Eric H. Roalson

Phylogenomics of C4 Photosynthesis in Sedges (Cyperaceae): Multiple Appearances and Genetic Convergence

C(4) photosynthesis is an adaptive trait conferring an advantage in warm and open habitats. It originated multiple times and is currently reported in 18 plant families. It has been recently shown that phosphoenolpyruvate carboxylase (PEPC), a key enzyme of the C(4) pathway, evolved through numerous independent but convergent genetic changes in grasses (Poaceae). To compare the genetics of multiple C(4) origins on a broader scale, we reconstructed the evolutionary history of the C(4) pathway in sedges (Cyperaceae), the second most species-rich C(4) family. A sedge phylogeny based on two plastome genes (rbcL and ndhF) has previously identified six fully C(4) clades. Here, a relaxed molecular clock was used to calibrate this tree and showed that the first C(4) acquisition occurred in this family between 19.6 and 10.1 Ma. According to analyses of PEPC-encoding genes (ppc), at least five distinct C(4) origins are present in sedges. Two C(4) Eleocharis species, which were unrelated in the plastid phylogeny, acquired their C(4)-specific PEPC genes from a single source, probably through reticulate evolution or a horizontal transfer event. Acquisitions of C(4) PEPC in sedges have been driven by positive selection on at least 16 codons (3.5% of the studied gene segment). These sites underwent parallel genetic changes across the five sedge C(4) origins. Five of these sites underwent identical changes also in grass and eudicot C(4) lineages, indicating that genetic convergence is most important within families but that identical genetic changes occurred even among distantly related taxa. These lines of evidence give new insights into the constraints that govern molecular evolution.

Evolutionary Switch and Genetic Convergence on rbcL following the Evolution of C4 Photosynthesis

Rubisco is responsible for the fixation of CO2 into organic compounds through photosynthesis and thus has a great agronomic importance. It is well established that this enzyme suffers from a slow catalysis, and its low specificity results into photorespiration, which is considered as an energy waste for the plant. However, natural variations exist, and some Rubisco lineages, such as in C4 plants, exhibit higher catalytic efficiencies coupled to lower specificities. These C4 kinetics could have evolved as an adaptation to the higher CO2 concentration present in C4 photosynthetic cells. In this study, using phylogenetic analyses on a large data set of C3 and C4 monocots, we showed that the rbcL gene, which encodes the large subunit of Rubisco, evolved under positive selection in independent C4 lineages. This confirms that selective pressures on Rubisco have been switched in C4 plants by the high CO2 environment prevailing in their photosynthetic cells. Eight rbcL codons evolving under positive selection in C4 clades were involved in parallel changes among the 23 independent monocot C4 lineages included in this study. These amino acids are potentially responsible for the C4 kinetics, and their identification opens new roads for human-directed Rubisco engineering. The introgression of C4-like high-efficiency Rubisco would strongly enhance C3 crop yields in the future CO2-enriched atmosphere.

Diversification of the Old World Salsoleae s.l. (Chenopodiaceae): Molecular Phylogenetic Analysis of Nuclear and Chloroplast Data Sets and a Revised Classification

A first comprehensive phylogenetic analysis of tribe Salsoleae s.l. (Salsoloideae: Chenopodiaceae) is presented based on maximum parsimony and maximum likelihood analysis of nuclear ribosomal internal transcribed spacer and chloroplast psbB‐psbH DNA sequences. Our data strongly support (1) the sister relationship of Camphorosmeae to the Salsoleae s.l.; (2) splitting of Salsoleae s.l. into two monophyletic tribes, Salsoleae s.s. and Caroxyloneae tribus nova; (3) the current status of most monotypic or oligotypic genera in Salsoleae; and (4) polyphyly of the Botschantzev and Freitag (among others) circumscriptions of Salsola, which falls into 10 (on average) monophyletic genera/lineages. Three well‐supported genera are described as new (Pyankovia, Kaviria, and Turania), and four previously described genera are resurrected (Caroxylon, Climacoptera, Kali, and Xylosalsola). Salsola s.s. include a group of central and southwest Asian and north African species that consists of Salsola sect. Salsola s.s., Salsola sect. Caroxylon subsect. Coccosalsola, Salsola sect. Obpyrifolia, Fadenia, Hypocylix, Seidlitzia, and Darniella. All species of tribe Caroxyloneae investigated so far have C4 photosynthesis of the NAD‐malic enzyme subtype, while the majority of the species of Salsoleae s.s. are known to be of the NADP‐malic enzyme subtype.

A Comparative Study in Ancestral Range Reconstruction Methods: Retracing the Uncertain Histories of Insular Lineages

