Organellar phylogenomics at the epidendroid orchid base, with a focus on the mycoheterotrophic Wullschlaegelia.

BACKGROUND AND AIMS: Heterotrophic plants have long been a challenge for systematists, exemplified by the base of the orchid subfamily Epidendroideae, which contains numerous mycoheterotrophic species. METHODS: Here we address the utility of organellar genomes in resolving relationships at the epidendroid base, specifically employing models of heterotachy, or lineage-specific rate variation over time. We further conduct comparative analyses of plastid genome evolution in heterotrophs and structural variation in matK. KEY RESULTS: We present the first complete plastid genomes (plastomes) of Wullschlaegelia, the sole genus of the tribe Wullschlaegelieae, revealing a highly reduced genome of 37 kilobases, which retains a fraction of the genes present in related autotrophs. Plastid phylogenomic analyses recovered a strongly supported clade composed exclusively of mycoheterotrophic species with long branches. We further analyzed mitochondrial gene sets, which recovered similar relationships to those in other studies using nuclear data, but the placement of Wullschlaegelia remains uncertain. We conducted comparative plastome analyses among Wullschlaegelia and other heterotrophic orchids, revealing a suite of correlated substitutional and structural changes relative to autotrophic species. Lastly, we investigated evolutionary and structural variation in matK, which is retained in Wullschlaegelia and a few other 'late stage' heterotrophs and found evidence for structural conservation despite rapid substitution rates in both Wullschlaegelia and the leafless Gastrodia. CONCLUSIONS: Our analyses reveal the limits of what the plastid genome can tell us on orchid relationships in this part of the tree, even when applying parameter-rich heterotachy models. Our study underscores the need for increased taxon sampling across all three genomes at the epidendroid base, and illustrates the need for further research on addressing heterotachy in phylogenomic analyses.

Plastid genome evolution in leafless members of the orchid subfamily Orchidoideae, with a focus on Degranvillea dermaptera.

PREMISE: Leafless, heterotrophic plants are prime examples of organismal modification, the genomic consequences of which have received considerable interest. In particular, plastid genomes (plastomes) are being sequenced at a high rate, allowing continual refinement of conceptual models of reductive evolution in heterotrophs. However, numerous sampling gaps exist, hindering the ability to conduct comprehensive phylogenomic analyses in these plants. METHODS: Using floral tissue from an herbarium specimen, we sequenced and analyzed the plastome of Degranvillea dermaptera, a rarely collected, leafless orchid species from South America about which little is known, including its phylogenetic affinities. RESULTS: The plastome is the most reduced of those sequenced among the orchid subfamily Orchidoideae. In Degranvillea, it has lost the majority of genes found in leafy autotrophic species, is structurally rearranged, and has similar gene content to the most reduced plastomes among the orchids. We found strong evidence for the placement of Degranvillea within the subtribe Spiranthinae using models that explicitly account for heterotachy, or lineage-specific evolutionary rate variation over time. We further found evidence of relaxed selection on several genes and of correlations among substitution rates and several other "traits" of the plastome among leafless members of orchid subfamily Orchidoideae. CONCLUSIONS: Our findings advance knowledge on the phylogenetic relationships and paths of plastid genome evolution among the orchids, which have experienced more independent transitions to heterotrophy than any other plant family. This study demonstrates the importance of herbarium collections in comparative genomics of poorly known species of conservation concern.

Lineage and role in integrative taxonomy of a heterotrophic orchid complex.

Lineage-based species definitions applying coalescent approaches to species delimitation have become increasingly popular. Yet, the application of these methods and the recognition of lineage-only definitions have recently been questioned. Species delimitation criteria that explicitly consider both lineages and evidence for ecological role shifts provide an opportunity to incorporate ecologically meaningful data from multiple sources in studies of species boundaries. Here, such criteria were applied to a problematic group of mycoheterotrophic orchids, the Corallorhiza striata complex, analysing genomic, morphological, phenological, reproductive-mode, niche, and fungal host data. A recently developed method for generating genomic polymorphism data-ISSRseq-demonstrates evidence for four distinct lineages, including a previously unidentified lineage in the Coast Ranges and Cascades of California and Oregon, USA. There is divergence in morphology, phenology, reproductive mode, and fungal associates among the four lineages. Integrative analyses, conducted in population assignment and redundancy analysis frameworks, provide evidence of distinct genomic lineages and a similar pattern of divergence in the extended data, albeit with weaker signal. However, none of the extended data sets fully satisfy the condition of a significant role shift, which requires evidence of fixed differences. The four lineages identified in the current study are recognized at the level of variety, short of comprising different species. This study represents the most comprehensive application of lineage + role to date and illustrates the advantages of such an approach.

