Settling a family feud: a high-level phylogenomic framework for the Gentianales based on 353 nuclear genes and partial plastomes.

PREMISE: Comprising five families that vastly differ in species richness-ranging from Gelsemiaceae with 13 species to the Rubiaceae with 13,775 species-members of the Gentianales are often among the most species-rich and abundant plants in tropical forests. Despite considerable phylogenetic work within particular families and genera, several alternative topologies for family-level relationships within Gentianales have been presented in previous studies. METHODS: Here we present a phylogenomic analysis based on nuclear genes targeted by the Angiosperms353 probe set for approximately 150 species, representing all families and approximately 85% of the formally recognized tribes. We were able to retrieve partial plastomes from off-target reads for most taxa and infer phylogenetic trees for comparison with the nuclear-derived trees. RESULTS: We recovered high support for over 80% of all nodes. The plastid and nuclear data are largely in agreement, except for some weakly to moderately supported relationships. We discuss the implications of our results for the order's classification, highlighting points of increased support for previously uncertain relationships. Rubiaceae is sister to a clade comprising (Gentianaceae + Gelsemiaceae) + (Apocynaceae + Loganiaceae). CONCLUSIONS: The higher-level phylogenetic relationships within Gentianales are confidently resolved. In contrast to recent studies, our results support the division of Rubiaceae into two subfamilies: Cinchonoideae and Rubioideae. We do not formally recognize Coptosapelteae and Luculieae within any particular subfamily but treat them as incertae sedis. Our framework paves the way for further work on the phylogenetics, biogeography, morphological evolution, and macroecology of this important group of flowering plants.

Chemotaxonomic investigation of Apocynaceae for retronecine-type pyrrolizidine alkaloids using HPLC-MS/MS.

Apocynaceae are well known for diverse specialized metabolites that are distributed in a phylogenetically informative manner. Pyrrolizidine alkaloids (PAs) have been reported sporadically in one lineage in the family, the APSA clade, but few species have been studied to date. We conducted the first systematic survey of Apocynaceae for retronecine-type PAs, sampling leaves from 231 species from 13 of 16 major lineages within the APSA clade using HPLC-MS/MS. We also followed up preliminary evidence for infra-specific variation of PA detectability in Echites umbellatus Jacq. Four precursor ion scans (PREC) were developed for a high-throughput survey for chemicals containing a structural moiety common to many PAs, the retronecine core. We identified with high confidence PAs in 7 of 8 sampled genera of tribe Echiteae, but not in samples from the closely related Odontadenieae and Mesechiteae, confirming the utility of PAs as a taxonomic character in tribal delimitation. Occurrence of PAs in Malouetieae is reported with moderate confidence in Galactophora schomburgkiana Woodson and Eucorymbia alba Stapf, but currently we have low confidence of their presence in Holarrena pubescens Wall. ex G. Don (the one Malouetieae species where they were previously reported), as well as in Holarrena curtisii King & Gamble and in Kibatalia macrophylla (Pierre ex Hua) Woodson. Candidate PAs in some species of Wrightia R. Br. (Wrightieae) and Marsdenia R. Br. (Marsdenieae) are proposed with moderate confidence, but a subset of the compounds in these taxa presenting with a PA-like fragmentation pattern are more likely to be aminobenzoyl glycosides. Candidate PAs are reported in species with predicted (VXXXD) and unexpected (IXXXN) amino acid motifs in their homospermidine synthase-like genes. Detectability of PAs varies among samples of Echites umbellatus and intra-individual plasticity contributes to this variation. Of toxicological importance, novel potential sources of human exposure to pro-toxic PAs were identified in the medicinal plant, Wrightia tinctoria R.Br., and the food plants, Marsdenia glabra Cost. and Echites panduratus A. DC., warranting immediate further research to elucidate the structures of the candidate PAs identified. Method development and limitations are discussed.

Enabling evolutionary studies at multiple scales in Apocynaceae through Hyb-Seq.

PREMISE: Apocynaceae is the 10th largest flowering plant family and a focus for study of plant-insect interactions, especially as mediated by secondary metabolites. However, it has few genomic resources relative to its size. Target capture sequencing is a powerful approach for genome reduction that facilitates studies requiring data from the nuclear genome in non-model taxa, such as Apocynaceae. METHODS: Transcriptomes were used to design probes for targeted sequencing of putatively single-copy nuclear genes across Apocynaceae. The sequences obtained were used to assess the success of the probe design, the intrageneric and intraspecific variation in the targeted genes, and the utility of the genes for inferring phylogeny. RESULTS: From 853 candidate nuclear genes, 835 were consistently recovered in single copy and were variable enough for phylogenomics. The inferred gene trees were useful for coalescent-based species tree analysis, which showed all subfamilies of Apocynaceae as monophyletic, while also resolving relationships among species within the genus Apocynum. Intraspecific comparison of Elytropus chilensis individuals revealed numerous single-nucleotide polymorphisms with potential for use in population-level studies. DISCUSSION: Community use of this Hyb-Seq probe set will facilitate and promote progress in the study of Apocynaceae across scales from population genomics to phylogenomics.

