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.

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.

Degradation of key photosynthetic genes in the critically endangered semi-aquatic flowering plant Saniculiphyllum guangxiense (Saxifragaceae).

BACKGROUND: Plastid gene loss and pseudogenization has been widely documented in parasitic and mycoheterotrophic plants, which have relaxed selective constraints on photosynthetic function. More enigmatic are sporadic reports of pseudogenization and loss of important photosynthesis genes in lineages thought to be fully photosynthetic. Here we report the complete plastid genome of Saniculiphyllum guangxiense, a critically endangered and phylogenetically isolated plant lineage, along with genomic evidence of reduced chloroplast function. We also report 22 additional plastid genomes representing the diversity of its containing clade Saxifragales, characterizing gene content and placing variation in a broader phylogenetic context. RESULTS: We find that the plastid genome of Saniculiphyllum has experienced pseudogenization of five genes of the ndh complex (ndhA, ndhB, ndhD, ndhF, and ndhK), previously reported in flowering plants with an aquatic habit, as well as the surprising pseudogenization of two genes more central to photosynthesis (ccsA and cemA), contrasting with strong phylogenetic conservatism of plastid gene content in all other sampled Saxifragales. These genes participate in photooxidative protection, cytochrome synthesis, and carbon uptake. Nuclear paralogs exist for all seven plastid pseudogenes, yet these are also unlikely to be functional. CONCLUSIONS: Saniculiphyllum appears to represent the greatest degree of plastid gene loss observed to date in any fully photosynthetic lineage, perhaps related to its extreme habitat specialization, yet plastid genome length, structure, and substitution rate are within the variation previously reported for photosynthetic plants. These results highlight the increasingly appreciated dynamism of plastid genomes, otherwise highly conserved across a billion years of green plant evolution, in plants with highly specialized life history traits.

The Planteome database: an integrated resource for reference ontologies, plant genomics and phenomics.

The Planteome project (http://www.planteome.org) provides a suite of reference and species-specific ontologies for plants and annotations to genes and phenotypes. Ontologies serve as common standards for semantic integration of a large and growing corpus of plant genomics, phenomics and genetics data. The reference ontologies include the Plant Ontology, Plant Trait Ontology and the Plant Experimental Conditions Ontology developed by the Planteome project, along with the Gene Ontology, Chemical Entities of Biological Interest, Phenotype and Attribute Ontology, and others. The project also provides access to species-specific Crop Ontologies developed by various plant breeding and research communities from around the world. We provide integrated data on plant traits, phenotypes, and gene function and expression from 95 plant taxa, annotated with reference ontology terms. The Planteome project is developing a plant gene annotation platform; Planteome Noctua, to facilitate community engagement. All the Planteome ontologies are publicly available and are maintained at the Planteome GitHub site (https://github.com/Planteome) for sharing, tracking revisions and new requests. The annotated data are freely accessible from the ontology browser (http://browser.planteome.org/amigo) and our data repository.

Biodiversity and the Species Concept-Lineages are not Enough.

The nature and definition of species continue to be matters of debate. Current views of species often focus on their nature as lineages-maximal reproductive communities through time. Whereas many authors point to the Evolutionary Species Concept as optimal, in its original form it stressed the ecological role of species as well as their history as lineages, but most recent authors have ignored the role aspect of the concept, making it difficult to apply unambiguously in a time-extended way. This trend has been exacerbated by the application of methods and concepts emphasizing the notion of monophyly, originally applied only at higher levels, to the level of individuals, as well as by the current emphasis on molecular data. Hence, some current authors recognize units that are no more than probable exclusive lineages as species. We argue that biodiversity is inherently a phenotypic concept and that role, as manifested in the organismal extended phenotype, is a necessary component of the species concept. Viewing species as historically connected populations with unique role brings together the temporal and phenotypic natures of species, providing a clear way to view species both in a time-limited and time-extended way. Doing so alleviates perceived issues with "paraphyletic species" and returns the focus of species to units that are most relevant for biodiversity.

Total duplication of the small single copy region in the angiosperm plastome: Rearrangement and inverted repeat instability in Asarum.

PREMISE OF THE STUDY: As more plastomes are assembled, it is evident that rearrangements, losses, intergenic spacer expansion and contraction, and syntenic breaks within otherwise functioning plastids are more common than was thought previously, and such changes have developed independently in disparate lineages. However, to date, the magnoliids remain characterized by their highly conserved plastid genomes (plastomes). METHODS: Illumina HiSeq and MiSeq platforms were used to sequence the plastomes of Saruma henryi and those of representative species from each of the six taxonomic sections of Asarum. Sequenced plastomes were compared in a phylogenetic context provided by maximum likelihood and parsimony inferences made using an additional 18 publicly available plastomes from early-diverging angiosperm lineages. KEY RESULTS: In contrast to previously published magnoliid plastomes and the newly sequenced Saruma henryi plastome published here, Asarum plastomes have undergone extensive disruption and contain extremely lengthy AT-repeat regions. The entirety of the small single copy region (SSC) of A. canadense and A. sieboldii var. sieboldii has been incorporated into the inverted repeat regions (IR), and the SSC of A. delavayi is only 14 bp long. All sampled Asarum plastomes share an inversion of a large portion of the large single copy region (LSC) such that trnE-UUC is adjacent to the LSC-IR boundary. CONCLUSIONS: Plastome divergence in Asarum appears to be consistent with trends seen in highly rearranged plastomes of the monocots and eudicots. We propose that plastome instability in Asarum is due to repetitive motifs that serve as recombinatory substrates and reduce genome stability.

Putative floral brood-site mimicry, loss of autonomous selfing, and reduced vegetative growth are significantly correlated with increased diversification in Asarum (Aristolochiaceae).