Digitized collections elucidate invasion history and patterns of awn polymorphism in Microstegium vimineum.

PREMISE: Digitized collections can help illuminate the mechanisms behind the establishment and spread of invasive plants. These databases provide a record of traits in space and time that allows for investigation of abiotic and biotic factors that influence invasive species. METHODS: Over 1100 digitized herbarium records were examined to investigate the invasion history and trait variation of Microstegium vimineum. Presence-absence of awns was investigated to quantify geographic patterns of this polymorphic trait, which serves several functions in grasses, including diaspore burial and dispersal to germination sites. Floret traits were further quantified, and genomic analyses of contemporary samples were conducted to investigate the history of M. vimineum's introduction and spread into North America. RESULTS: Herbarium records revealed similar patterns of awn polymorphism in native and invaded ranges of M. vimineum, with awned forms predominating at higher latitudes and awnless forms at lower latitudes. Herbarium records and genomic data suggested initial introduction and spread of the awnless form in the southeastern United States, followed by a putative secondary invasion and spread of the awned form from eastern Pennsylvania. Awned forms have longer florets, and floret size varies significantly with latitude. There is evidence of a transition zone with short-awned specimens at mid-latitudes. Genomic analyses revealed two distinct clusters corresponding to awnless and awned forms, with evidence of admixture. CONCLUSIONS: Our results demonstrate the power of herbarium data to elucidate the invasion history of a problematic weed in North America and, together with genomic data, reveal a possible key trait in introduction success: presence or absence of an awn.

Ancient Mitochondrial Gene Transfer between Fungi and the Orchids.

The mitochondrial genomes (mitogenomes) of plants are known to incorporate and accumulate DNA from intra- and extracellular donors. Despite the intimate relationships formed between flowing plants (angiosperms) and fungi, lengthy fungal-like sequence has not been identified in angiosperm mitogenomes to date. Here, we present multiple lines of evidence documenting horizontal gene transfer (HGT) between the mitogenomes of fungi and the ancestors of the orchids, plants that are obligate parasites of fungi during their early development. We show that the ancestor of the orchids acquired an ∼270-bp fungal mitogenomic region containing three transfer RNA genes. We propose that the short HGT was later replaced by a second HGT event transferring >8 kb and 14 genes from a fungal mitogenome to that of the ancestor of the largest orchid subfamily, Epidendroideae. Our results represent the first evidence of genomic-scale HGT between fungal and angiosperm mitogenomes and demonstrate that the length intergenic spacer regions of angiosperm mitogenomes can effectively fossilize the genomic remains of ancient, nonplant organisms.

Unprecedented Parallel Photosynthetic Losses in a Heterotrophic Orchid Genus.

Heterotrophic plants are evolutionary experiments in genomic, morphological, and physiological change. Yet, genomic sampling gaps exist among independently derived heterotrophic lineages, leaving unanswered questions about the process of genome modification. Here, we have sequenced complete plastid genomes for all species of the leafless orchid genus Hexalectris, including multiple individuals for most, and leafy relatives Basiphyllaea and Bletia. Our objectives are to determine the number of independent losses of photosynthesis and to test hypotheses on the process of genome degradation as a result of relaxed selection. We demonstrate four to five independent losses of photosynthesis in Hexalectris based on degradation of the photosynthetic apparatus, with all but two species displaying evidence of losses, and variation in gene loss extending below the species level. Degradation in the atp complex is advanced in Hexalectris warnockii, whereas only minimal degradation (i.e., physical loss) has occurred among some "housekeeping" genes. We find genomic rearrangements, shifts in Inverted Repeat boundaries including complete loss in one accession of H. arizonica, and correlations among substitutional and genomic attributes. Our unprecedented finding of multiple, independent transitions to a fully mycoheterotrophic lifestyle in a single genus reveals that the number of such transitions among land plants is likely underestimated. This study underscores the importance of dense taxon sampling, which is highly informative for advancing models of genome evolution in heterotrophs. Mycoheterotrophs such as Hexalectris provide forward-genetic opportunities to study the consequences of radical genome evolution beyond what is possible with mutational studies in model organisms alone.