The diversity and evolution of pollination systems in large plant clades: Apocynaceae as a case study.

Background and Aims: Large clades of angiosperms are often characterized by diverse interactions with pollinators, but how these pollination systems are structured phylogenetically and biogeographically is still uncertain for most families. Apocynaceae is a clade of >5300 species with a worldwide distribution. A database representing >10 % of species in the family was used to explore the diversity of pollinators and evolutionary shifts in pollination systems across major clades and regions. Methods: The database was compiled from published and unpublished reports. Plants were categorized into broad pollination systems and then subdivided to include bimodal systems. These were mapped against the five major divisions of the family, and against the smaller clades. Finally, pollination systems were mapped onto a phylogenetic reconstruction that included those species for which sequence data are available, and transition rates between pollination systems were calculated. Key Results: Most Apocynaceae are insect pollinated with few records of bird pollination. Almost three-quarters of species are pollinated by a single higher taxon (e.g. flies or moths); 7 % have bimodal pollination systems, whilst the remaining approx. 20 % are insect generalists. The less phenotypically specialized flowers of the Rauvolfioids are pollinated by a more restricted set of pollinators than are more complex flowers within the Apocynoids + Periplocoideae + Secamonoideae + Asclepiadoideae (APSA) clade. Certain combinations of bimodal pollination systems are more common than others. Some pollination systems are missing from particular regions, whilst others are over-represented. Conclusions: Within Apocynaceae, interactions with pollinators are highly structured both phylogenetically and biogeographically. Variation in transition rates between pollination systems suggest constraints on their evolution, whereas regional differences point to environmental effects such as filtering of certain pollinators from habitats. This is the most extensive analysis of its type so far attempted and gives important insights into the diversity and evolution of pollination systems in large clades.

HPLC-MS detection of pyrrolizidine alkaloids and their N-oxides in herbarium specimens dating back to the 1850s.

PREMISE OF THE STUDY: Understanding the phylogenetic distribution of defensive plant secondary metabolites is essential to the macroevolutionary study of chemically mediated plant-animal interactions. The chemical ecology of pyrrolizidine alkaloids (PAs) has been extensively studied in a number of plant-herbivore systems, including Apocynaceae (the milkweed and dogbane family) and Danainae (the milkweed and clearwing butterflies). A systematic survey is necessary to establish a detailed understanding of their occurrence across Apocynaceae. A survey of this species-rich, mainly tropical and subtropical family will rely heavily on small tissue samples removed from herbarium specimens, some of which will be very old and/or preserved with alcohols or mercuric chloride. METHODS: We optimized PA extraction methods from small leaf fragments of recently collected silica-dried leaves of the PA-positive Echites umbellatus, varying crushing and extraction time. We then applied our optimized method to leaf fragments from 70-167-year-old herbarium specimens of E. umbellatus. To simulate the effect of alcohol treatment on PA detectability in herbarium specimens, we incubated freshly collected leaves of the PA-positive Parsonsia alboflavescens in three different alcohols before drying and compared PA recovery to freshly dried controls. PAs were quantified using high-performance liquid chromatography-mass spectrometry analysis. X-ray fluorescence was used to identify mercury-containing specimens. RESULTS: Fifteen seconds of leaf crushing followed by 24 h of extraction were optimal for PA free-base and N-oxide recovery. This method yielded ~50-fold greater PA recovery than prior methods. Half of the herbarium specimens (13 of 23), including the oldest, tested positive for PAs; leaf age did not correlate with success in PA extraction. Treatment of fresh leaves with alcohol before drying did not diminish PA recovery; mercury was observed in both PA-positive and PA-negative specimens. CONCLUSIONS: PAs can be reliably detected in small tissue samples from herbarium specimens up to 167 years old, including specimens that had been treated with alcohol or mercury salts. The variability of PA presence among herbarium specimens of E. umbellatus indicates that multiple specimens will need to be tested before a particular species is determined to lack PAs.

Evolution on the backbone: Apocynaceae phylogenomics and new perspectives on growth forms, flowers, and fruits.

