Evolutionary trends of reproductive phenotype in Cycadales: an analysis of morphological evolution in Ceratozamia.

BACKGROUND AND AIMS: The size and shape of reproductive structures is especially relevant in evolution because these characters are directly related to the capacity of pollination and seed dispersal, a process that plays a basic role in evolutionary patterns. The evolutionary trajectories of reproductive phenotypes in gymnosperms have received special attention in terms of pollination and innovations related to the emergence of the Spermatophytes. However, variability of reproductive structures, evolutionary trends and the role of environment in the evolution of cycad species have not been well documented and explored. This study considered this topic under an explicitly phylogenetic and evolutionary approach that included a broad sampling of reproductive structures in the genus Ceratozamia. METHODS: We sampled 1400 individuals of 36 Ceratozamia species to explore evolutionary pattern and identify and evaluate factors that potentially drove their evolution. We analyzed characters for both pollen and ovulate strobili within a phylogenetic framework using different methods and characters (i. e., molecular and both quantitative and qualitative morphological) to infer phylogenetic relationships. Using this phylogenetic framework, evolutionary models of trait evolution for strobilar size were evaluated. In addition, quantitative morphological variation and its relation to environmental variables across species were analyzed. KEY RESULTS: We found contrasting phylogenetic signals between characters of pollen and ovulate strobili. These structures exhibited high morphological disparity in several characters related to size. Results of analyses of evolutionary trajectories suggested a stabilizing selection model. In regards to phenotype-environment, the analysis produced mixed results and differences for groups in the vegetation type where the species occur; however, a positive relationship with climatic variables was found. CONCLUSIONS: The integrated approach synthesized reproductive phenotypic variation with current phylogenetic hypotheses and provided explicit statements of character evolution. The characters of volume for ovulate strobili were the most informative, which could provide a reference for further study of the evolutionary complexity in Ceratozamia. Finally, heterogeneous environments, which are under changing weather conditions, promote variability of reproductive structures.

The genome of the Wollemi pine, a critically endangered "living fossil" unchanged since the Cretaceous, reveals extensive ancient transposon activity.

We present the genome of the living fossil, Wollemia nobilis, a southern hemisphere conifer morphologically unchanged since the Cretaceous. Presumed extinct until rediscovery in 1994, the Wollemi pine is critically endangered with less than 60 wild adults threatened by intensifying bushfires in the Blue Mountains of Australia. The 12 Gb genome is among the most contiguous large plant genomes assembled, with extremely low heterozygosity and unusual abundance of DNA transposons. Reduced representation and genome re-sequencing of individuals confirms a relictual population since the last major glacial/drying period in Australia, 120 ky BP. Small RNA and methylome sequencing reveal conservation of ancient silencing mechanisms despite the presence of thousands of active and abundant transposons, including some transferred horizontally to conifers from arthropods in the Jurassic. A retrotransposon burst 8-6 my BP coincided with population decline, possibly as an adaptation enhancing epigenetic diversity. Wollemia, like other conifers, is susceptible to Phytophthora, and a suite of defense genes, similar to those in loblolly pine, are targeted for silencing by sRNAs in leaves. The genome provides insight into the earliest seed plants, while enabling conservation efforts.

Developmental Morphology and Anatomy Shed Light on Both Parallel and Convergent Evolution of the Umbellate Inflorescence in Monocots, Underlain by a New Variant of Metatopy.

Inflorescence structure is very diverse and homoplasious, yet the developmental basis of their homoplasy is poorly understood. To gain an understanding of the degree of homology that these diverse structures share, we characterize the developmental morphology and anatomy of various umbellate inflorescences across the monocots and analyzed them in an evolutionary context. To characterize branching order, we characterized the developmental morphology of multiple inflorescences with epi-illumination, and vascular anatomy with Laser Ablation Tomography, a novel high-throughput method to reconstruct three-dimensional vasculature. We used these approaches to analyze the umbellate inflorescences in five instances of presumed homoplasy: in three members of the Amaryllidaceae; in three members of the Asparagaceae, including a putatively derived raceme in Dichelostemma congestum; in Butomus umbellatus (Alismataceae), in Tacca chantrieri (Dioscoreaceae), and in umbellate structure in Fritillaria imperialis (Liliaceae). We compare these with racemes found in three members of the subfamily Scilliioideae (Asparagaceae). We find there are three convergent developmental programs that generate umbellate inflorescences in the monocots, bostryx-derived, cincinnus-derived and raceme-derived. Additionally, among the bostryx-derived umbellate inflorescence, there are three instances of parallel evolution found in the Amaryllidaceae, in two members of Brodiaeoideae (Asparagaceae), and Butomus umbellatus, all of which share the same generative developmental program. We discuss the morphological modifications necessary to generate such complex and condensed structures and use these insights to describe a new variant of metatopy, termed horizontal concaulesence. We contextualize our findings within the broader literature of monocot inflorescence development, with a focus on synthesizing descriptive developmental morphological studies.