Dense infraspecific sampling reveals rapid and independent trajectories of plastome degradation in a heterotrophic orchid complex.

Heterotrophic plants provide excellent opportunities to study the effects of altered selective regimes on genome evolution. Plastid genome (plastome) studies in heterotrophic plants are often based on one or a few highly divergent species or sequences as representatives of an entire lineage, thus missing important evolutionary-transitory events. Here, we present the first infraspecific analysis of plastome evolution in any heterotrophic plant. By combining genome skimming and targeted sequence capture, we address hypotheses on the degree and rate of plastome degradation in a complex of leafless orchids (Corallorhiza striata) across its geographic range. Plastomes provide strong support for relationships and evidence of reciprocal monophyly between C. involuta and the endangered C. bentleyi. Plastome degradation is extensive, occurring rapidly over a few million years, with evidence of differing rates of genomic change among the two principal clades of the complex. Genome skimming and targeted sequence capture differ widely in coverage depth overall, with depth in targeted sequence capture datasets varying immensely across the plastome as a function of GC content. These findings will help to fill a knowledge gap in models of heterotrophic plastid genome evolution, and have implications for future studies in heterotrophs.

Monocot plastid phylogenomics, timeline, net rates of species diversification, the power of multi-gene analyses, and a functional model for the origin of monocots.

PREMISE OF THE STUDY: We present the first plastome phylogeny encompassing all 77 monocot families, estimate branch support, and infer monocot-wide divergence times and rates of species diversification. METHODS: We conducted maximum likelihood analyses of phylogeny and BAMM studies of diversification rates based on 77 plastid genes across 545 monocots and 22 outgroups. We quantified how branch support and ascertainment vary with gene number, branch length, and branch depth. KEY RESULTS: Phylogenomic analyses shift the placement of 16 families in relation to earlier studies based on four plastid genes, add seven families, date the divergence between monocots and eudicots+Ceratophyllum at 136 Mya, successfully place all mycoheterotrophic taxa examined, and support recognizing Taccaceae and Thismiaceae as separate families and Arecales and Dasypogonales as separate orders. Only 45% of interfamilial divergences occurred after the Cretaceous. Net species diversification underwent four large-scale accelerations in PACMAD-BOP Poaceae, Asparagales sister to Doryanthaceae, Orchidoideae-Epidendroideae, and Araceae sister to Lemnoideae, each associated with specific ecological/morphological shifts. Branch ascertainment and support across monocots increase with gene number and branch length, and decrease with relative branch depth. Analysis of entire plastomes in Zingiberales quantifies the importance of non-coding regions in identifying and supporting short, deep branches. CONCLUSIONS: We provide the first resolved, well-supported monocot phylogeny and timeline spanning all families, and quantify the significant contribution of plastome-scale data to resolving short, deep branches. We outline a new functional model for the evolution of monocots and their diagnostic morphological traits from submersed aquatic ancestors, supported by convergent evolution of many of these traits in aquatic Hydatellaceae (Nymphaeales).

Plastid genomes reveal support for deep phylogenetic relationships and extensive rate variation among palms and other commelinid monocots.

Despite progress based on multilocus, phylogenetic studies of the palms (order Arecales, family Arecaceae), uncertainty remains in resolution/support among major clades and for the placement of the palms among the commelinid monocots. Palms and related commelinids represent a classic case of substitution rate heterogeneity that has not been investigated in the genomic era. To address questions of relationships, support and rate variation among palms and commelinid relatives, 39 plastomes representing the palms and related family Dasypogonaceae were generated via genome skimming and integrated within a monocot-wide matrix for phylogenetic and molecular evolutionary analyses. Support was strong for 'deep' relationships among the commelinid orders, among the five palm subfamilies, and among tribes of the subfamily Coryphoideae. Additionally, there was extreme heterogeneity in the plastid substitution rates across the commelinid orders indicated by model based analyses, with c. 22 rate shifts, and significant departure from a global clock. To date, this study represents the most comprehensively sampled matrix of plastomes assembled for monocot angiosperms, providing genome-scale support for phylogenetic relationships of monocot angiosperms, and lays the phylogenetic groundwork for comparative analyses of the drivers and correlates of such drastic differences in substitution rates across a diverse and significant clade.

