Revisiting the Zingiberales: Using Multiplexed Exon Capture to Resolve Ancient and Recent Phylogenetic Splits in a Charismatic Plant Lineage

The Zingiberales are an iconic order of monocotyledonous plants comprising eight families with distinctive and diverse floral morphologies and representing an important ecological element of tropical and subtropical forests. While the eight families are demonstrated to be monophyletic, phylogenetic relationships among these families remain unresolved. Neither combined morphological and molecular studies nor recent attempts to resolve family relationships using sequence data from whole plastomes has resulted in a well-supported, family-level phylogenetic hypothesis of relationships. Here we approach this challenge by leveraging the complete genome of one member of the order, Musa acuminata, together with transcriptome information from each of the other seven families to design a set of nuclear loci that can be enriched from highly divergent taxa with a single array-based capture of indexed genomic DNA. A total of 494 exons from 418 nuclear genes were captured for 53 ingroup taxa. The entire plastid genome was also captured for the same 53 taxa. Of the total genes captured, 308 nuclear and 68 plastid genes were used for phylogenetic estimation. The concatenated plastid and nuclear dataset supports the position of Musaceae as sister to the remaining seven families. Moreover, the combined dataset recovers known intra- and inter-family phylogenetic relationships with generally high bootstrap support. This is a flexible and cost effective method that gives the broader plant biology community a tool for generating phylogenomic scale sequence data in non-model systems at varying evolutionary depths

The phylogeny of Heliconia (Heliconiaceae) and the evolution of floral presentation

© 2016 Elsevier Inc. Heliconia (Heliconiaceae, order Zingiberales) is among the showiest plants of the Neotropical rainforest and represent a spectacular co-evolutionary radiation with hummingbirds. Despite the attractiveness and ecological importance of many Heliconia, the genus has been the subject of limited molecular phylogenetic studies. We sample seven markers from the plastid and nuclear genomes for 202 samples of Heliconia. This represents ca. 75% of accepted species and includes coverage of all taxonomic subgenera and sections. We date this phylogeny using fossils associated with other families in the Zingiberales; in particular we review and evaluate the Eocene fossil Ensete oregonense. We use this dated phylogenetic framework to evaluate the evolution of two components of flower orientation that are hypothesized to be important for modulating pollinator discrimination and pollen placement: resupination and erect versus pendant inflorescence habit. Our phylogenetic results suggest that the monophyletic Melanesian subgenus Heliconiopsis and a small clade of Ecuadorian species are together the sister group to the rest of Heliconia. Extant diversity of Heliconia originated in the Late Eocene (39 Ma) with rapid diversification through the Early Miocene, making it the oldest known clade of hummingbird-pollinated plants. Most described subgenera and sections are not monophyletic, though closely related groups of species, often defined by shared geography, mirror earlier morphological cladistic analyses. Evaluation of changes in resupination and inflorescence habit suggests that these characters are more homoplasious than expected, and this largely explains the non-monophyly of previously circumscribed subgenera, which were based on these characters. We also find strong evidence for the correlated evolution of resupination and inflorescence habit. The correlated model suggests that the most recent common ancestor of all extant Heliconia had resupinate flowers and erect inflorescences. Finally, we note our nearly complete species sampling and dated phylogeny allow for an assessment of taxonomic history in terms of phylogenetic diversity. We find approximately half of the currently recognized species, corresponding to half of the phylogenetic diversity, have been described since 1975, highlighting the continued importance of basic taxonomic research and conservation initiatives to preserve both described and undiscovered species of Heliconia

Towards integrative taxonomy in Neotropical botany: Disentangling the Pagamea guianensis species complex (Rubiaceae)

Species complexes are common in the Neotropical flora, and the Pagamea guianensis complex is one of the most widespread groups of species in the Amazonian white-sand flora. Previous analyses suggested the occurrence of ten species in this group, but species limits remained unclear due to poor sampling, morphological overlap and low molecular variation. Here we present the most comprehensive population and molecular sampling across the geographical distribution of the P. guianensis complex to date in order to test the monophyly of this group and to clarify species limits. Using a high-throughput DNA sequencing approach, we sequenced 431 loci (> 34 M bases) for 179 individuals. We applied phylogenetic and species tree analyses to resolve phylogenetic relationships among the sampled individuals. Species delimitation was inferred based on genomic data, and we tested whether hypothesized species could be differentiated using morphological, ecological and near-infrared spectroscopy data. We confirm the monophyly of the P. guianensis complex and accept 15 distinct and well-supported lineages, here proposed as 14 species and one subspecies. Our findings highlight the importance of multiple lines of evidence from independent datasets in the process of species delimitation and species discovery in species complexes in the Neotropics. © The Botanical Journal of the Linnean Society 2018

