Remarkable variation in androecial morphology is closely associated with corolla traits in Western Hemisphere Justiciinae (Acanthaceae: Justicieae).

BACKGROUND AND AIMS: Few studies of angiosperms have focused on androecial evolution in conjunction with evolutionary shifts in corolla morphology and pollinator relationships. The Western Hemisphere clade of Justiciinae (Acanthaceae) presents the rare opportunity to examine remarkable diversity in staminal morphology. We took a phylogenetically informed approach to examine staminal diversity in this hypervariable group and asked whether differences in anther thecae separation is associated with phylogenetically informed patterns of variation in corolla morphology. We further discuss evidence for associations between anther diversity and pollinators in this lineage. METHODS: For the Dianthera/Sarotheca/Plagiacanthus (DSP) clade of Western Hemisphere Justiciinae, we characterized floral diversity based on a series of corolla measurements and using a model-based clustering approach. We then tested for correlations between anther thecae separation and corolla traits, and for shifts in trait evolution, including evidence for convergence. KEY RESULTS: There is evolutionary vagility in corolla and anther traits across the DSP clade with little signal of phylogenetic constraint. Floral morphology clusters into four distinct groups that are, in turn, strongly associated with anther thecae separation, a novel result in Acanthaceae and, to our knowledge, across flowering plants. These cluster groups are marked by floral traits that strongly point to associations with pollinating animals. Specifically, species that are known or likely to be hummingbird pollinated have stamens with parallel thecae, whereas those that are likely bee or fly pollinated have stamens with offset, divergent thecae. CONCLUSIONS: Our results suggest that anther thecae separation is likely under selection in concert with other corolla characters. Significant morphological shifts detected by our analyses corresponded to putative shifts from insect to hummingbird pollination. Results from this study support the hypothesis that floral structures function in an integrated manner and are likely subject to selection as a suite. Further, these changes can be hypothesized to represent adaptive evolution.

A RADseq phylogeny of Barleria (Acanthaceae) resolves fine-scale relationships.

Barleria is a genus of approximately 300 species of herbs, shrubs or, rarely, trees, that is broadly distributed across the Paleotropics. The genus is especially diverse in Tanzania, Angola, and Madagascar. A recent molecular study sampled 53 Barleria species and gathered data for five molecular markers (i.e., four chloroplast loci and the nuclear nrITS) to find support for the recognition of two subgenera previously circumscribed based on morphology, subg. Barleria and subg. Prionitis. That study further reconstructed four previously recognized sections (i.e., Fissimura, Prionitis, Somalia, Stellatohirta) as monophyletic, while three others (i.e., Barleria, Cavirostrata, Chrysothrix) were recovered as para- or polyphyletic. The present study aimed to reconstruct phylogenetic relationships within Barleria based on a broader sample of taxa and many more characters. We sampled 190 accessions representing 184 taxa, including varieties and subspecies. The dataset includes 167 of the ca. 300 species currently recognized or about 56% of total species diversity. We relied heavily on herbarium specimens to sample across the taxonomic breadth and geographic range of Barleria. Single nucleotide polymorphism data were generated using double-digest restriction-site associated DNA sequencing (ddRADseq). The maximum likelihood phylogeny corroborated the topology estimated from the chloroplast and nrITS data, but with greatly increased resolution and support for fine-scale relationships. A coalescent analysis failed to resolve distant evolutionary relationships across Barleria and between Barleria and outgroups, but recovered the same or similar topologies within each Barleria section. Importantly, the ddRADseq phylogeny recovered seven major lineages within subg. Barleria and resolved a polytomy that included B. cristata, the type species of the genus. The topology suggests at least four independent dispersal events to Madagascar followed by three subsequent radiations. Our results broadly inform our understanding of diversity and evolution in one of the largest genera of Acanthaceae, representing an important step towards a stable subgeneric classification for the genus.

Phylosymbiosis in the Rhizosphere Microbiome Extends to Nitrogen Cycle Functional Potential.

Most plants rely on specialized root-associated microbes to obtain essential nitrogen (N), yet not much is known about the evolutionary history of the rhizosphere-plant interaction. We conducted a common garden experiment to investigate the plant root-rhizosphere microbiome association using chloridoid grasses sampled from around the world and grown from seed in a greenhouse. We sought to test whether plants that are more closely related phylogenetically have more similar root bacterial microbiomes than plants that are more distantly related. Using metagenome sequencing, we found that there is a conserved core and a variable rhizosphere bacterial microbiome across the chloridoid grasses. Additionally, phylogenetic distance among the host plant species was correlated with bacterial community composition, suggesting the plant hosts prefer specific bacterial lineages. The functional potential for N utilization across microbiomes fluctuated extensively and mirrored variation in the microbial community composition across host plants. Variation in the bacterial potential for N fixation was strongly affected by the host plants' phylogeny, whereas variation in N recycling, nitrification, and denitrification was unaffected. This study highlights the evolutionary linkage between the N fixation traits of the microbial community and the plant host and suggests that not all functional traits are equally important for plant-microbe associations.

Phylogenomics of Salvia L. subgenus Calosphace (Lamiaceae).

