enrichment via hybridization-based sequence capture can use customized or universal probes (short nucleotide fragments between 80 and 120 bp in length, also called baits). This versatile and powerful approach relies on probes to capture complementary sets of genomic targets from whole genome DNA or cDNA samples using solid-phase or in-solution hybridization (Gnirke et al., 2009; Mamanova et al., 2010; Cronn et al., 2012). To account for frequent (ancient) whole genome duplication events, plant systematists can identify sets of consistently low-to-single-copy markers that balance two common issues: ensuring recovery across a broad range of taxa, while containing sufficient phylogenetic informativeness. Target regions can be customized to address specific research questions, including targeting genes common to a metabolic pathway (Gardner et al., 2016; Folk et al., 2021) or genes of agronomic interest (Witek et al., 2016; Soto Gomez et al., 2019). The targets are often complete genes or exonic sequences, but non-coding DNA such as introns or untranslated regions can also be enriched. In-solution capture of the probe targets depends on the thermodynamics of hybridization. This process is governed, in part, by the DNA complexity of the bait and the probe length. Longer probes tolerate more mismatches, making them more suitable for capture across different species. Higher tiling density (the degree to which probes overlap, e.g., 75%) can compensate for failures of specific probes to hybridize or for low-quality input material, such as those stemming from historical herbarium specimens, so-called herbariomic approaches (Brewer et al., 2019). While overlapping probes increase the chance of capturing the target of interest via neighboring probes, there is a trade-off, as a higher tiling density tends to reduce the number of loci that can be examined per capture kit. Targeted enrichment of nuclear genes also overcomes several potential drawbacks of estimating phylogenies from organelle data alone. The single-locus behavior of organellar DNAs means that only a single history is provided: the entire plastid genome is expected to function as a single linkage group (Birky, 2001; but see Zhang et al., 2020). Organellar data are also prone to high rates of genetic drift in small population sizes, which could lead to a less accurate estimate of lineage differentiation, compared with large sets of nuclear genes (reviewed in Wicke and Schneeweiss, 2015). Although target capture data provide many more gene histories, analysis of these data requires specific bioinformatic approaches, including assembly of gene sequences without genomic references and detection of local gene duplication events that violate assumptions of orthology. In this regard, the plant systematics community has been innovative in addressing the related computational challenges, resulting in the wide adoption of targeted sequencing for plant systematics in recent years. Angiosperms353 is a tool for enabling phylogenomic-level analysis of any angiosperm group at any scale; the targeted genes and associated probes (Johnson et al., 2019) were developed from a set of low-copy nuclear genes used to infer the phylogeny of all green plants (One Thousand Plant Transcriptomes Initiative et al., 2019) to facilitate the collection of phylogenomic data in a relatively cost-effective and repeatable way. When considering Angiosperms353, it is important to distinguish between the low-copy orthologous genes found in all angiosperms and the 120-bp probe sequences used to enrich genomic libraries. The genes were selected based on copy number (ideally, genes that are single copy across more than 1400 available plant transcriptomes) and contain useful phylogenetic signal at deep and recent time scales. The probe sequences were designed from ~600 angiosperm transcriptomes and genomes, and were selected to maximize sequence similarity to other angiosperms, while minimizing the number of sequences used for probe design. Distinguishing the genes from the probes is important when considering the advantages, limitations, and future directions for Angiosperms353 in phylogenetic systematics, as discussed below. Although initial reports of the utility of Angiosperms353 genes and probes have been promising (Dodsworth et al., 2019), a few key questions remain: (1) Can Angiosperms353 probes truly be used to reliably recover orthologous genes across all groups of flowering plants?; (2) Is an angiosperm-wide probe design the most efficient for all targeted sequencing projects?; (3) What new bioinformatics tools are needed to apply Angiosperms353 genes to previously intractable research questions?; and (4) Are Angiosperms353 genes variable enough within species to extend the utility of the probes to population genetic studies? In this special issue and its companion issue in the American Journal of Botany (Baker et al., 2021b), the papers collected address these questions and demonstrate a number of ways in which research employing Angiosperms353 will benefit future angiosperm phylogenomic studies. This issue of Applications in Plant Sciences is focused on a variety