Nuclear phylogenomics of the palm subfamily Arecoideae (Arecaceae).

Palms (Arecaceae) include economically important species such as coconut, date palm, and oil palm. Resolution of the palm phylogeny has been problematic due to rapid diversification and slow rates of molecular evolution. The focus of this study is on relationships of the 14 tribes of subfamily Arecoideae and their inferred ancestral areas. A targeted sequencing approach was used to generate a data set of 168 single/low copy nuclear genes for 34 species representing the Arecoideae tribes and the other palm subfamilies. Species trees from the concatenated and coalescent based analyses recovered largely congruent topologies. Three major tribal clades were recovered: the POS clade (Podococceae, Oranieae, Sclerospermeae), the RRC clade (Roystoneeae, Reinhardtieae, Cocoseae), and the core arecoid clade (Areceae, Euterpeae, Geonomateae, Leopoldinieae, Manicarieae, Pelagodoxeae). Leopoldinieae was sister to the rest of the core arecoids (Geonomateae, Manicarieae+Pelagodoxeae, and Areceae+Euterpeae). The nuclear phylogeny supported a North American origin for subfamily Arecoideae, with most tribal progenitors diversifying within the Americas. The POS clade may have dispersed from the Americas into Africa, with tribe Oranieae subsequently spreading into the Indo-Pacific. Two independent dispersals into the Indo-Pacific were inferred for two tribes within the core arecoids (tribes Areceae and Pelagodoxeae).

Plastid phylogenomics and molecular evolution of Alismatales.

Past phylogenetic studies of the monocot order Alismatales left several higher-order relationships unresolved. We addressed these uncertainties using a nearly complete genus-level sampling of whole plastid genomes (gene sets representing 83 protein-coding and ribosomal genes) from members of the core alismatid families, Tofieldiaceae and additional taxa (Araceae and other angiosperms). Parsimony and likelihood analyses inferred generally highly congruent phylogenetic relationships within the order, and several alternative likelihood partitioning schemes had little impact on patterns of clade support. All families with multiple genera were resolved as monophyletic, and we inferred strong bootstrap support for most inter- and intrafamilial relationships. The precise placement of Tofieldiaceae in the order was not well supported. Although most analyses inferred Tofieldiaceae to be the sister-group of the rest of the order, one likelihood analysis indicated a contrasting Araceae-sister arrangement. Acorus (Acorales) was not supported as a member of the order. We also investigated the molecular evolution of plastid NADH dehydrogenase, a large enzymatic complex that may play a role in photooxidative stress responses. Ancestral-state reconstructions support four convergent losses of a functional NADH dehydrogenase complex in Alismatales, including a single loss in Tofieldiaceae.

Drastic reduction of plastome size in the mycoheterotrophic Thismia tentaculata relative to that of its autotrophic relative Tacca chantrieri.

PREMISE OF THE STUDY: Heterotrophic angiosperms tend to have reduced plastome sizes relative to those of their autotrophic relatives because genes that code for proteins involved in photosynthesis are lost. However, some plastid-encoded proteins may have vital nonphotosynthetic functions, and the plastome therefore may be retained after the loss of photosynthesis. METHODS: We sequenced the plastome of the mycoheterotrophic species Thismia tentaculata and a representative of its sister genus, Tacca chantrieri, using next-generation technology, and we compared sequences and structures of genes and genomes of these species. KEY RESULTS: The plastome of Tacca chantrieri is similar to those of other autotrophic taxa of Dioscoreaceae, except in a few local rearrangements and one gene loss. The plastome of Thismia tentaculata is ca. 16 kbp long with a quadripartite structure and is among the smallest known plastomes. Synteny is minimal between the plastomes of Tacca chantrieri and Thismia tentaculata. The latter includes only 12 candidate genes, with all except accD involved in protein synthesis. Of the 12 genes, trnE, trnfM, and accD are frequently among the few that remain in depauperate plastomes. CONCLUSIONS: The plastome of Thismia tentaculata, like those of most other heterotrophic plants, includes a small number of genes previously suggested to be essential to plastome survival.