Monograph of Ceratozamia (Zamiaceae, Cycadales): an endangered genus.

Ceratozamia (Zamiaceae, Cycadales), is a member one of the most endangered seed plant groups. Species of Ceratozamia grow from near sea level up to 2,100 meters in Mexico and Mesoamerica. We present a modern taxonomic treatment of Ceratozamia, based on fieldwork combined with detailed study of herbarium specimens in and from Mexico and Central America. This new revision is based on incorporation of morphological, molecular and biogeographic data that have been previously published in circumscriptions of species complexes by our group. Detailed morphological descriptions of the 36 species of Ceratozamia are provided and relevant characters for the genus are discussed and described. A majority are endemic to Mexico and are concentrated at high elevations in mountainous areas. Synonymies, lectotypifications, etymologies, taxonomic notes, distribution maps, illustrations and detailed species-level comparisons are included, as well as a dichotomous key for identification of all species. Data on distributional ranges and habitats of all species are summarized. Ceratozamiaosbornei D.W.Stev., Mart.-Domínguez & Nic.-Mor., sp. nov. is described from evergreen tropical forests of Belize and we highlight new populations and distributional ranges for C.subroseophylla Mart.-Domínguez & Nic.-Mor. and C.vovidesii Pérez-Farr. & Iglesias in the Mesoamerican region.

A novelty in Ceratozamia (Zamiaceae, Cycadales) from the Sierra Madre del Sur, Mexico: biogeographic and morphological patterns, DNA barcoding and phenology.

Ceratozamia is a genus of cycads occurring in eastern Mexico and Central America. In this study, we describe a new species from the Pacific region of Mexico in Guerrero state. This locality represents the most northwestern Mexico distribution for the genus. We focus the comparison of this species with the most geographically proximate and phenotypically relevant lineages for this taxon. We followed an integrative taxonomy approach to evaluate the classification of these species, including geographic location, morphology, DNA barcoding and phenology as primary sources of systematic data. Within the morphological dataset, reproductive structures are described in detail and new characters are proposed for microsporophylls. The comparative morphology of these structures facilitated the elucidation of differences in forms and species for identification. The two chosen DNA barcoding markers - namely, the chloroplast genome coding region matK and the nuclear ribosomal internal transcribed spacer (ITS) region - had low divergence, allowing only 61% of species identification, suggesting slow molecular evolutionary rates. Besides employing these three basic sources of evidence, we introduced phenology as additional information for species circumscription. In addition, this work includes a brief review of the genus at the species-level. This is therefore the most recent review for Ceratozamia across its full geographic range (latitudinal and elevational). Overall, this work further contributes to a comprehensive framework for systematic studies in Mexican cycads.

Taxonomic review of Ceratozamia (Zamiaceae) in the Sierra Madre Oriental, Mexico.

The genus Ceratozamia is revised for the Sierra Madre Oriental in Mexico. This region is one of the biogeographic areas with the greatest diversity of species in this genus. These species are highly variable morphologically and this variability has led to a complex taxonomic history with many synonyms, particularly with reference to C. mexicana. We present a comprehensive taxonomic revision with history of nomenclature and the morphology, relationships, distribution and use of these species. We also introduce a key for their identification, descriptions, full synonymy, nomenclatural notes, etymologies and neotypes as well as taxonomic comments describing relevant taxonomic changes. We recognise fourteen species in this biogeographic province: C. brevifrons, C. chamberlainii, C. decumbens, C. delucana, C. fuscoviridis, C. hildae, C. kuesteriana, C. latifolia, C. mexicana, C. morettii, C. sabatoi, C. tenuis, C. totonacorum and C. zaragozae. This study provides a foundation for future taxonomic work in Neotropical species of Ceratozamia.