of methodological issues, including comparisons between the Angiosperms353 probe set and taxon-specific probe sets, the development of new tools for specific lineages, a new lab technique to enrich libraries using Angiosperms353 and taxon-specific probe sets simultaneously, and bioinformatic approaches to facilitate careful assembly and analysis of data obtained using the Angiosperms353 kit. Low-to-single-copy nuclear markers have been an important tool in evolutionary studies of angiosperms (e.g., Small et al., 2004; Wu et al., 2006; Duarte et al., 2010). The identification of a set of hundreds of nuclear markers shared across angiosperms has facilitated the design of probes for targeted enrichment and subsequent high-throughput sequencing (Johnson et al., 2019). In this issue, three papers address the application and expansion of the Angiosperms353 loci. These papers build on existing resources to design new lineage-specific probes that contain the Angiosperms353 loci (Eserman et al., 2021; Ufimov et al., 2021), generate careful comparisons of the performance of an existing lineage-specific probe set with the Angiosperms353 probe set (Siniscalchi et al., 2021), and investigate the efficacy of simultaneous enrichment of libraries using universal and lineage-specific probes in a single hybridization reaction (Hendriks et al., 2021). Eserman et al. (2021) describe the development of Orchidaceae963, a new bait set that incorporates 254 of the Angiosperms353 loci, which they tested on members of three subfamilies of Orchidaceae. This resource will facilitate systematic work in the family and is also expected to be useful in population and conservation genomic studies. The incorporation of the Angiosperms353 loci along with custom marker genes is becoming more common (Yardeni et al., 2019; Jantzen et al., 2020; Christe et al., 2021; Ogutcen et al., 2021), and will allow collaboration between research teams and foster data sharing across studies. Comparative analyses of the custom sets and the Angiosperms353 set will naturally follow (e.g., see Larridon et al., 2020; Shah et al., 2021). For example, Ufimov et al. (2021) found a similar amount of parsimony informative sites using the Angiosperms353 probe set and a custom probe set in their study of Rosaceae subtribe Malinae. They also showed that the custom probes allowed for improved locus recovery rates across samples and lower levels of gene tree discordance (but see Yardeni et al., 2019). Siniscalchi et al. (2021) compared their lineage-specific Compositae1061 probes with the Angiosperms353 probes in eight members of the sunflower family. Although they were able to recover more loci on average with the lineage-specific probes, they found lower levels of paralogy in the gene trees based on the Angiosperms353 loci, which may simplify downstream analyses of the latter versus the former. Hendriks et al. (2021) performed a test case for combining both lineage-specific (i.e., Brassicaceae) and universal probes in a single hybridization reaction to simultaneously enrich libraries for both sets of loci. They found high levels of enrichment and locus recovery for both sets of targets across 26 Brassicaceae samples that included 16 samples from a single tribe and 10 samples reflecting broader sampling across the family. Their study shows that it is possible to generate data for multiple probe sets with little extra cost or work per sample. Taken together, these papers highlight that researchers have several options when choosing loci for target capture in flowering plants. The Angiosperms353 genes provide a ready set of loci that are low copy and reliably recovered using the universal Angiosperms353 probes, or by using custom probes designed with orthologs of the Angiosperms353 genes from the target taxa. The feasibility of combining probe sets during hybridization suggests additional flexibility. There is a trade-off, as custom probe designs will have higher per-sample costs and require existing sequence data in the focal group. However, far fewer probes will be needed for the Angiosperms353 genes in a custom design, allowing for additional loci for a given number of probes. Whichever choice is made, by including Angiosperms353 in a target capture project, researchers will benefit the entire phylogenetics community by enabling meta-analysis that parallels the broad adoption of single-gene markers in past decades. The development of Angiosperms353 into a common tool for molecular phylogenetics and population genetics has been paralleled by the need for new analytical workflows. One of the most active areas of tool development is in using extensions to existing tools to facilitate analysis of Angiosperms353 data. In this issue, three tools specifically tie in with HybPiper (Johnson et al., 2016), a popular tool for assembly and recovery of genes and flanking regions from target-capture sequencing reads. A common issue identified with Angiosperms353 data assembled using HybPiper is relatively low sequence recovery (e.g., Gaynor et al., 2020), but it is often unclear whether the issue lies with the molecular tools (i.e., poor hybridization between