Investigating the path of plastid genome degradation in an early-transitional clade of heterotrophic orchids, and implications for heterotrophic angiosperms.

Parasitic organisms exemplify morphological and genomic reduction. Some heterotrophic, parasitic plants harbor drastically reduced and degraded plastid genomes resulting from relaxed selective pressure on photosynthetic function. However, few studies have addressed the initial stages of plastome degradation in groups containing both photosynthetic and nonphotosynthetic species. Corallorhiza is a genus of leafless, heterotrophic orchids that contains both green, photosynthetic species and nongreen, putatively nonphotosynthetic species, and represents an ideal system in which to assess the beginning of the transition to a "minimal plastome." Complete plastomes were generated for nine taxa of Corallorhiza using Illumina paired-end sequencing of genomic DNA to assess the degree of degradation among taxa, and for comparison with a general model of degradation among angiosperms. Quantification of total chlorophyll suggests that nongreen Corallorhiza still produce chlorophyll, but at 10-fold lower concentrations than green congeners. Complete plastomes and partial nuclear rDNA cistrons yielded a fully resolved tree for Corallorhiza, with at least two independent losses of photosynthesis, evidenced by gene deletions and pseudogenes in Co. striata and nongreen Co. maculata. All Corallorhiza show some evidence of degradation in genes of the NAD(P)H dehydrogenase complex. Among genes with open reading frames, photosynthesis-related genes displayed evidence of neutral evolution in nongreen Corallorhiza, whereas genes of the ATP synthase complex displayed some evidence of positive selection in these same groups, though for reasons unknown. Corallorhiza spans the early stages of a general model of plastome degradation and has added critical insight for understanding the process of plastome evolution in heterotrophic angiosperms.

Resolving relationships within the palm subfamily Arecoideae (Arecaceae) using plastid sequences derived from next-generation sequencing.

PREMISE OF THE STUDY: Several studies have incorporated molecular and morphological data to study the phylogeny of the palms (Arecaceae), but some relationships within the family remain ambiguous-particularly those within Arecoideae, the most diverse subfamily including coconut and oil palm. Here, two next-generation, targeted plastid-enrichment methods were compared and used to elucidate Arecoideae phylogeny. METHODS: Next-generation sequencing techniques were used to generate a plastid genome data set. Long range PCR and hybrid gene capture were used to enrich for chloroplast targets. Ten taxa were enriched using both methods for comparison. Chloroplast sequence data were generated for 31 representatives of the 14 Arecoideae tribes and five outgroup taxa. The phylogeny was reconstructed using maximum likelihood, maximum parsimony, and Bayesian analyses. KEY RESULTS: Long range PCR and hybrid gene capture both enriched the plastid genome and provided similar sequencing coverage. Subfamily Arecoideae was resolved as monophyletic with tribe Chamaedoreeae as the earliest-diverging lineage, implying that the development of flowers in triads defines a synapomorphy for the Arecoideae clade excluding Chamaedoreeae. Three major clades within this group were recovered: Roystoneeae/Reinhardtieae/Cocoseae (RRC), Areceae/Euterpeae/Geonomateae/Leopoldinieae/Manicarieae/Pelagodoxeae (core arecoids), and Podococceae/Oranieae/Sclerospermeae (POS). An Areceae + Euterpeae clade was resolved within the core arecoids. The POS clade was sister to a RRC + core arecoids clade, implying a shared ancestral area in South America for these three clades. CONCLUSIONS: The plastome phylogeny recovered here provides robust resolution of previously ambiguous studies and new insights into palm evolution.

Resolving ancient radiations: can complete plastid gene sets elucidate deep relationships among the tropical gingers (Zingiberales)?