Data supporting the nuclear phylogenomics of the palm subfamily Arecoideae (Arecaceae).

This data article provides data and supplemental materials referenced in "Nuclear phylogenomics of the palm subfamily Arecoideae (Arecaceae)" (Comer et al., 2016) [1]. Raw sequence reads generated for this study are available through the Sequence Read Archive (SRA Study Accession: SRP061467). An aligned supermatrix of 168 nuclear genes for 35 taxa (34 palms and one outgroup taxon) is provided. Also provided are individual maximum likelihood gene trees used for the coalescent based analyses, output from the maximum parsimony analyses, and two figures.

A Phylogenomic Assessment of Ancient Polyploidy and Genome Evolution across the Poales.

Comparisons of flowering plant genomes reveal multiple rounds of ancient polyploidy characterized by large intragenomic syntenic blocks. Three such whole-genome duplication (WGD) events, designated as rho (ρ), sigma (σ), and tau (τ), have been identified in the genomes of cereal grasses. Precise dating of these WGD events is necessary to investigate how they have influenced diversification rates, evolutionary innovations, and genomic characteristics such as the GC profile of protein-coding sequences. The timing of these events has remained uncertain due to the paucity of monocot genome sequence data outside the grass family (Poaceae). Phylogenomic analysis of protein-coding genes from sequenced genomes and transcriptome assemblies from 35 species, including representatives of all families within the Poales, has resolved the timing of rho and sigma relative to speciation events and placed tau prior to divergence of Asparagales and the commelinids but after divergence with eudicots. Examination of gene family phylogenies indicates that rho occurred just prior to the diversification of Poaceae and sigma occurred before early diversification of Poales lineages but after the Poales-commelinid split. Additional lineage-specific WGD events were identified on the basis of the transcriptome data. Gene families exhibiting high GC content are underrepresented among those with duplicate genes that persisted following these genome duplications. However, genome duplications had little overall influence on lineage-specific changes in the GC content of coding genes. Improved resolution of the timing of WGD events in monocot history provides evidence for the influence of polyploidization on functional evolution and species diversification.

Nuclear phylogenomics of the palm subfamily Arecoideae (Arecaceae).

Palms (Arecaceae) include economically important species such as coconut, date palm, and oil palm. Resolution of the palm phylogeny has been problematic due to rapid diversification and slow rates of molecular evolution. The focus of this study is on relationships of the 14 tribes of subfamily Arecoideae and their inferred ancestral areas. A targeted sequencing approach was used to generate a data set of 168 single/low copy nuclear genes for 34 species representing the Arecoideae tribes and the other palm subfamilies. Species trees from the concatenated and coalescent based analyses recovered largely congruent topologies. Three major tribal clades were recovered: the POS clade (Podococceae, Oranieae, Sclerospermeae), the RRC clade (Roystoneeae, Reinhardtieae, Cocoseae), and the core arecoid clade (Areceae, Euterpeae, Geonomateae, Leopoldinieae, Manicarieae, Pelagodoxeae). Leopoldinieae was sister to the rest of the core arecoids (Geonomateae, Manicarieae+Pelagodoxeae, and Areceae+Euterpeae). The nuclear phylogeny supported a North American origin for subfamily Arecoideae, with most tribal progenitors diversifying within the Americas. The POS clade may have dispersed from the Americas into Africa, with tribe Oranieae subsequently spreading into the Indo-Pacific. Two independent dispersals into the Indo-Pacific were inferred for two tribes within the core arecoids (tribes Areceae and Pelagodoxeae).

Resolving relationships within the palm subfamily Arecoideae (Arecaceae) using plastid sequences derived from next-generation sequencing.

