J. Travis Columbus

Anatomical enablers and the evolution of C4 photosynthesis in grasses

C4 photosynthesis is a series of anatomical and biochemical modifications to the typical C3 pathway that increases the productivity of plants in warm, sunny, and dry conditions. Despite its complexity, it evolved more than 62 times independently in flowering plants. However, C4 origins are absent from most plant lineages and clustered in others, suggesting that some characteristics increase C4 evolvability in certain phylogenetic groups. The C4 trait has evolved 22–24 times in grasses, and all origins occurred within the PACMAD clade, whereas the similarly sized BEP clade contains only C3 taxa. Here, multiple foliar anatomy traits of 157 species from both BEP and PACMAD clades are quantified and analyzed in a phylogenetic framework. Statistical modeling indicates that C4 evolvability strongly increases when the proportion of vascular bundle sheath (BS) tissue is higher than 15%, which results from a combination of short distance between BS and large BS cells. A reduction in the distance between BS occurred before the split of the BEP and PACMAD clades, but a decrease in BS cell size later occurred in BEP taxa. Therefore, when environmental changes promoted C4 evolution, suitable anatomy was present only in members of the PACMAD clade, explaining the clustering of C4 origins in this lineage. These results show that key alterations of foliar anatomy occurring in a C3 context and preceding the emergence of the C4 syndrome by millions of years facilitated the repeated evolution of one of the most successful physiological innovations in angiosperm history.

Phylogenetic Relationships in Cariceae (Cyperaceae) Based on ITS (nrDNA) and trnT-L-F (cpDNA) Region Sequences: Assessment of Subgeneric and Sectional Relationships in Carex with Emphasis on Section Acrocystis

Abstract With approximately 2,000 species, Carex is the largest genus in the Cyperaceae and is one of the most widespread genera in the world. Relationships within Carex and among the genera of the Cariceae (Carex, Cymophyllus, Kobresia, Schoenoxiphium, and Uncinia) are unclear. For this reason, a molecular phylogenetic study employing nrDNA ITS and cpDNA trnT-L-F spacer sequences was undertaken. In addition to creating hypotheses of relationship for the Cariceae and testing classifications of this tribe, a primary goal of this study was to assess relationships within Carex section Acrocystis and identify a monophyletic group for more detailed study. These analyses suggest that Cymophyllus, Kobresia, Schoenoxiphium, and Uncinia are nested within Carex. Three primary clades are suggested: a Carex subgenus Vignea clade, a clade including Carex subgenus Primocarex (for the most part) and the other genera of Cariceae, and a clade predominately comprised of Carex subgenera Carex and Indocarex. A large part of Carex section Acrocystis forms a monophyletic group but several Eurasian species are more closely related to other groups rather than to this core clade. Assessment of chromosome number variation across the Cariceae clade suggests that the ancestor of the Cariceae had a moderate to high chromosome number. In addition, these analyses suggest the sister group of the Cariceae is a clade including Scirpus sensu stricto, Amphiscirpus, and Dulichium. Communicating Editor: Jeff H. Rettig

Molecular phylogenetics of suborder Cactineae (Caryophyllales), including insights into photosynthetic diversification and historical biogeography.