The evolutionary relationships of Salvia have been difficult to estimate. In this study, we used the Next Generation Sequencing method Hyb-Seq to evaluate relationships among 90 Lamiaceae samples, including representatives of Mentheae, Ocimeae, Salvia subgenera Audibertia, Leonia, Salvia, and 69 species of subgenus Calosphace, representing 32 of Epling's sections. A bait set was designed in MarkerMiner using available transcriptome data to enrich 119 variable nuclear loci. Nuclear and chloroplast loci were assembled with hybphylomaker (HPM), followed by coalescent approach analyses for nuclear data (ASTRAL, BEAST) and a concatenated Maximum Likelihood analysis of chloroplast loci. The HPM assembly had an average of 1,314,368 mapped reads for the sample and 527 putative exons. Phylogenetic inferences resolved strongly supported relationships for the deep-level nodes, agreeing with previous hypotheses which assumed that subgenus Audibertia is sister to subgenus Calosphace. Within subgenus Calosphace, we recovered eight monophyletic sections sensu Epling, Cardinalis, Hastatae, Incarnatae, and Uricae in all the analyses (nDNA and cpDNA), Biflorae, Lavanduloideae, and Sigmoideae in nuclear analyses (ASTRAL, BEAST) and Curtiflorae in ASTRAL trees. Network analysis supports deep node relationships, some of the main clades, and recovers reticulation within the core Calosphace. The chloroplast phylogeny resolved deep nodes and four monophyletic Calosphace sections. Placement of S. axillaris is distinct in nuclear evidence and chloroplast, as sister to the rest of the S. subg. Calosphace in chloroplast and a clade with "Hastatae clade" sister to the rest of the subgenus in nuclear evidence. We also tested the monophyly of S. hispanica, S. polystachia, S. purpurea, and S. tiliifolia, including two samples of each, and found that S. hispanica and S. purpurea are monophyletic. Our baits can be used in future studies of Lamiaceae phylogeny to estimate relationships between genera and among species. In this study, we presented a Hyb-Seq phylogeny for complex, recently diverged Salvia, which could be implemented in other Lamiaceae.

Evolutionary history of chloridoid grasses estimated from 122 nuclear loci.

Chloridoideae (chloridoid grasses) are a subfamily of ca. 1700 species with high diversity in arid habitats. Until now, their evolutionary relationships have primarily been studied with DNA sequences from the chloroplast, a maternally inherited organelle. Next-generation sequencing is able to efficiently recover large numbers of nuclear loci that can then be used to estimate the species phylogeny based upon bi-parentally inherited data. We sought to test our chloroplast-based hypotheses of relationships among chloridoid species with 122 nuclear loci generated through targeted-enrichment next-generation sequencing, sometimes referred to as hyb-seq. We targeted putative single-copy housekeeping genes, as well as genes that have been implicated in traits characteristic of, or particularly labile in, chloridoids: e.g., drought and salt tolerance. We recovered ca. 70% of the targeted loci (122 of 177 loci) in all 47 species sequenced using hyb-seq. We then analyzed the nuclear loci with Bayesian and coalescent methods and the resulting phylogeny resolves relationships between the four chloridoid tribes. Several novel findings with this data were: the sister lineage to Chloridoideae is unresolved; Centropodia+Ellisochloa are excluded from Chloridoideae in phylogenetic estimates using a coalescent model; Sporobolus subtilis is more closely related to Eragrostis than to other species of Sporobolus; and Tragus is more closely related to Chloris and relatives than to a lineage of mainly New World species. Relationships in Cynodonteae in the nuclear phylogeny are quite different from chloroplast estimates, but were not robust to changes in the method of phylogenetic analysis. We tested the data signal with several partition schemes, a concatenation analysis, and tests of alternative hypotheses to assess our confidence in this new, nuclear estimate of evolutionary relationships. Our work provides markers and a framework for additional phylogenetic studies that sample more densely within chloridoid tribes. These results represent progress towards a robust classification of this important subfamily of grasses, as well as proof-of-concept for hyb-seq next-generation sequencing as a method to generate sequences for phylogenetic analyses in grasses and other plant families.

Independent allopolyploidization events preceded speciation in the temperate and tropical woody bamboos.

The objectives of the current study were to investigate the origin of polyploidy in the woody bamboos and examine putative hybrid relationships in one major lineage (the temperate woody bamboos, tribe Arundinarieae). Phylogenetic analyses were based on sequence data from three nuclear loci and 38 species in 27 genera. We identify six ancestral genome donors for contemporary bamboo lineages: temperate woody bamboos (tribe Arundinarieae) contain genomes A and B, tropical woody bamboos (tribe Bambuseae) contain genomes C and D, and herbaceous bamboos (tribe Olyreae) contain genome H; some hexaploid paleotropical bamboos contain genome E in addition to C and D. Molecular data indicate that allopolyploidy arose independently in temperate (AABB) and tropical woody lineages (CCDD and CCDDEE), and speciation occurred subsequent to polyploidization. Moreover, hybridization has played a surprising and recurrent role in bamboo evolution, generating allohexaploid species in the paleotropical clade and intergeneric hybrids among the allotetraploid temperate bamboos. We suggest this complex history of reticulate evolution is at least partially responsible for the taxonomic difficulty associated with the woody bamboos. This newly-resolved phylogenetic framework reflects a major step forward in our understanding of bamboo biodiversity and has important implications for the interpretation of bamboo phylogenomics.

The D4 set: primers that target highly variable intron loops in plant chloroplast genomes.

Chloroplast group II introns offer high-quality, rapidly evolving single-copy loci for comparative sequence analysis. These introns feature diagnostic secondary structures with loops that are among the least evolutionarily constrained sequence in plastomes. We exploited these structures to develop universal primers that amplify and sequence the large Domain IV (D4) loop in several angiosperm introns. With a single sequence read, we recover 300-600 nucleotides of highly variable sequence across angiosperms, with rates of change that are equal to or higher than many of the best known intergenic spacers in plant chloroplast genomes.