Search

Phylogenomic datasets using genomes and transcriptomes provide rich opportunities beyond resolving bifurcating phylogenetic relationships. Monkeyflower (Phrymaceae) is a model system for evolutionary ecology. However, it lacks a well-supported phylogeny as a basis for a stable taxonomy and for macroevolutionary comparisons.We sampled 24 genomes and transcriptomes in Phrymaceae and closely related families, including eight newly sequenced transcriptomes. We reconstructed the phylogeny using IQ-TREE and ASTRAL, evaluated gene tree discordance using PhyParts, Quartet Sampling, and a cloudogram, and carried out reticulation analyses using PhyloNet and HyDe. We searched for whole genome duplication (WGD) events using chromosome numbers, synonymous distances, and gene duplication events as evidence.Most gene trees support the monophyly of Phrymaceae and each of its tribes. Most gene trees also support tribe Mimuleae being sister to Phrymeae + Diplaceae + Leucocarpeae, with extensive gene tree discordance among the latter three. Despite the discordance, the monophyly of Mimulus s.l. is rejected, and no individual reticulation event among the Phrymaceae tribes is well-supported. Reticulation likely occurred among Erythranthe bicolor and closely related species. No ancient WGD was detected in Phrymaceae. Instead, small-scale duplications are among potential drivers of macroevolutionary diversification of Phrymaceae.We show that analysis of reticulate evolution is sensitive to taxon sampling and methods used. We also demonstrate that phylogenomic datasets using genomes and transcriptomes present rich opportunities to investigate gene family evolution and genome duplication events involved in lineage diversification and adaptation.

Premise Among the sophisticated trap types in carnivorous plants, the underground eel traps of corkskrew plants (Genlisea spp., Lentibulariaceae) are probably the least understood in terms of their functional principle. Here, we provide a detailed analysis of structural and hydraulic features of G. hispidula traps, contributing to the ongoing debate on whether these traps can actively generate water streams to promote prey capture. Methods Anatomical and hydraulic traits of detached traps, including inner trap diameters, chamber line element, hair length, glandular pattern, and hydraulic conductivity, were investigated quantitatively using light and electron microscopy, x‐ray microtomography, and hydraulic measurements. Results Hydraulic resistivity in the neck of the trap, from the trap mouth toward the vesicle (digestive chamber) was 10 times lower than in the opposite direction. The comparison of measured and theoretical flow rates suggests that the retrorse hairs inside trap necks also provide considerable resistance against movement of matter toward the vesicle. Hairs showed a gradient in length along the neck, with the shortest hairs near the vesicle. Co‐occurrence of quadrifid and bifid glands was limited to a small part of the neck, with quadrifids near the vesicle and bifids toward the trap mouth. Conclusions The combination of structural gradients with hydraulic anisotropy suggests the trap is a highly fine‐tuned system based on likely trade‐offs between efficient prey movement in the trap interior toward the vesicle, prey retention, and spatial digestion capacities and is not counter to the generation of water streams., publishedVersion

PREMISE OF THE STUDY The Neotropical Gesneriaceae is a lineage known for its colorful and diverse flowers, as well as an extensive history of intra- and intergeneric hybridization, particularly among Achimenes (the magic flowers) and other members of subtribe Gloxiniinae. Despite numerous studies seeking to elucidate the evolutionary relationships of these lineages, relatively few have sought to infer specific patterns of gene flow despite evidence of widespread hybridization. METHODS To explore the utility of phylogenomic data for reassessing phylogenetic relationships and inferring patterns of gene flow among species of Achimenes, we sequenced 12 transcriptomes. We used a variety of methods to infer the species tree, examine gene tree discordance, and infer patterns of gene flow. KEY RESULTS Phylogenomic analyses resolve clade relationships at the crown of the lineage with strong support. In contrast to previous analyses, we recovered strong support for several new relationships despite a significant amount of gene tree discordance. We present evidence for at least two introgression events between two species pairs that share pollinators, and suggest that the species status of Achimenes admirabilis be reexamined. CONCLUSIONS Our study demonstrates the utility of transcriptome data for phylogenomic analyses, and inferring patterns of gene flow despite gene tree discordance. Moreover, these data provide another example of prevalent interspecific gene flow among Neotropical plants that share pollinators.