UNLABELLED PREMISE OF THE STUDY Phylogenetic relationships were investigated among the eight families (Anacampserotaceae, Basellaceae, Cactaceae, Didiereaceae, Halophytaceae, Montiaceae, Portulacaceae, Talinaceae) that form suborder Cactineae (= Portulacineae) of the Caryophyllales. In addition, photosynthesis diversification and historical biogeography were addressed. • METHODS Chloroplast DNA sequences, mostly noncoding, were used to estimate the phylogeny. Divergence times were calibrated using two Hawaiian Portulaca species, due to the lack of an unequivocal fossil record for Cactineae. Photosynthetic pathways were determined from carbon isotope ratios (δ(13)C) and leaf anatomy. • KEY RESULTS Maximum likelihood and Bayesian analyses were consistent with previous studies in that the suborder, almost all families, and the ACPT clade (Anacampserotaceae, Cactaceae, Portulacaceae, Talinaceae) were strongly supported as monophyletic; however, relationships among families remain uncertain. The age of Cactineae was estimated to be 18.8 Myr. Leaf anatomy and δ(13)C and were congruent in most cases, and inconsistencies between these pointed to photosynthetic intermediates. Reconstruction of photosynthesis diversification showed C(3) to be the ancestral pathway, a shift to C(4) in Portulacaceae, and five independent origins of Crassulacean acid metabolism (CAM). Cactineae were inferred to have originated in the New World. • CONCLUSIONS Although the C(3) pathway is inferred as the ancestral state in Cactineae, some CAM activity has been reported in the literature in almost every family of the suborder, leaving open the possibility that CAM may have one origin in the group. Incongruence among loci could be due to internal short branches, which possibly represent rapid radiations in response to increasing aridity in the Miocene.

Molecular phylogenetics, historical biogeography, and chromosome number evolution of Portulaca (Portulacaceae)

Portulaca is the only genus in Portulacaceae and has ca. 100 species distributed worldwide, mainly in the tropics and subtropics. Molecular data place the genus as one of the closest relatives of Cactaceae, but phylogenetic relationships within Portulaca are barely known. This study samples 59 species of Portulaca, 10 infraspecific taxa, and three cultivars, including multiple samples of widespread species. The sampled taxa represent all subgenera in the classifications of von Poellnitz (1934), Legrand (1958), and Geesink (1969) and come from around the world. Nuclear ITS and chloroplast ndhF, trnT-psbD intergenic spacer, and ndhA intron DNA sequences were analyzed using maximum likelihood and Bayesian methods to produce a hypothesis of relationships within Portulaca. Divergence times were estimated using Hawaiian endemics for calibration, and biogeographical patterns were examined using a Bayes-DIVA approach. In addition, the evolution of chromosome numbers in the genus was investigated using probabilistic models. The analyses strongly support the monophyly of Portulaca, with an age of the most recent common ancestor (MRCA) of 23 Myr. Within Portulaca are two major lineages: the OL clade (comprising opposite-leaved species) distributed in Africa, Asia, and Australia, and the AL clade (comprising alternate to subopposite-leaved species), which is more widespread and originated in the New World. Sedopsis, a genus sometimes recognized as distinct from Portulaca based on a long corolla tube, is nested within the OL clade and does not merit taxonomic recognition. Samples of Portulaca grandiflora, Portulaca halimoides, and Portulaca oleracea were found to be non-monophyletic. It is hypothesized that the ancestral distribution area of Portulaca included southern hemisphere continents and Asia. The OL clade remained restricted to the Old World (except Portulaca quadrifida, a pantropical weed), while the AL clade, with a South American origin, was able to disperse multiple times to other continents. The base chromosome number for Portulaca is inferred to be x=9, although the analysis was primarily based on the available data for the AL clade. A number of chromosome number change events (polyploidization, demi-polyploidization, gain, and loss) were shown to have occurred in the genus, especially within the Oleracea clade.

Proposal for an Expanded Distichlis (Poaceae, Chloridoideae): Support from Molecular, Morphological, And Anatomical Characters

Abstract The Distichlis clade comprises Distichlis (7 species), Monanthochloë (2), and Reederochloa (1). All species except D. distichophylla (endemic to Australia) and D. spicata (widespread in the New World) are restricted either to North or South America. We investigated phylogenetic relationships within the clade using chloroplast (trnL–F and ndhF) and nuclear ribosomal (internal transcribed spacers and 5.8S) DNA sequences. We also studied lemma micromorphology, leaf blade anatomy, macromorphology, and biogeography in a phylogenetic context. The Distichlis clade is strongly supported in the molecular analyses. A morphological synapomorphy for the clade is the presence of a single papilla on the center of each subsidiary cell of lemma stomata. Other diagnostic features include dioecy, rhizomes or stolons, conspicuously distichous leaves, 5–13 lemma nerves, dumbbell- or flask-shaped bicellular microhairs with sunken basal cells, and growth in alkaline or saline soils. The nuclear and chloroplast phylogenies indicate that Monanthochloë and Reederochloa are nested within a paraphyletic Distichlis, and a number of structural characters, including leaf blade length, number of spikelets per inflorescence, and number of florets per spikelet, also fall within the range of variation in Distichlis. Therefore, we propose expanding the circumscription of Distichlis to include Monanthochloë and Reederochloa, and make the following new combinations: Distichlis acerosa, D. eludens , and D. littoralis . Biogeographical analysis revealed that the group likely originated in North America followed by a number of long-distance dispersal events, including back dispersals.