PREMISE OF THE STUDY: We provide the largest phylogenetic analyses to date of Apocynaceae in terms of taxa and molecular data as a framework for analyzing the evolution of vegetative and reproductive traits. METHODS: We produced maximum-likelihood phylogenies of Apocynaceae using 21 plastid loci sampled from 1045 species (nearly 25% of the family) and complete plastomes from 73 species. We reconstructed ancestral states and used model comparisons in a likelihood framework to analyze character evolution across Apocynaceae. KEY RESULTS: We obtained a well-supported phylogeny of Apocynaceae, resolving poorly understood tribal and subtribal relationships (e.g., among Amsonieae and Hunterieae, within Asclepiadeae), rejecting monophyly of Melodineae and Odontadenieae, and placing previously unsampled and enigmatic taxa (e.g., Pycnobotrya). We provide new insights into the evolution of Apocynaceae, including frequent shifts between herbaceousness and woodiness, reversibility of twining, integrated evolution of the corolla and gynostegium, and ancestral baccate fruits. CONCLUSIONS: Increased sampling and selection of best-fitting models of evolution provide more resolved and robust estimates of phylogeny and character evolution than obtained in previous studies. Evolutionary inferences are sensitive to choice of phylogenetic frameworks and models.

Using citizen science to bridge taxonomic discovery with education and outreach.

PREMISE OF THE STUDY: Biological collections are uniquely poised to inform the stewardship of life on Earth in a time of cataclysmic biodiversity loss. Efforts to fully leverage collections are impeded by a lack of trained taxonomists and a lack of interest and engagement by the public. We provide a model of a crowd-sourced data collection project that produces quality taxonomic data sets and empowers citizen scientists through real contributions to science. Entitled MicroPlants, the project is a collaboration between taxonomists, citizen science experts, and teachers and students from universities and K-12. METHODS: We developed an online tool that allows citizen scientists to measure photographs of specimens of a hyper-diverse group of liverworts from a biodiversity hotspot. RESULTS: Using the MicroPlants online tool, citizen scientists are generating high-quality data, with preliminary analysis indicating non-expert data can be comparable to expert data. DISCUSSION: More than 11,000 users from both the website and kiosk versions have contributed to the data set, which is demonstrably aiding taxonomists working toward establishing conservation priorities within this group. MicroPlants provides opportunities for public participation in authentic science research. The project's educational component helps move youth toward engaging in scientific thinking and has been adopted by several universities into curriculum for both biology and non-biology majors.

Evolution of pyrrolizidine alkaloid biosynthesis in Apocynaceae: revisiting the defence de-escalation hypothesis.

Plants produce specialized metabolites for their defence. However, specialist herbivores adapt to these compounds and use them for their own benefit. Plants attacked predominantly by specialists may be under selection to reduce or eliminate production of co-opted chemicals: the defence de-escalation hypothesis. We studied the evolution of pyrrolizidine alkaloids (PAs) in Apocynaceae, larval host plants for PA-adapted butterflies (Danainae, milkweed and clearwing butterflies), to test if the evolutionary pattern is consistent with de-escalation. We used the first PA biosynthesis specific enzyme (homospermidine synthase, HSS) as tool for reconstructing PA evolution. We found hss orthologues in diverse Apocynaceae species, not all of them known to produce PAs. The phylogenetic analysis showed a monophyletic origin of the putative hss sequences early in the evolution of one Apocynaceae lineage (the APSA clade). We found an hss pseudogene in Asclepias syriaca, a species known to produce cardiac glycosides but no PAs, and four losses of an HSS amino acid motif. APSA clade species are significantly more likely to be Danainae larval host plants than expected if all Apocynaceae species were equally likely to be exploited. Our findings are consistent with PA de-escalation as an adaptive response to specialist attack.

Phylogenetic signal detection from an ancient rapid radiation: Effects of noise reduction, long-branch attraction, and model selection in crown clade Apocynaceae.

Crown clade Apocynaceae comprise seven primary lineages of lianas, shrubs, and herbs with a diversity of pollen aggregation morphologies including monads, tetrads, and pollinia, making them an ideal group for investigating the evolution and function of pollen packaging. Traditional molecular systematic approaches utilizing small amounts of sequence data have failed to resolve relationships along the spine of the crown clade, a likely ancient rapid radiation. The previous best estimate of the phylogeny was a five-way polytomy, leaving ambiguous the homology of aggregated pollen in two major lineages, the Periplocoideae, which possess pollen tetrads, and the milkweeds (Secamonoideae plus Asclepiadoideae), which possess pollinia. To assess whether greatly increased character sampling would resolve these relationships, a plastome sequence data matrix was assembled for 13 taxa of Apocynaceae, including nine newly generated complete plastomes, one partial new plastome, and three previously reported plastomes, collectively representing all primary crown clade lineages and outgroups. The effects of phylogenetic noise, long-branch attraction, and model selection (linked versus unlinked branch lengths among data partitions) were evaluated in a hypothesis-testing framework based on Shimodaira-Hasegawa tests. Discrimination among alternative crown clade resolutions was affected by all three factors. Exclusion of the noisiest alignment positions and topologies influenced by long-branch attraction resulted in a trichotomy along the spine of the crown clade consisting of Rhabdadenia+the Asian clade, Baisseeae+milkweeds, and Periplocoideae+the New World clade. Parsimony reconstruction on all optimal topologies after noise exclusion unambiguously supports parallel evolution of aggregated pollen in Periplocoideae (tetrads) and milkweeds (pollinia). Our phylogenomic approach has greatly advanced the resolution of one of the most perplexing radiations in Apocynaceae, providing the basis for study of convergent floral morphologies and their adaptive value.