Premise Closely related species occurring in sympatry may experience the negative consequences of interspecific pollen transfer if reproductive isolation (RI) barriers are not in place. We evaluated the importance of pre- and post-pollination RI barriers in three sympatric species of Achimenes (Gesneriaceae), including ecogeographic, phenological, floral isolation, self-pollination, and hybrid viability (fruit and seed set). Methods We recorded geographic distribution throughout species ranges and assessed flowering phenology and pollinator visitation at one site in central Mexico. In the greenhouse, we measured floral traits involved in RI and quantified fruit and seed set for from self, intraspecific, and interspecific crosses. Results Ecogeographic barriers were important in RI, but under sympatry, phenological and floral barriers contributed more to total RI. Phenological RI varied between species and years, while floral RI was 100% effective at preventing interspecific visitation. Species showed differences in floral morphology, color, and scents associated with specialized pollination systems (A. antirrhina-hummingbirds, A. flava-bees, A. patens-butterflies); heterospecific visitation events were restricted to rare secondary pollinators. Hybrid crosses consistently yielded progeny in lower numbers than intraspecific crosses. Conclusions This study indicated that neither autogamy nor early post-pollination barriers prevent interspecific pollen flow between Achimenes species. However, floral isolation, acting through a combination of attraction and reward traits, consistently ensures specificity of the pollination system. These results suggest that selection on floral traits to reduce the costs of hybrid progeny production may have played a role in evolution or maintenance of specialized pollination systems in Achimenes.

PREMISE Gesneria pauciflora is a rare, threatened plant in riparian forests. Periodic disturbances, expected in this habitat, could influence demographic dynamics on plant populations, yet their impact may not be the same across the watershed. We hypothesized that differences in disturbances between the main channel and tributaries may lead to spatial dissimilarities in population growth rate (λ), structure, and fecundity. METHODS In the Maricao River Watershed in Puerto Rico, 1277 plants were tagged and monitored for 1.5 years. Every 6 months, we measured plant size and recorded survival, fecundity, and appearance of seedlings. These variables were used in integral projection models to assess the population status of G. pauciflora. RESULTS Plants in the main channel were smaller but more likely to flower and fruit than those in the tributaries. Overall mortality was greater in the main channel and greater during the rainy season. At both sites, λ ranged from 0.9114 to 0.9865, and survival/growth of larger plants had a greater effect on λ (>0.90) regardless of site. CONCLUSIONS Values for population growth rates suggest that G. pauciflora is declining across the watershed. Higher mortality rates in the main channel (more-perturbed sites) might drive G. pauciflora to reproduce at smaller sizes, while tributaries (less-perturbed sites) might be better for growth and lead to larger plant sizes. Extreme climatic events are expected to increase in the Caribbean and might decrease the population if the population is left unmanaged. Management strategies that reduce the time plants require to reach larger sizes might be necessary to increase λ, and reintroduction using cuttings might be a possible solution.

Premise of the study Bignoniaceae is an important component of neotropical forests and a model for evolutionary and biogeographical studies. A previous combination of molecular markers and morphological traits improved the phylogeny of the group. Here we demonstrate the value of next-generation sequencing (NGS) to assemble the chloroplast genome of eight Anemopaegma species and solve taxonomic problems. Methods Three NGS platforms were used to sequence total DNA of Anemopaegma species. After genome assembly and annotation, we compared chloroplast genomes within Anemopaegma, with other Lamiales species, and the evolutionary rates of protein-coding genes using Tanaecium tetragonolobum as the outgroup. Phylogenetic analyses of Anemopaegma with different data sets were performed. Key results Chloroplast genomes of Anemopaegma species ranged from 167,413 bp in A. foetidum to 168,987 bp in A. acutifolium ("typical" form). They exhibited a characteristic quadripartite structure with a large single-copy region (75,070-75,761 bp), a small single-copy region (12,766-12,817 bp) and a pair of inverted repeat regions (IRs) (39,480-40,481) encoding an identical set of 112 genes. An inversion of a fragment with ca. 8 kb, located in the IRs and containing the genes trnI-AAU, ycf2, and trnL-CAA, was observed in these chloroplast genomes when compared with those of other Lamiales. Conclusions Anemopaegma species have the largest genomes within the Lamiales possibly due to the large amount of repetitive sequences and IR expansion. Variation was higher in coding regions than in noncoding regions, and some genes were identified as markers for differentiation between species. The use of the entire chloroplast genome gave better phylogenetic resolution of the taxonomic groups. We found that two forms of A. acutifolium result from different maternal lineages.