PREMISE OF THE STUDY: Several studies have incorporated molecular and morphological data to study the phylogeny of the palms (Arecaceae), but some relationships within the family remain ambiguous-particularly those within Arecoideae, the most diverse subfamily including coconut and oil palm. Here, two next-generation, targeted plastid-enrichment methods were compared and used to elucidate Arecoideae phylogeny. METHODS: Next-generation sequencing techniques were used to generate a plastid genome data set. Long range PCR and hybrid gene capture were used to enrich for chloroplast targets. Ten taxa were enriched using both methods for comparison. Chloroplast sequence data were generated for 31 representatives of the 14 Arecoideae tribes and five outgroup taxa. The phylogeny was reconstructed using maximum likelihood, maximum parsimony, and Bayesian analyses. KEY RESULTS: Long range PCR and hybrid gene capture both enriched the plastid genome and provided similar sequencing coverage. Subfamily Arecoideae was resolved as monophyletic with tribe Chamaedoreeae as the earliest-diverging lineage, implying that the development of flowers in triads defines a synapomorphy for the Arecoideae clade excluding Chamaedoreeae. Three major clades within this group were recovered: Roystoneeae/Reinhardtieae/Cocoseae (RRC), Areceae/Euterpeae/Geonomateae/Leopoldinieae/Manicarieae/Pelagodoxeae (core arecoids), and Podococceae/Oranieae/Sclerospermeae (POS). An Areceae + Euterpeae clade was resolved within the core arecoids. The POS clade was sister to a RRC + core arecoids clade, implying a shared ancestral area in South America for these three clades. CONCLUSIONS: The plastome phylogeny recovered here provides robust resolution of previously ambiguous studies and new insights into palm evolution.

Resolving ancient radiations: can complete plastid gene sets elucidate deep relationships among the tropical gingers (Zingiberales)?

BACKGROUND AND AIMS: Zingiberales comprise a clade of eight tropical monocot families including approx. 2500 species and are hypothesized to have undergone an ancient, rapid radiation during the Cretaceous. Zingiberales display substantial variation in floral morphology, and several members are ecologically and economically important. Deep phylogenetic relationships among primary lineages of Zingiberales have proved difficult to resolve in previous studies, representing a key region of uncertainty in the monocot tree of life. METHODS: Next-generation sequencing was used to construct complete plastid gene sets for nine taxa of Zingiberales, which were added to five previously sequenced sets in an attempt to resolve deep relationships among families in the order. Variation in taxon sampling, process partition inclusion and partition model parameters were examined to assess their effects on topology and support. KEY RESULTS: Codon-based likelihood analysis identified a strongly supported clade of ((Cannaceae, Marantaceae), (Costaceae, Zingiberaceae)), sister to (Musaceae, (Lowiaceae, Strelitziaceae)), collectively sister to Heliconiaceae. However, the deepest divergences in this phylogenetic analysis comprised short branches with weak support. Additionally, manipulation of matrices resulted in differing deep topologies in an unpredictable fashion. Alternative topology testing allowed statistical rejection of some of the topologies. Saturation fails to explain observed topological uncertainty and low support at the base of Zingiberales. Evidence for conflict among the plastid data was based on a support metric that accounts for conflicting resampled topologies. CONCLUSIONS: Many relationships were resolved with robust support, but the paucity of character information supporting the deepest nodes and the existence of conflict suggest that plastid coding regions are insufficient to resolve and support the earliest divergences among families of Zingiberales. Whole plastomes will continue to be highly useful in plant phylogenetics, but the current study adds to a growing body of literature suggesting that they may not provide enough character information for resolving ancient, rapid radiations.

A functional phylogenomic view of the seed plants.

A novel result of the current research is the development and implementation of a unique functional phylogenomic approach that explores the genomic origins of seed plant diversification. We first use 22,833 sets of orthologs from the nuclear genomes of 101 genera across land plants to reconstruct their phylogenetic relationships. One of the more salient results is the resolution of some enigmatic relationships in seed plant phylogeny, such as the placement of Gnetales as sister to the rest of the gymnosperms. In using this novel phylogenomic approach, we were also able to identify overrepresented functional gene ontology categories in genes that provide positive branch support for major nodes prompting new hypotheses for genes associated with the diversification of angiosperms. For example, RNA interference (RNAi) has played a significant role in the divergence of monocots from other angiosperms, which has experimental support in Arabidopsis and rice. This analysis also implied that the second largest subunit of RNA polymerase IV and V (NRPD2) played a prominent role in the divergence of gymnosperms. This hypothesis is supported by the lack of 24nt siRNA in conifers, the maternal control of small RNA in the seeds of flowering plants, and the emergence of double fertilization in angiosperms. Our approach takes advantage of genomic data to define orthologs, reconstruct relationships, and narrow down candidate genes involved in plant evolution within a phylogenomic view of species' diversification.