A multi-step comparison of short-read full plastome sequence assembly methods in grasses

Technological advances have allowed phylogenomic studies of plants, such as full chloroplast genome (plastome) analysis, to become increasingly popular and economically feasible. Although next-generation short-read sequencing allows for full plastomes to be sequenced relatively rapidly, it requires additional attention using software to assemble these reads into comprehensive sequences. Here we compare the use of three de novo assemblers combined with three contig assembly methods. Seven plastome sequences were analyzed. Three of these were Sanger-sequenced. The other four were assembled from short, single-end read files generated from next-generation libraries. These plastomes represented a total of six grass species (Poaceae), one of which was sequenced in duplicate by the two methods to allow direct comparisons for accuracy. Enumeration of missing sequence and ambiguities allowed for assessments of completeness and efficiency. All methods that used de Bruijn-based de novo assemblers were shown to produce assemblies comparable to the Sanger-sequenced plastomes but were not equally efficient. Contig assembly methods that utilized automatable and repeatable processes were generally more efficient and advantageous when applied to larger scale projects with many full plastomes. However, contig assembly methods that were less automatable and required more manual attention did show utility in determining plastomes with lower read depth that were not able to be assembled when automatable procedures were implemented. Although the methods here were used exclusively to generate grass plastomes, these could be applied to other taxonomic groups if previously sequenced plastomes were available. In addition to comparing sequencing methods, a supplemental guide for short-read plastome assembly and applicable scripts were generated for this study.

Evolution of leaf anatomy and photosynthetic pathways in Portulacaceae.

PREMISE OF THE STUDY Portulacaceae is a family with a remarkable diversity in photosynthetic pathways. This lineage not only has species with different C4 biochemistry (NADP-ME and NAD-ME types) and C3-C4 intermediacy, but also displays different leaf anatomical configurations. Here we addressed the evolutionary history of leaf anatomy and photosynthetic pathways in Portulacaceae. METHODS Photosynthetic pathways were assessed based on leaf anatomy and carbon isotope ratios. Information on the NADP-ME and NAD-ME C4 variants was obtained from the literature. The evolutionary relationships and trait evolution were estimated under a Bayesian framework, and divergence times were calibrated using the ages obtained in a previous study. KEY RESULTS C4 photosynthesis is the main pathway in Portulacaceae. One clade (Cryptopetala), however, includes species that have non-Kranz anatomy and C3 type isotope values, two of which are C3-C4 intermediates. The ancestral leaf anatomy for the family is uncertain. The analysis showed one origin of the C4 pathway, which was lost in the Cryptopetala clade. Nevertheless, when a second analysis was performed taking into account the limited number of species with NAD-ME and NADP-ME data, a secondary gain of the C4 pathway from a C3-C4 intermediate was inferred. CONCLUSIONS The C4 pathway evolved ca. 23 Myr in the Portulacaceae. The number of times that the pathway evolved in the family is uncertain. The diversity of leaf anatomical types and C4 biochemical variants suggest multiple independent origins of C4 photosynthesis. Evidence for a switch from C4 to C3-C4 intermediacy supports the hypothesis that intermediates represent a distinct successful strategy.