DNA Barcoding in the Cycadales: Testing the Potential of Proposed Barcoding Markers for Species Identification of Cycads

Barcodes are short segments of DNA that can be used to uniquely identify an unknown specimen to species, particularly when diagnostic morphological features are absent. These sequences could offer a new forensic tool in plant and animal conservation—especially for endangered species such as members of the Cycadales. Ideally, barcodes could be used to positively identify illegally obtained material even in cases where diagnostic features have been purposefully removed or to release confiscated organisms into the proper breeding population. In order to be useful, a DNA barcode sequence must not only easily PCR amplify with universal or near-universal reaction conditions and primers, but also contain enough variation to generate unique identifiers at either the species or population levels. Chloroplast regions suggested by the Plant Working Group of the Consortium for the Barcode of Life (CBoL), and two alternatives, the chloroplast psbA-trnH intergenic spacer and the nuclear ribosomal internal transcribed spacer (nrITS), were tested for their utility in generating unique identifiers for members of the Cycadales. Ease of amplification and sequence generation with universal primers and reaction conditions was determined for each of the seven proposed markers. While none of the proposed markers provided unique identifiers for all species tested, nrITS showed the most promise in terms of variability, although sequencing difficulties remain a drawback. We suggest a workflow for DNA barcoding, including database generation and management, which will ultimately be necessary if we are to succeed in establishing a universal DNA barcode for plants

Diversification of the Core Bromelioids with a focus on the genus Aechmea: phylogeny, morphology, and climate tolerance

Understanding patterns of speciation is of fundamental importance to evolutionary biologists. Species diversity can be maintained and generated through processes originating from interactions with climate, biota, and geography. In order to investigate the role of these factors on diversity in a species-rich group of plants, we developed a phylogeny of the core Bromelioideae including Aechmea and related genera with the specific goals of investigating monophyly of genera, biogeographic history, morphological trait evolution, and environmental niche diversification. The phylogenetic estimation encompassed 20 genera and 4 genomic regions including chloroplast, low-copy, and ribosomal nuclear genes. Subsequently, morphologies were re-explored to understand species relationships that had not been previously recognized and to determine if morphological synapomorphies could define well supported clades. Species were coded as present or absent in 16 geographic regions to reconstruct ancestral areas. Finally, to understand how species within this group divided climatic niche space, research was focused on two clades that diversified independently in Central America. Niche models were generated for each species and for the clades as a whole and were compared for overlap. The distribution of each environmental variable for all occurrence records was compared at each node of divergence within each clade to understand divergence in environmental conditions in a method free from model uncertainty. From these analyses we found: (1) Lack of monophyly in many genera and subgenera (2) Closely related species often were found in geographic sympatry (at the scale of co-occurrence in the same country or state) such that taxonomic groups that had been thought to include geographically disjunct species, actually were representative of distinct evolutionary histories (3) Morphological traits that had been used to assign species to genera were often homoplastic (through convergence or reversals of plesiomorphic states) (4) Although a single unique synapomorphy for each clade was not found, morphological traits were often consistent within clades, providing independent support for the molecular phylogenetic estimation and a basis for future taxonomic work (5) Most well supported clades diversified within one of eight geographic areas, although the ancestral areas for several larger clades remains uncertain (6) Both clades native to Central America are found in a unique environmental niche (i.e. environmental niche models of each clade do not overlap and there is significant divergence of climatic variables between the clades) (7) Species within the clade composed of species with greater morphological variation had more environmental niche overlap than the clade with little morphological variation. These findings add to the evolutionary knowledge of the Bromeliaceae and suggest that geographic range should be taken into account when assigning species to genera. Centers of diversity of the core Bromelioids are found within eastern and Amazonian Brazil, northern south America and Central America and each radiation could have been influenced by climate in a variety of ways. In the two studied examples, species divergence is coincident with divergence in environmental niche occupation. However, environmental niche evolution was limited such that each clade as a whole was not modeled in the niche of the other clade, suggesting either some degree of niche conservatism or competition preventing parapatric niche expansion

Diversification of the Core Bromelioids with a focus on the genus Aechmea: phylogeny, morphology, and climate tolerance