A well-resolved species level phylogeny is critically important in studying organismal evolution (e.g., hybridization, polyploidization, adaptive speciation). Lack of appropriate molecular markers that give sufficient resolution to gene trees is one of the major impediments to inferring species level phylogenies. In addition, sampling multiple independent loci is essential to overcome the lineage sorting problem. The availability of nuclear loci has often been a limiting factor in plant species-level phylogenetic studies. Here the two PHOT loci were developed as new sources of nuclear gene trees. The PHOT1 and PHOT2 gene trees of the Verbena complex (Verbenaceae) are well resolved and have good clade support. These gene trees are consistent with each other and previously generated chloroplast and nuclear waxy gene trees in most of the phylogenetic backbone as well as some terminal relationships, but are incongruent in some other relationships. Locus-specific primers were optimized for amplifying and sequencing these two loci in all Lamiales. Comparing intron size in the context of the gene trees shows dramatic variation within the Verbena complex, particularly at the PHOT1 locus. These variations are largely caused by invasions of short transposable elements and frequent long deletions and insertions of unknown causes. In addition, inspection of DNA sequences and phylogenetic analyses unmask a clear footprint of ancestral recombination in one species.

2,4 PREMISE OF THE STUDY: CYCLOIDEA2 ( CYC2 )-like and RADIALIS ( RAD )-like genes are needed for the normal development of corolla bilateral symmetry in Antirrhinum majus L. (snapdragon, Plantaginaceae, Lamiales). However, if and how changes in expression of CYC2 -like and RAD -like genes correlate with the origin of corolla bilateral symmetry early in Lamiales remains largely unknown. The asymmetrical expression of CYC2 -like and/or RAD -like genes during fl oral meristem development could be ancestral or derived in Plantaginaceae. METHODS: We used in situ RNA localization to examine the expression of CYC2 -like and RAD -like genes in two early-diverging Lamiales. KEY RESULTS: CYC2 -like and RAD -like genes are expressed broadly in the fl oral meristems in early-diverging Lamiales with radially symmetrical corollas, in contrast to their restricted expression in adaxial/lateral regions in core Lamiales. The expression pattern of CYC2 -like genes has evolved in stepwise fash- ion, in that CYC2 -like genes are likely expressed briefl y in the fl oral meristem during fl ower development in sampled Oleaceae; prolonged expression of CYC2 -like genes in petals originated in the common ancestor of Tetrachondraceae and core Lamiales, and asymmetrical expression in adaxial/lateral pet- als appeared later, in the common ancestor of the core Lamiales. Likewise, expression of RAD -like genes in petals appeared in early-diverging Lamiales or earlier; asymmetrical expression in adaxial/lateral petals then appeared in core Lamiales. CONCLUSIONS: These data plus published reports of CYC2 -like and RAD -like genes show that asymmetrical expression of these two genes is likely derived and correlates with the origins of corolla bilateral symmetry.

Chloroplast trnL/F and nuclear ribosomal ITS and ETS sequence data were used to analyze phylogenetic relationships among members of tribe Mimuleae (Scrophulariaceae) and other closely related families in Lamiales. The results of these analyses led to the following conclusions. (1) The Australian genera Glossostigma and Peplidium and the taxonomically isolated Phryma join four genera of tribe Mimuleae to form a well-supported clade that is distinct from other families in the Lamiales. We refer to that clade as the subfamily Phrymoideae. (2) The genera Mazus and Lancea (tribe Mimuleae) together form a well-supported clade that we recognize as the subfamily Mazoideae. Mazoideae is weakly supported as sister to Phrymoideae. We assign Mazoideae and Phrymoideae to a redefined family Phrymaceae. (3) Mimulus is not monophyletic, because members of at least six other genera have been derived from within it. In light of the molecular evidence, it is clear that species of Phrymaceae (about 190 species) have undergone two geographically distinct radiations; one in western North America (about 130 species) and another in Australia (about 30 species). Phylogenetic interpretations of morphological evolution and biogeographical patterns are discussed.