probes and target DNA) or bioinformatic tools (i.e., poor recovery in HybPiper or other tools). To test this, McLay et al. (2021) developed new target files to improve the mapping frequency of reads to Angiosperms353 target genes, a result independently confirmed by Slimp et al. (2021) and Lee et al. (2021). McLay et al. (2021) also provide a workflow that uses a hidden Markov model to choose clade-specific target sequences from a database of potential Angiosperms353 sequences. Their results indicate that the sequence divergence between the Angiosperms353 probe sequences and the target DNA of angiosperms during nucleic acid hybridization and subsequent sequencing is more forgiving than the effect of the same divergence on gene recovery during bioinformatic analysis. For researchers struggling with poor target enrichment efficiency using Angiosperms353, McLay et al. provide a potential bioinformatic solution that may reduce the need to re-enrich libraries. Their approach is to improve the target file used to recover genes in HybPiper by selecting more appropriate orthologs of the Angiosperms353 genes. Nauheimer et al. (2021) describe HybPhaser, a method for detecting hybrid taxa using HybPiper assemblies. Using an Angiosperms353 data set collected for Nepenthes, they employ an innovative two-step strategy in which an initial phylogeny is first used to identify representatives of different clades that have low heterozygosity (and are thus presumably not hybrids). In the second step, reads from putative hybrids are mapped to these chosen clade representatives. Differential mapping information is then used to phase hybrid sequences, which can then be placed on a multiply-labeled tree to identify hybrid origins. The HybPhaser method also has promise for addressing a common issue in target-capture phylogenetics: the identification of paralogous genes. This builds upon previous work using clade references to identify paralogs (Gardner et al., 2021), but the development of a more generalizable workflow in HybPhaser will be of use to many researchers unsure about whether target-captured genes are single copy in their data. Slimp et al. (2021) also describe new methods for downstream analysis of HybPiper output, for use in population genomic studies and the calculation of within-species demographic parameters. Their method adapts previously described workflows (Kates et al., 2018) to use HybPiper "supercontigs" (targeted coding regions and flanking non-coding regions) as reference sequences within species. Within-species variants can then be assessed jointly and used with standard population genomics programs (also see Beck et al., 2021 and Wenzell et al., 2021). Slimp et al. further demonstrate that the Angiosperms353 loci are variable within populations of several species, which suggests that genome-scale estimation of population parameters is now feasible without the need to develop taxon-specific methods. Since its initial publication, there has been a rapid and substantial uptake of Angiosperms353 to address systematics questions. The papers in this issue and its companion American Journal of Botany issue further illustrate that the utility of data generated through target enrichment extends beyond phylogenomics, with promising applications into population and conservation genomics. Of course, with the adaptability of data comes a requirement for flexible and innovative methods to process and analyze these data. While new methods and novel applications are already emerging, we recognize that there are some challenges and improvements that lie ahead. The need to evaluate the efficiency of recovering the targeted loci, both in vitro and in silico, is reflected in several papers in this issue, highlighting the distinction between targeted Angiosperms353 genes and the probes used to recover them. The papers presented here and in the companion issue build further evidence that the Angiosperms353 genes can be reliably recovered across the diversity of flowering plant species, with strong phylogenetic signal at scales ranging from deep time to the population level. Several papers in this issue describe innovations in how these target genes are recovered. Custom probe sets can still provide additional phylogenetic resolution within focal taxa by capturing more genes with the same number of probes, but the inclusion of Angiosperms353 loci along with custom probe sets will enable meta-analyses to compare species across flowering plants. In silico, the use of enhanced target files offers potential improvements for the assembly of enriched sequence libraries and illustrates that newly available genomic data can be used to complement the original Angiosperms353 target file and probe set. In this regard, the availability of an ever-increasing number of complete flowering plant genome and transcriptome sequences (e.g., One Thousand Plant Transcriptomes Initiative et al., 2019), and the massive increase in available sequence data facilitated by Angiosperms353, should allow for future refinement of the probes and choice of target sequences to better represent the diversity of angiosperms and maximize