Understanding patterns of speciation is of fundamental importance to evolutionary biologists. Species diversity can be maintained and generated through processes originating from interactions with climate, biota, and geography. In order to investigate the role of these factors on diversity in a species-rich group of plants, we developed a phylogeny of the core Bromelioideae including Aechmea and related genera with the specific goals of investigating monophyly of genera, biogeographic history, morphological trait evolution, and environmental niche diversification. The phylogenetic estimation encompassed 20 genera and 4 genomic regions including chloroplast, low-copy, and ribosomal nuclear genes. Subsequently, morphologies were re-explored to understand species relationships that had not been previously recognized and to determine if morphological synapomorphies could define well supported clades. Species were coded as present or absent in 16 geographic regions to reconstruct ancestral areas. Finally, to understand how species within this group divided climatic niche space, research was focused on two clades that diversified independently in Central America. Niche models were generated for each species and for the clades as a whole and were compared for overlap. The distribution of each environmental variable for all occurrence records was compared at each node of divergence within each clade to understand divergence in environmental conditions in a method free from model uncertainty. From these analyses we found: (1) Lack of monophyly in many genera and subgenera (2) Closely related species often were found in geographic sympatry (at the scale of co-occurrence in the same country or state) such that taxonomic groups that had been thought to include geographically disjunct species, actually were representative of distinct evolutionary histories (3) Morphological traits that had been used to assign species to genera were often homoplastic (through convergence or reversals of plesiomorphic states) (4) Although a single unique synapomorphy for each clade was not found, morphological traits were often consistent within clades, providing independent support for the molecular phylogenetic estimation and a basis for future taxonomic work (5) Most well supported clades diversified within one of eight geographic areas, although the ancestral areas for several larger clades remains uncertain (6) Both clades native to Central America are found in a unique environmental niche (i.e. environmental niche models of each clade do not overlap and there is significant divergence of climatic variables between the clades) (7) Species within the clade composed of species with greater morphological variation had more environmental niche overlap than the clade with little morphological variation. These findings add to the evolutionary knowledge of the Bromeliaceae and suggest that geographic range should be taken into account when assigning species to genera. Centers of diversity of the core Bromelioids are found within eastern and Amazonian Brazil, northern south America and Central America and each radiation could have been influenced by climate in a variety of ways. In the two studied examples, species divergence is coincident with divergence in environmental niche occupation. However, environmental niche evolution was limited such that each clade as a whole was not modeled in the niche of the other clade, suggesting either some degree of niche conservatism or competition preventing parapatric niche expansion

Data from: 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 substitution 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 fill a knowledge gap in models of heterotrophic plastid genome evolution, and have implications for future studies in heterotrophs

Development of Single-Copy Nuclear Intron Markers for Species-Level Phylogenetics: Case Study with Paullinieae (Sapindaceae)

Premise of the study:We developed a bioinformatic pipeline that leverages a publicly available genome and published transcriptomes to design primers in conserved coding sequences flanking targeted introns of single-copy nuclear loci. Paullinieae (Sapindaceae) is used to demonstrate the pipeline.Methods and Results:Transcriptome reads phylogenetically closer to the lineage of interest are aligned to the closest genome. Single-nucleotide polymorphisms are called, generating a “pseudoreference” closer to the lineage of interest. Several filters are applied to meet the criteria of single-copy nuclear loci with introns of a desired size. Primers are designed in conserved coding sequences flanking introns. Using this pipeline, we developed nine single-copy nuclear intron markers for Paullinieae.Conclusions:This pipeline is highly flexible and can be used for any group with available genomic and transcriptomic resources. This pipeline led to the development of nine variable markers for phylogenetic study without generating sequence data de novo

Development of Single-Copy Nuclear Intron Markers for Species-Level Phylogenetics: Case Study with Paullinieae (Sapindaceae)

Premise of the study:We developed a bioinformatic pipeline that leverages a publicly available genome and published transcriptomes to design primers in conserved coding sequences flanking targeted introns of single-copy nuclear loci. Paullinieae (Sapindaceae) is used to demonstrate the pipeline.Methods and Results:Transcriptome reads phylogenetically closer to the lineage of interest are aligned to the closest genome. Single-nucleotide polymorphisms are called, generating a “pseudoreference” closer to the lineage of interest. Several filters are applied to meet the criteria of single-copy nuclear loci with introns of a desired size. Primers are designed in conserved coding sequences flanking introns. Using this pipeline, we developed nine single-copy nuclear intron markers for Paullinieae.Conclusions:This pipeline is highly flexible and can be used for any group with available genomic and transcriptomic resources. This pipeline led to the development of nine variable markers for phylogenetic study without generating sequence data de novo

Corallorhiza_all

Nexus format alignment of captured plastomes of the Corallorhiza striata complex, with Corallorhiza trifida and Calypso bulbosa as outgroup tax