The drivers of angiosperm diversity have long been sought and the flower-arthropod association has often been invoked as the most powerful driver of the angiosperm radiation. We now know that features that influence arthropod interactions cannot only affect the diversification of lineages, but also expedite or constrain their rate of extinction, which can equally influence the observed asymmetric richness of extant angiosperm lineages. The genus Asarum (Aristolochiaceae; ∼100 species) is widely distributed in north temperate forests, with substantial vegetative and floral divergence between its three major clades, Euasarum, Geotaenium, and Heterotropa. We used Binary-State Speciation and Extinction Model (BiSSE) Net Diversification tests of character state distributions on a Maximum Likelihood phylogram and a Coalescent Bayesian species tree, inferred from seven chloroplast markers and nuclear rDNA, to test for signal of asymmetric diversification, character state transition, and extinction rates of floral and vegetative characters. We found that reduction in vegetative growth, loss of autonomous self-pollination, and the presence of putative fungal-mimicking floral structures are significantly correlated with increased diversification in Asarum. No significant difference in model likelihood was identified between symmetric and asymmetric rates of character state transitions or extinction. We conclude that the flowers of the Heterotropa clade may have converged on some aspects of basidiomycete sporocarp morphology and that brood-site mimicry, coupled with a reduction in vegetative growth and the loss of autonomous self-pollination, may have driven diversification within Asarum.

Phylogenetic relationships in Asarum: Effect of data partitioning and a revised classification.

PREMISE OF THE STUDY: Generic boundaries and infrageneric relationships among the charismatic temperate magnoliid Asarum sensu lato (Aristolochiaceae) have long been uncertain. Previous molecular phylogenetic analyses used either plastid or nuclear loci alone and varied greatly in their taxonomic implications for the genus. We analyzed additional molecular markers from the nuclear and plastid genomes, reevaluated the possibility of a derived loss of autonomous self-pollination, and investigated the topological effects of matrix-partitioning-scheme choice. METHODS: We sequenced seven plastid regions and the nuclear ITS1-ITS2 region of 58 individuals representing all previously recognized Asarum s.l. segregate genera and the monotypic genus Saruma. Matrices were partitioned using common a priori partitioning schemes and PartitionFinder. KEY RESULTS: Topologies that were recovered using a priori partitioning of matrices differed from those recovered using a PartitionFinder-selected scheme, and by analysis method. We recovered six monophyletic groups that we circumscribed into three subgenera and six sections. Putative fungal mimic characters served as synapomorphies only for subgenus Heterotropa. Subgenus Geotaenium, a new subgenus, was recovered as sister to the remainder of Asarum by ML analyses of highly partitioned datasets. Section Longistylis, also newly named, is sister to section Hexastylis. CONCLUSIONS: Our analyses do not unambiguously support a single origin for all fungal-mimicry characters. Topologies recovered through the analysis of PartitionFinder-optimized matrices can differ drastically from those inferred from a priori partitioned matrices, and by analytical method. We recommend that investigators evaluate the topological effects of matrix partitioning using multiple methods of phylogenetic reconstruction.

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.

Freshwater mussels (Unionidae) brought into captivity exhibit up-regulation of genes involved in stress and energy metabolism.

Approximately two thirds of freshwater mussel species in the United States and Canada are imperiled, and populations are declining rapidly. Translocation and captive management are commonly used to mitigate losses of freshwater mussel biodiversity, but these conservation tools may result in decreased growth and increased mortality. This study uses RNA-Seq to determine how translocation into captivity affects gene expression in Amblema plicata. Mussels were collected from the Muskingum River in Ohio, USA and brought into a captive holding facility. RNA was extracted from gill tissue 11 months post translocation from mussels in captivity and the Muskingum River on the same day. RNA was sequenced on an Illumina HiSeq 2500, and differential expression analysis was performed on de novo assembled transcripts. More than 1200 transcripts were up-regulated in captive mussels, and 246 were assigned functional annotations. Many up-regulated transcripts were involved in energy metabolism and the stress response, such as heat shock proteins and antioxidants. More than 500 transcripts were down-regulated in captive mussels, and 41 were assigned functional annotations. We observed an over-representation of down-regulated transcripts associated with immune response. Our work suggests that A. plicata experienced moderate levels of stress and altered energy metabolism and immune response for at least 11 months post translocation into captivity.

Ancient Polyploidy and Genome Evolution in Palms.

Mechanisms of genome evolution are fundamental to our understanding of adaptation and the generation and maintenance of biodiversity, yet genome dynamics are still poorly characterized in many clades. Strong correlations between variation in genomic attributes and species diversity across the plant tree of life suggest that polyploidy or other mechanisms of genome size change confer selective advantages due to the introduction of genomic novelty. Palms (order Arecales, family Arecaceae) are diverse, widespread, and dominant in tropical ecosystems, yet little is known about genome evolution in this ecologically and economically important clade. Here, we take a phylogenetic comparative approach to investigate palm genome dynamics using genomic and transcriptomic data in combination with a recent, densely sampled, phylogenetic tree. We find conclusive evidence of a paleopolyploid event shared by the ancestor of palms but not with the sister clade, Dasypogonales. We find evidence of incremental chromosome number change in the palms as opposed to one of recurrent polyploidy. We find strong phylogenetic signal in chromosome number, but no signal in genome size, and further no correlation between the two when correcting for phylogenetic relationships. Palms thus add to a growing number of diverse, ecologically successful clades with evidence of whole-genome duplication, sister to a species-poor clade with no evidence of such an event. Disentangling the causes of genome size variation in palms moves us closer to understanding the genomic conditions facilitating adaptive radiation and ecological dominance in an evolutionarily successful, emblematic tropical clade.