• Premise of the study: The Lamiidae, a clade composed of approximately 15% of all flowering plants, consists of five orders: Boraginales, Gentianales, Garryales, Lamiales, and Solanales; and four families unplaced in an order: Icacinaceae, Metteniusiaceae, Oncothecaceae, and Vahliaceae. Our understanding of the phylogenetic relationships of Lamiidae has improved significantly in recent years, however, relationships among the orders and unplaced families of the clade remain partly unresolved. Here, we present a phylogenetic analysis of the Lamiidae based on an expanded sampling, including all families together, for the first time, in a single phylogenetic analyses.• Methods: Phylogenetic analyses were conducted using maximum parsimony, maximum likelihood, and Bayesian approaches. Analyses included nine plastid regions (atpB, matK, ndhF, psbBTNH, rbcL, rps4, rps16, trnL-F, and trnV-atpE) and the mitochondrial rps3 region, and 129 samples representing all orders and unplaced families of Lamiidae.• Key results: Maximum Likelihood (ML) and Bayesian trees provide good support for Boraginales sister to Lamiales, with successive outgroups (Solanales + Vahlia) and Gentianales, together comprising the core Lamiidae. Early branching patterns are less well supported, with Garryales only poorly supported as sister to the above ‘core’ and a weakly supported clade composed of Icacinaceae, Metteniusaceae, and Oncothecaceae sister to all other Lamiidae.• Conclusions: Our phylogeny of Lamiidae reveals increased resolution and support for internal relationships that have remained elusive. Within Lamiales, greater resolution also is obtained, but some family interrelationships remain a challenge.

A molecular systematic study of Scrophulariaceae sensu lato using DNA sequences of three plastid genes (rbcL, ndhF, and rps2) revealed at least five distinct monophyletic groups. Thirty-nine genera representing 24 tribes of the Scrophulariaceae s.l. (sensu lato) were analyzed along with representatives of 15 other families of Lamiales. The Scrophulariaceae s.s. (sensu stricto) include part or all of tribes Aptosimeae, Hemimerideae, Leucophylleae, Manuleae, Selagineae, and Verbasceae (5 Scrophularieae) and the conventional families Buddlejaceae and Myoporaceae. Veronicaceae includes all or part of tribes Angelonieae, Antirrhineae, Cheloneae, Digitaleae, and Gratioleae and the conventional families Callitrichaceae, Globulariaceae, Hippuridaceae, and Plantaginaceae. The Orobanchaceae include tribes Buchnereae, Rhinantheae, and the conventional Orobanchaceae. All sampled members of Orobanchaceae are parasitic, except Lindenbergia, which is sister to the rest of the family. Family Calceolariaceae Olmstead is newly erected herein to recognize the phylogenetic distinctiveness of tribe Calceolarieae. The Calceolariaceae are close to the base of the Lamiales. The Stilbaceae are expanded by the inclusion of Halleria. Mimulus does not belong in any of these five groups.

We used sequence data from the intron and spacer of the trnL-trnF chloroplast region to study phylogenetic relationships among Acanthaceae. This region is more variable than other chloroplast loci that have been sequenced for members of Acanthaceae (rbcL and ndhF), is more prone to length mutations, and is less homoplasious than these genes. Our results indicate that this region is likely to be useful in addressing phylogenetic questions among but not within genera in these and related plants. In terms of phylogenetic relationships, Elytraria (representing Nelsonioideae) is more distantly related to Acanthaceae sensu stricto (s.s.) than Thunbergia and Mendoncia. These last two genera are strongly supported as sister taxa. Molecular evidence does not support monophyly of Acanthaceae s.s., although there is strong morphological evidence for this relationship. There is strong support for monophyly of four major lineages within Acanthaceae s.s.: the Acanthus, Barleria, Ruellia, and Justicia lineages as here defined. The last three of these comprise a strongly supported monophyletic group, and there is weaker evidence linking the Ruellia and Justicia lineages as closest relatives. Within the Acanthus lineage, our results confirm the existence of monophyletic lineages representing Aphelandreae and Acantheae. Lastly, within the Justicia lineage, we develop initial hypotheses regarding the definition of sublineages; some of these correspond to earlier ideas, whereas others do not. All of these hypotheses need to be tested against more data. Acanthaceae are a large family (.4000 species; Mabberley, 1987), the members of which present a rich diversity of morphological and ecological characteristics. The family is part of Lamiales s.l. (sensu lato) (i.e., sensu Olmstead et al., 1993). Unlike a number of family-level taxa in Lamiales (e.g., Lamiaceae, Verbenaceae, Scrophulariaceae), there seems little doubt that Acanthaceae s.s. (sensu stricto, see below) are monophyletic. This hypothesis is supported by the shared presence of a fruit type that is unique among angiosperms: a few-seeded, explosively dehiscent capsule within which the seeds are borne on retinacula (the lignified derivatives of funiculae). The precise delimitation of the family, however, has been controversial due to three small lineages that do not 1