BACKGROUND AND AIMS: Zingiberales comprise a clade of eight tropical monocot families including approx. 2500 species and are hypothesized to have undergone an ancient, rapid radiation during the Cretaceous. Zingiberales display substantial variation in floral morphology, and several members are ecologically and economically important. Deep phylogenetic relationships among primary lineages of Zingiberales have proved difficult to resolve in previous studies, representing a key region of uncertainty in the monocot tree of life. METHODS: Next-generation sequencing was used to construct complete plastid gene sets for nine taxa of Zingiberales, which were added to five previously sequenced sets in an attempt to resolve deep relationships among families in the order. Variation in taxon sampling, process partition inclusion and partition model parameters were examined to assess their effects on topology and support. KEY RESULTS: Codon-based likelihood analysis identified a strongly supported clade of ((Cannaceae, Marantaceae), (Costaceae, Zingiberaceae)), sister to (Musaceae, (Lowiaceae, Strelitziaceae)), collectively sister to Heliconiaceae. However, the deepest divergences in this phylogenetic analysis comprised short branches with weak support. Additionally, manipulation of matrices resulted in differing deep topologies in an unpredictable fashion. Alternative topology testing allowed statistical rejection of some of the topologies. Saturation fails to explain observed topological uncertainty and low support at the base of Zingiberales. Evidence for conflict among the plastid data was based on a support metric that accounts for conflicting resampled topologies. CONCLUSIONS: Many relationships were resolved with robust support, but the paucity of character information supporting the deepest nodes and the existence of conflict suggest that plastid coding regions are insufficient to resolve and support the earliest divergences among families of Zingiberales. Whole plastomes will continue to be highly useful in plant phylogenetics, but the current study adds to a growing body of literature suggesting that they may not provide enough character information for resolving ancient, rapid radiations.

Evolution of Whirly1 in the angiosperms: sequence, splicing, and expression in a clade of early transitional mycoheterotrophic orchids.

The plastid-targeted transcription factor Whirly1 (WHY1) has been implicated in chloroplast biogenesis, plastid genome stability, and fungal defense response, which together represent characteristics of interest for the study of autotrophic losses across the angiosperms. While gene loss in the plastid and nuclear genomes has been well studied in mycoheterotrophic plants, the evolution of the molecular mechanisms impacting genome stability is completely unknown. Here, we characterize the evolution of WHY1 in four early transitional mycoheterotrophic orchid species in the genus Corallorhiza by synthesizing the results of phylogenetic, transcriptomic, and comparative genomic analyses with WHY1 genomic sequences sampled from 21 orders of angiosperms. We found an increased number of non-canonical WHY1 isoforms assembled from all but the greenest Corallorhiza species, including intron retention in some isoforms. Within Corallorhiza, phylotranscriptomic analyses revealed the presence of tissue-specific differential expression of WHY1 in only the most photosynthetically capable species and a coincident increase in the number of non-canonical WHY1 isoforms assembled from fully mycoheterotrophic species. Gene- and codon-level tests of WHY1 selective regimes did not infer significant signal of either relaxed selection or episodic diversifying selection in Corallorhiza but did so for relaxed selection in the late-stage full mycoheterotrophic orchids Epipogium aphyllum and Gastrodia elata. Additionally, nucleotide substitutions that most likely impact the function of WHY1, such as nonsense mutations, were only observed in late-stage mycoheterotrophs. We propose that our findings suggest that splicing and expression changes may precede the selective shifts we inferred for late-stage mycoheterotrophic species, which therefore does not support a primary role for WHY1 in the transition to mycoheterotrophy in the Orchidaceae. Taken together, this study provides the most comprehensive view of WHY1 evolution across the angiosperms to date.

Plastid genomes and deep relationships among the commelinid monocot angiosperms.