The impact of outgroup choice and missing data on major seed plant phylogenetics using genome-wide EST data.

BACKGROUND: Genome level analyses have enhanced our view of phylogenetics in many areas of the tree of life. With the production of whole genome DNA sequences of hundreds of organisms and large-scale EST databases a large number of candidate genes for inclusion into phylogenetic analysis have become available. In this work, we exploit the burgeoning genomic data being generated for plant genomes to address one of the more important plant phylogenetic questions concerning the hierarchical relationships of the several major seed plant lineages (angiosperms, Cycadales, Gingkoales, Gnetales, and Coniferales), which continues to be a work in progress, despite numerous studies using single, few or several genes and morphology datasets. Although most recent studies support the notion that gymnosperms and angiosperms are monophyletic and sister groups, they differ on the topological arrangements within each major group. METHODOLOGY: We exploited the EST database to construct a supermatrix of DNA sequences (over 1,200 concatenated orthologous gene partitions for 17 taxa) to examine non-flowering seed plant relationships. This analysis employed programs that offer rapid and robust orthology determination of novel, short sequences from plant ESTs based on reference seed plant genomes. Our phylogenetic analysis retrieved an unbiased (with respect to gene choice), well-resolved and highly supported phylogenetic hypothesis that was robust to various outgroup combinations. CONCLUSIONS: We evaluated character support and the relative contribution of numerous variables (e.g. gene number, missing data, partitioning schemes, taxon sampling and outgroup choice) on tree topology, stability and support metrics. Our results indicate that while missing characters and order of addition of genes to an analysis do not influence branch support, inadequate taxon sampling and limited choice of outgroup(s) can lead to spurious inference of phylogeny when dealing with phylogenomic scale data sets. As expected, support and resolution increases significantly as more informative characters are added, until reaching a threshold, beyond which support metrics stabilize, and the effect of adding conflicting characters is minimized.

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.

Nuclear genome size in Selaginella.

Estimates of nuclear genome size for 9 Selaginella species were obtained using flow cytometry, and measurements for 7 of these species are reported for the first time. Estimates range from 0.086 to 0.112 pg per holoploid genome (84-110 Mb). The data presented here agree with the previously published flow cytometric results for S. moellendorffii. Within the 9 species sampled here, chromosome number varies from 2n = 16 to 2n = 27. Nuclear genome size appears to be strongly correlated with chromosome number (Spearman's rank correlation; p = 0.00003725). Cultivated S. moellendorffii lacks sexual reproduction--manifest by the production of abortive megasporangia. Flow cytometric data generated from a herbarium specimen of a fertile wild-collected S. moellendorffii are virtually indistinguishable from the data generated from fresh material (0.088 vs. 0.089 pg/1C). Therefore, the limited fertility observed in cultivated plants is probably not the result of abnormal chromosome number (e.g., induced by interspecific hybridization).

A comparison of algorithms for the identification of specimens using DNA barcodes: examples from gymnosperms.

In order to use DNA sequences for specimen identification (e.g., barcoding, fingerprinting) an algorithm to compare query sequences with a reference database is needed. Precision and accuracy of query sequence identification was estimated for hierarchical clustering (parsimony and neighbor joining), similarity methods (BLAST, BLAT and megaBLAST), combined clustering/similarity methods (BLAST/parsimony and BLAST/neighbor joining), diagnostic methods (DNA-BAR and DOME ID), and a new method (ATIM). We offer two novel alignment-free algorithmic solutions (DOME ID and ATIM) to identify query sequences for the purposes of DNA barcoding. Publicly available gymnosperm nrITS 2 and plastid matK sequences were used as test data sets. On the test data sets, almost all of the methods were able to accurately identify sequences to genus; however, no method was able to accurately identify query sequences to species at a frequency that would be considered useful for routine specimen identification (42-71% unambiguously correct). Clustering methods performed the worst (perhaps due to alignment issues). Similarity methods, ATIM, DNA-BAR, and DOME ID all performed at approximately the same level. Given the relative precision of the algorithms (median = 67% unambiguous), the low accuracy of species-level identification observed could be ascribed to the lack of correspondence between patterns of allelic similarity and species delimitations. Application of DNA barcoding to sequences of CITES listed cycads (Cycadopsida) provides an example of the potential application of DNA barcoding to enforcement of conservation laws.