Phylogenomics and Plastome Evolution of the Chloridoid Grasses (Chloridoideae: Poaceae)

Premise of research. Studies of complete plastomes have proven informative for our understanding of the molecular evolution and phylogenomics of grasses, but subfamily Chloridoideae has not been included in this research. In previous multilocus studies, specific deep branches, as in the large clade corresponding to Cynodonteae, are not uniformly well supported. Methodology. In this study, a plastome phylogenomic analysis sampled 14 species representing 4 tribes and 10 genera of Chloridoideae. One species was Sanger sequenced, and 14 other species, including outgroups, were sequenced with next-generation sequencing-by-synthesis methods. Plastomes from next-generation sequences were assembled by de novo methods, and the unambiguously aligned coding and noncoding sequences of the entire plastomes were analyzed phylogenetically. Pivotal results. Complete plastomes showed rare genomic changes in Distichlis, Centropodia, and Eragrostis tef that were of potential phylogenomic significance. Phylogenomic analyses showed uniformly strong support for all ingroup relationships except one node in Cynodonteae in which a short internal branch connected long terminal branches. Resolution within this clade was found to be taxon dependent and possibly subject to long-branch attraction artifacts. Conclusions. Our study indicates that the increase in phylogenetic information in sequences of entire plastomes well resolves and strongly supports relationships among tribes and genera of chloridoid grasses. Sampling more species, especially in the Centropodia + Ellisochloa clade and Cynodonteae, will further address relationships in these groups and clarify the evolutionary origins of the subfamily.

Molecular Phylogeny of Dissanthelium (Poaceae: Pooideae) and its Taxonomic Implications

Abstract We investigated the phylogeny of the New World grass Dissanthelium to explore its monophyly, to examine relationships within the genus, and to investigate its relationship with Poa. Molecular phylogenetic analyses including a thorough sampling of Dissanthelium (seventeen of the 20 species) and DNA sequences from the nuclear ribosomal ITS and plastid trnT-trnL-trnF regions suggest that Dissanthelium is not monophyletic and is nested within Poa. However, ten species form a strongly supported clade (the Dissanthelium clade) in the ITS tree. We propose treating Dissanthelium and Tovarochloa as taxonomic synonyms of Poa. We erect two new sections in Poa: sect. Dissanthelium, comprising the Dissanthelium clade and D. peruvianum, and the monotypic sect. Tovarochloa. The necessary new combinations (Poa aequalis, P. amplivaginata, P. calycina var. mathewsii, P. gigantea, P. macusaniensis, P. rahuii, and P. trollii) and new names (Poa sections Dissanthelium and Tovarochloa, P. apiculata, P. arcuata, P. boliviana, P. congesta, P. deminuta, P. linearifolia, P. parvifolia, P. serpaiana, P. swallenii, and P. thomasii) are effected herein.

C3 photosynthesis in Aristida longifolia: Implication for photosynthetic diversification in Aristidoideae (Poaceae).

Only a small percentage of plant species undergo C(4) photosynthesis. Despite its rarity, the C(4) pathway has evolved numerous times from C(3) ancestors, with as many as 18 independent origins in grasses alone. We report non-Kranz (C(3)) anatomy in Aristida longifolia, a species in a genus of ca. 300 species previously thought to possess only Kranz (C(4)) anatomy. Leaf blade transections of A. longifolia show widely spaced vascular bundles, nonradiate chlorenchyma, and few or no chloroplasts in cells of the sheaths surrounding the vascular bundle, all features indicative of C(3) photosynthesis. Carbon isotope ratios range from -27.68 to -29.71%, likewise indicative of C(3) photosynthesis. We also reconstruct the phylogeny of Aristidoideae, comprising Aristida, Sartidia (C(3)), and Stipagrostis (C(4)), using a sample of 11 species, including A. longifolia, and DNA sequences of the nuclear ribosomal internal transcribed spacer region and the chloroplast rpl16 intron and trnL-trnF region. Sartidia and Stipagrostis resolve as sisters, and sister to this clade is Aristida. Aristida longifolia resolves as sister to the remaining species in the genus. C(3) photosynthesis is hypothesized to be ancestral in Aristidoideae, which means the C(4) pathway evolved twice in the subfamily-in Stipagrostis and early in the diversification of the Aristida clade.