Duplication of some floral regulatory genes has occurred repeatedly in angiosperms, whereas others are thought to be single-copy in most lineages. We selected three genes that interact in a pathway regulating floral development conserved among higher tricolpates (LFY/FLO, UFO/FIM, and AP3/DEF) and screened for copy number among families of Lamiales that are closely related to the model species Antirrhinum majus. We show that two of three genes have duplicated at least twice in the Lamiales. Phylogenetic analyses of paralogs suggest that an ancient whole genome duplication shared among many families of Lamiales occurred after the ancestor of these families diverged from the lineage leading to Veronicaceae (including the single-copy species A. majus). Duplication is consistent with previous patterns among angiosperm lineages for AP3/DEF, but this is the first report of functional duplicate copies of LFY/FLO outside of tetraploid species. We propose Lamiales taxa will be good models for understanding mechanisms of duplicate gene preservation and how floral regulatory genes may contribute to morphological diversity.

Acanthaceae (Asteridae; Lamiales) include ∼4000 species and encompass a range of morphological diversity, habitats, and biogeographic patterns. Although they are important components of tropical and subtropical habitats worldwide, inadequate knowledge of the family's phylogenetic framework has impeded comparative research. In this study, we sampled all known lineages of Acanthaceae including Andrographideae. Also included were eight of 13 genera whose relationships remain enigmatic. We used sequence data from nrITS and four chloroplast noncoding regions, and parsimony and Bayesian methods of analysis. Results strongly support most aspects of relationships including inclusion of Avicennia in Acanthaceae. Excepting Neuracanthus, newly sampled taxa are placed with strong support; Kudoacanthus is in Justicieae, Tetramerium lineage, and the remaining enigmatic genera are in Whitfieldieae or Barlerieae, and Andrographideae are sister to Barlerieae. This last result is unanticipated, but placement of Andrographideae based on structural characters has been elusive. Neuracanthus is monophyletic but placement relative to (Whitfieldieae (Andrographideae + Barlerieae)) is weakly supported. Many clades have clear morphological synapomorphies, but nonmolecular evidence for some remains elusive. Results suggest an Old World origin with multiple dispersal events to the New World. This study informs future work by clarifying sampling strategy and identifying aspects of relationships that require further study.

Evaluating the morphological relationships of angiosperm families that still remain unplaced in the current systems of classifi cation is challenging because it requires comparative data across a broad phylogenetic range. The small neotropical family Metteniusaceae was recently placed within the lamiids, as sister to either the enigmatic Oncothecaceae or the clade (Boraginaceae + Gentianales + Lamiales + Solanales + Vahliaceae). We examined the development of two of the primary diagnostic traits of Metteniusaceae, the moniliform anthers and the unilocular gynoecium. The gynoecium is 5-carpellate, and contains two ovules with a massive, vascularized integument. Late sympetaly and unitegmic ovules support placement of Metteniusaceae in the lamiids. The 5-carpellate gynoecium is consistent with a sister-group relationship between Metteniusaceae and Oncothecaceae. The gynoecium of Metteniusaceae is unusual in that it is monosymmetric throughout ontogeny, which indicates pseudomonomery; the fi ve carpel initials are congenitally fused by their margins and form a single locule; the two ovules develop from the two smallest and most poorly developed lateral carpels. Comparisons with other pseudomonomerous taxa allow us to propose division of the complex processes leading to pseudomonomery into eight characters, including carpel number and fusion, gynoecial symmetry, timing of carpel reduction, and number and position of nonfertile carpels.