Cataphylls of the Middle Triassic cycad Antarcticycas schopfii and new insights into cycad evolution.

Cataphylls associated with the Middle Triassic stem genus Antarcticycas are described, and their impact on understanding cycad evolution is discussed. The cataphylls of Antarcticycas are triangular in outline and flattened adaxially with lateral flanges. The outer surfaces are covered with a ramentum of filamentous hairs, the epidermis is a single cell layer thick, and the ground tissue is parenchymatous with mucilage canals and sclereids. Vascular bundles form a distinct inverted omega-shaped pattern characteristic of the Cycadales observed in petioles of extant species. The structures in Antarcticycas are interpreted as cataphylls based on overall morphology, presence of straight vascular strands in the cortex of the associated stem, and lack of fascicular cambia in the vascular bundles. Because much of the overall diversity of Cycadales is represented by fossils, integrating fossil taxa into explicit phylogenetic hypotheses is important for understanding cycad evolution. Therefore, character and minimum age mapping were performed on a phylogeny of extant and fossil taxa including Antarcticycas. The results suggest that major extant lineages of Cycadales had diverged by the Permian to Triassic and that certain synapomorphies for Cycadales had evolved by the Permian. Evidence of insect feeding on Antarcticycas suggests that associations between cycads and insects are ancient.

Abstracts of the 22nd Annual Meeting of the Willi Hennig Society.

The 22nd annual meeting of the Willi Hennig Society was held at the New York Botanical Garden in the Bronx, New York from July 20-24. There were 154 participants representing 15 countries and a total of 65 talks, 16 posters, and four software presentations. There were five symposia ranging from sequence alignment to worm biology to biogeography. As with Hennig 21 at Helsinki, there was strong student participation. Student posters represented 13 of the total 16 posters and 23 of 65 oral presentations were by students. Student presentations were part of all symposia. The strength and future of the Willi Hennig Society resides in the students and recent meetings indicate a healthy society for the future. The Student Awards Committee consisted of Jonathan A. Coddington, Arnold G. Kluge and Gunilla Ståhls. The winners were Taran Grant, the Hennig Prize ($1000) for "Insertions and deletions in the evolution of equal-length DNA fragments"; Johannes Bergsten, the Brundin Prize ($500) for "Acilius Phylogeny (Coleoptera: Dytiscidae), Problems with Long-branch attraction and Morphological Intersexual Coevolution"; and A. R. Lindgren, the Rosen Prize ($250) for "A New Phylogeny of the Cephalopoda Using Total Evidence".

Phylogenetic relationships among Poaceae and related families as inferred from morphology, inversions in the plastid genome, and sequence data from the mitochondrial and plastid genomes.

A phylogenetic analysis of the Poales was conducted to assess relationships among Poaceae and allied families. The analysis included 40 taxa, representing all families of the Poales as circumscribed by the Angiosperm Phylogeny Group (APG), plus five of the six unplaced Commelinid families in the APG system. The data matrix included 98 informative characters representing variation in morphology and chloroplast genome structure (including three inversions in the chloroplast genome), and 563 informative characters derived from rbcL and atpA nucleotide sequences. Ecdeiocolea has the 6-kilobase (kb) chloroplast genome inversion previously reported in Joinvillea and Poaceae, and like Joinvillea it lacks the trnT inversion that occurs in grasses. Analysis of the morphological data places Poaceae in an unresolved relationship relative to several other taxa, including Joinvillea and Ecdeiocolea, while analysis of the molecular and combined data resolves Ecdeiocolea as sister of Poaceae, with Joinvillea the sister of this group. Although the 6-kb and trnT inversions are non-homoplasious in the phylogenies obtained in this study, the 28-kb inversion is optimized as having originated twice (once in Restionaceae and another time in the most recent common ancestor of Ecdeiocolea, Joinvillea, and the grasses); an alternative interpretation is that it arose once and was later lost in Anarthria. Ecdeiocolea shares with Poaceae the presence of operculate, annulate pollen that lacks scrobiculi, and a dry, indehiscent fruit.