The commelinid monocots comprise the orders Arecales (A), Commelinales (C), Poales sensu Angiosperm Phylogeny Group III (APGIII) (P), Zingiberales (Z), plus the unplaced family Dasypogonaceae (D), collectively containing numerous economically and ecologically important species and encompassing enormous morphological diversity. Commelinids are supported as monophyletic based on anatomy and molecular data; however, relationships among major commelinid groupings conflict among previous studies, representing a long-standing problem in monocot systematics, with major implications for interpretations of character evolution. In more recent analyses, with whole-plastome sampling largely focused on Poales, areas of conflict remain, suggesting the need for closer investigation of relationships and support. Here, we increased sampling of plastomes among non-Poalean commelinid orders to investigate deep nodal support. Analysis of 83 plastid genes recovered relationships as ((A, D) (ZC, P)) with robust support, regardless of reconstruction method (parsimony/likelihood). However, conflict among genes was evident when grouped by genomic region. Cumulative analyses of genes ranked by decreasing numbers of informative characters indicated continued fluctuation in support, even as small genes were added to a nearly complete matrix, contrary to the expected pattern of stabilization in support. Topology tests among major commelinid groups suggested that the data were not powerful enough to reject all alternatives. This study provides clues to the limits of the plastid genome for resolving deep relationships among the commelinid monocots.

A lack of population structure characterizes the invasive Lonicera japonica in West Virginia and across eastern North America.

Invasive plant species cause massive ecosystem damage globally, yet represent powerful case studies in population genetics and rapid adaptation to new habitats. The availability of digitized herbarium collections data, and the ubiquity of invasive species across the landscape make them highly accessible for studies of invasion history and population dynamics associated with their introduction, establishment, spread, and ecological interactions. Here we focus on Lonicera japonica, one of the most damaging invasive vine species in North America. We leveraged digitized collections data and contemporary field collections to reconstruct the invasion history and characterize patterns of genomic variation in the eastern USA, using a straightforward method for generating nucleotide polymorphism data and a recently published, chromosome-level genome for the species. We found an overall lack of population structure among sites in northern West Virginia, USA, as well as across sites in the central and eastern USA. Heterozygosity and population differentiation were both low based on Fst, analysis of molecular variance, principal components analysis, and cluster-based analyses. We also found evidence of high inbreeding coefficients and significant linkage disequilibrium, in line with the ability of this otherwise outcrossing, perennial species to propagate vegetatively. Our findings corroborate earlier studies based on allozyme data, and suggest that intentional, human-assisted spread explains the lack of population structure, as this species was planted for erosion control and as an ornamental, escaping cultivation repeatedly across the USA. Finally, we discuss how plant invasion genomics can be incorporated into experiential undergraduate education as a way to integrate teaching and research.

Mitochondrial genome sequencing and analysis of the invasive Microstegium vimineum: a resource for systematics, invasion history, and management.

Premise of the Research: Plants remain underrepresented among species with sequenced mitochondrial genomes (mitogenomes), due to the difficulty in assembly with short-read technology. Invasive species lag behind crops and other economically important species in this respect, representing a lack of tools for management and land conservation efforts. Methodology: The mitogenome of Microstegium vimineum, one of the most damaging invasive plant species in North America, was sequenced and analyzed using long-read data, providing a resource for biologists and managers. We conducted analyses of genome content, phylogenomic analyses among grasses and relatives based on mitochondrial coding regions, and an analysis of mitochondrial single nucleotide polymorphism in this invasive grass species. Pivotal Results: The assembly is 478,010 bp in length and characterized by two large, inverted repeats, and a large, direct repeat. However, the genome could not be circularized, arguing against a "master circle" structure. Long-read assemblies with data subsets revealed several alternative genomic conformations, predominantly associated with large repeats. Plastid-like sequences comprise 2.4% of the genome, with further evidence of Class I and Class II transposable element-like sequences. Phylogenetic analysis placed M. vimineum with other Microstegium species, excluding M. nudum, but with weak support. Analysis of polymorphic sites across 112 accessions of M. vimineum from the native and invasive ranges revealed a complex invasion history. Conclusions: We present an in-depth analysis of mitogenome structure, content, phylogenetic relationships, and range-wide genomic variation in M. vimineum's invasive US range. The mitogenome of M. vimineum is typical of other andropogonoid grasses, yet mitochondrial sequence variation across the invasive and native ranges is extensive. Our findings suggest multiple introductions to the US over the last century, with subsequent spread, secondary contact, long-distance dispersal, and possibly post-invasion selection on awn phenotypes. Efforts to produce genomic resources for invasive species, including sequenced mitochondrial genomes, will continue to provide tools for their effective management, and to help predict and prevent future invasions.