the length of sequences recovered across a broader set of taxa, including those with extremely altered lifestyles that affect the rate of sequence evolution, such as in heterotrophic taxa (e.g., Lam et al., 2018). Of course, the utility of Angiosperms353 data, or any other set of target enrichment data, relies on the accessibility of these genomic resources. Currently, there is no central data repository specifically tailored to access target enrichment data in a way that is not taxon specific. The Kew Tree of Life Explorer (https://treeoflife.kew.org/) was recently launched (Baker et al., 2021a) to make freely available pre- and post-publication Angiosperms353 data for thousands of taxa. The continued development of accessible tools to store and distribute these data will accelerate the progress that has been made thus far. For example, the ability to download a specific set of genes for a specific set of taxa from a central repository, rather than having to download and then filter multiple data sets from publication-specific repositories, would significantly improve the ability to incorporate existing data for addressing new research questions. Similarly, a centralized repository for uploading assembled and annotated target enrichment data generated for any taxon across the entire tree of life facilitates standardization and accessibility. A streamlined set of tools for identifying Angiosperms353 homologs for building custom probe sets would also increase the efficiency of data collection, much like how MarkerMiner (Chamala et al., 2015) provides a way to identify orthologs from a starting data set. The adoption of Angiosperms353 and target enrichment more generally in angiosperm systematics should cultivate further growth in data and in methods, as we have seen in this special issue. As more people use these data and methods, the opportunities for positive developments will only increase further, and improvements to existing probe sets, the development of new probe sets, opportunities to create solutions to challenges such as the resolution of paralogs, and the establishment of companion databases are all likely to be addressed head-on and embraced by the angiosperm systematics community. We thank all authors and reviewers for their valuable contributions to this special issue of Applications in Plant Sciences. We also acknowledge the tireless work of the APPS publications team, especially Managing Editor Beth Parada and Editor-in-Chief Theresa Culley.

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.

The unveiling of the angiosperm (flowering plant) tree of life over the past three decades has been one of the great success stories of modern plant biology. Flowering plants underpin most terrestrial biomes: they fix vast amounts of terrestrial carbon, in turn producing a substantial fraction of planetary oxygen, and drive major biogeochemical cycles. The bulk of human calories are derived either directly (crops) or indirectly (fodder) from angiosperms, as are many medicines, fuel, dyes, beverages, timber, fibers, and other materials. Countless indispensable and mundane items that impact human existence find their origins in flowering plants, and without them, life would be decidedly drearier— imagine a world without herbs, spices, or garden flowers, for example. In this context, the importance of a comprehensive understanding of the angiosperm tree of life cannot be overstated. The tree of life is the fundamental, biological roadmap to the evolution and properties of plants (e.g., Wong et al., 2020). For evolutionary biologists, phylogenies allow us to better understand the spectacular rise of the flowering plants to dominance over the past 140 million or so years (e.g., Lutzoni et al., 2018; RamírezBarahona et al., 2020). Information about angiosperm phylogenetic relationships also underpins modern angiosperm classification (e.g., APG IV, 2016), and helps us to better understand species origins and boundaries (e.g., Fazekas et al., 2009). Today, tree of life research is undergoing a renaissance due to the development of powerful, new phylogenomic methods (Dodsworth et al., 2019). In this special issue of the American Journal of Botany, together with a companion issue of Applications in Plant Sciences, we gather a set of papers that focus on a new, common phylogenomic toolkit, the Angiosperms353 probe set (Johnson et al., 2019), and illustrate its potential for evolutionary synthesis by promoting open collaboration across our community. Floweringplant systematists and evolutionary biologists worked cooperatively from the earliest days of molecular systematics, making major discoveries that were fuelled by communitywide agreement on common sets of DNA markers (e.g., Chase et al., 1993; CBOL Plant Working Group, 2009). Until fairly recently, most researchers focused their efforts on sequencing a handful of common genes from the plastid genome and the nuclear ribosomal cistron, as these were readily retrievable using standard laboratory R E S E A R C H A R T I C L E

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.

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.