Climate niches of milkweeds with plesiomorphic traits (Secamonoideae; Apocynaceae) and the milkweed sister group link ancient African climates and floral evolution.

PREMISE OF THE STUDY: Climate change that increases mortality of plants and pollinators can create mate-finding Allee effects and thus act as a strong selective force on floral morphology. Milkweeds (Secamonoideae and Asclepiadoideae; Apocynaceae) are typically small plants of seasonally dry habitats, with pollinia and high pollen-transfer efficiency. Their sister group (tribe Baisseeae and Dewevrella) is mostly comprised of giant lianas of African rainforests, with pollen in monads. Comparison of the two groups motivated a new hypothesis: milkweeds evolved in the context of African aridification and the shifting of rainforest to dry forest. Pollinia and high pollen-transfer efficiency may have been adaptations that alleviated mate-finding Allee effects generated by high mortality during droughts. We formally tested whether milkweeds have a drier climate niche by comparing milkweeds with plesiomorphic traits (Secamonoideae) and the milkweed sister group in continental Africa. METHODS: We georeferenced specimens of the milkweed sister group and Secamonoideae in continental Africa, extracted 19 climatic variables from the Worldclim model, conducted factor analysis to identify correlated suites of variables, and compared the frequency distributions of the two lineages relative to each factor. KEY RESULTS: The distributions of Secamonoideae and the milkweed sister group differed significantly relative to four factors, each correlated with a distinct suite of climate parameters: (1) air temperature (Secamonoideae: cooler), (2) total and (3) summer precipitation (Secamonoideae: drier), and (4) temperature seasonality and isothermality (Secamonoideae: more seasonal and less isothermal). CONCLUSIONS: Secamonoideae in continental Africa inhabit drier, cooler sites than do the milkweed sister group, consistent with a shift from rainforests to dry forests in a cooling climate.

Building a model: developing genomic resources for common milkweed (Asclepias syriaca) with low coverage genome sequencing.

BACKGROUND: Milkweeds (Asclepias L.) have been extensively investigated in diverse areas of evolutionary biology and ecology; however, there are few genetic resources available to facilitate and compliment these studies. This study explored how low coverage genome sequencing of the common milkweed (Asclepias syriaca L.) could be useful in characterizing the genome of a plant without prior genomic information and for development of genomic resources as a step toward further developing A. syriaca as a model in ecology and evolution. RESULTS: A 0.5× genome of A. syriaca was produced using Illumina sequencing. A virtually complete chloroplast genome of 158,598 bp was assembled, revealing few repeats and loss of three genes: accD, clpP, and ycf1. A nearly complete rDNA cistron (18S-5.8S-26S; 7,541 bp) and 5S rDNA (120 bp) sequence were obtained. Assessment of polymorphism revealed that the rDNA cistron and 5S rDNA had 0.3% and 26.7% polymorphic sites, respectively. A partial mitochondrial genome sequence (130,764 bp), with identical gene content to tobacco, was also assembled. An initial characterization of repeat content indicated that Ty1/copia-like retroelements are the most common repeat type in the milkweed genome. At least one A. syriaca microread hit 88% of Catharanthus roseus (Apocynaceae) unigenes (median coverage of 0.29×) and 66% of single copy orthologs (COSII) in asterids (median coverage of 0.14×). From this partial characterization of the A. syriaca genome, markers for population genetics (microsatellites) and phylogenetics (low-copy nuclear genes) studies were developed. CONCLUSIONS: The results highlight the promise of next generation sequencing for development of genomic resources for any organism. Low coverage genome sequencing allows characterization of the high copy fraction of the genome and exploration of the low copy fraction of the genome, which facilitate the development of molecular tools for further study of a target species and its relatives. This study represents a first step in the development of a community resource for further study of plant-insect co-evolution, anti-herbivore defense, floral developmental genetics, reproductive biology, chemical evolution, population genetics, and comparative genomics using milkweeds, and A. syriaca in particular, as ecological and evolutionary models.