Island systems have long been useful models for understanding lineage diversification in a geographic context, especially pertaining to the importance of dispersal in the origin of new clades. Here we use a well-resolved phylogeny of the flowering plant genus Cyrtandra (Gesneriaceae) from the Pacific Islands to compare four methods of inferring ancestral geographic ranges in islands: two developed for character-state reconstruction that allow only single-island ranges and do not explicitly associate speciation with range evolution (Fitch parsimony [FP; parsimony-based] and stochastic mapping [SM; likelihood-based]) and two methods developed specifically for ancestral range reconstruction, in which widespread ranges (spanning islands) are integral to inferences about speciation scenarios (dispersal-vicariance analysis [DIVA; parsimony-based] and dispersal-extinction-cladogenesis [DEC; likelihood-based]). The methods yield conflicting results, which we interpret in light of their respective assumptions. FP exhibits the least power to unequivocally reconstruct ranges, likely due to a combination of having flat (uninformative) transition costs and not using branch length information. SM reconstructions generally agree with a prior hypothesis about dispersal-driven speciation across the Pacific, despite the conceptual mismatch between its character-based model and this mode of range evolution. In contrast with narrow extant ranges for species of Cyrtandra, DIVA reconstructs broad ancestral ranges at many nodes. DIVA results also conflict with geological information on island ages; we attribute these conflicts to the parsimony criterion not considering branch lengths or time, as well as vicariance being the sole means of divergence for widespread ancestors. DEC analyses incorporated geological information on island ages and allowed prior hypotheses about range size and dispersal rates to be evaluated in a likelihood framework and gave more nuanced inferences about range evolution and the geography of speciation than other methods tested. However, ancestral ranges at several nodes could not be conclusively resolved, due possibly to uncertainty in the phylogeny or the relative complexity of the underlying model. Of the methods tested, SM and DEC both converge on plausible hypotheses for area range histories in Cyrtandra, due in part to the consideration of branch lengths and/or timing of events. We suggest that DEC model-based methods for ancestral range inference could be improved by adopting a Bayesian SM approach, in which stochastic sampling of complete geographic histories could be integrated over alternative phylogenetic topologies. Likelihood-based estimates of ancestral ranges for Cyrtandra suggest a major dispersal route into the Pacific through the islands of Fiji and Samoa, motivating future biogeographic investigation of this poorly known region.

Phylogenetic analysis of the myostatin gene sub-family and the differential expression of a novel member in zebrafish

Summary The myostatin (MSTN)‐null phenotype in mammals is characterized by extreme gains in skeletal muscle mass or “double muscling” as the cytokine negatively regulates skeletal muscle growth. Recent attempts, however, to reproduce a comparable phenotype in zebrafish have failed. Several aspects of MSTN biology in the fishes differ significantly from those in mammals and at least two distinct paralogs have been identified in some species, which possibly suggests functional divergence between the different vertebrate classes or between fish paralogs. We therefore conducted a phylogenetic analysis of the entire MSTN gene sub‐family. Maximum likelihood, Bayesian inference, and bootstrap analyses indicated a monophyletic distribution of all MSTN genes with two distinct fish clades: MSTN‐1 and ‐2. These analyses further indicated that all Salmonid genes described are actually MSTN‐1 orthologs and that additional MSTN‐2 paralogs may be present in most, if not all, teleosts. An additional zebrafish homolog was identified by BLAST searches of the zebrafish Hierarchical Tets Generation System database and was subsequently cloned. Comparative sequence analysis of both genes (zebrafish MSTN (zfMSTN)‐1 and ‐2) revealed many differences, primarily within the latency‐associated peptide regions, but also within the bioactive domains. The 2‐kb promoter region of zfMSTN‐2 contained many putative cis regulatory elements that are active during myogenesis, but are lacking in the zfMSTN‐1 promoter. In fact, zfMSTN‐2 expression was limited to the early stages of somitogenesis, whereas zfMSTN‐1 was expressed throughout embryogenesis. These data suggest that zfMSTN‐2 may be more closely associated with skeletal muscle growth and development. They also resolve the previous ambiguity in classification of fish MSTN genes.

The Evolution of Chromosome Arrangements in Carex (Cyperaceae)

Sedges (Carex: Cyperaceae) exhibit remarkable agmatoploid chromosome series between and within species. This chromosomal diversity is due in large part to the structure of the holocentric chromosomes: fragments that would not be heritable in organisms with monocentric chromosomes have the potential to produce viable gametes in organisms with holocentric chromosomes. The rapid rate of chromosome evolution in the genus and high species diversification rate in the order (Cyperales Hutch., sensu Dahlgren) together suggest that chromosome evolution may play an important role in the evolution of species diversity in Carex. Yet the other genera of the Cyperaceae and their sister group, the Juncaceae, do not show the degree of chromosomal variation found in Carex, despite the fact that diffuse centromeres are a synapomorphy for the entire clade. Moreover, fission and fusion apparently account for the majority of chromosome number changes in Carex, with relatively little duplication of whole chromosomes, whereas polyploidy is relatively important in the other sedge genera. In this paper, we review the cytologic and taxonomic literature on chromosome evolution in Carex and identify unanswered questions and directions for future research. In the end, an integration of biosystematic, cytogenetic, and genomic studies across the Cyperaceae will be needed to address the question of what role chromosome evolution plays in species diversification within Carex and the Cyperaceae as a whole.

Repeated evolution of net venation and fleshy fruits among monocots in shaded habitats confirms a priori predictions: evidence from an ndhF phylogeny

We present a well-resolved, highly inclusive phylogeny for monocots, based on ndhF sequence variation, and use it to test a priori hypotheses that net venation and vertebrate-dispersed fleshy fruits should undergo concerted convergence, representing independent but often concurrent adaptations to shaded conditions. Our data demonstrate that net venation arose at least 26 times and was lost eight times over the past 90 million years; fleshy fruits arose at least 21 times and disappeared 11 times. Both traits show a highly significant pattern of concerted convergence (p<10−9), arising 16 times and disappearing four times in tandem. This phenomenon appears driven by even stronger tendencies for both traits to evolve in shade and be lost in open habitats (p<10−13–10−29). These patterns are among the strongest ever demonstrated for evolutionary convergence in individual traits and the predictability of evolution, and the strongest evidence yet uncovered for concerted convergence. The rate of adaptive shifts per taxon has declined exponentially over the past 90 million years, as expected when large-scale radiations fill adaptive zones.

A Synopsis of Chromosome Number Variation in the Cyperaceae

The Cyperaceae are well known for having a large amount of variation in chromosome numbers both within and among genera. Most of this variation has been previously attributed to agmatoploid or qualitative aneuploid chromosome number change. To date there have been 4,231 reported chromosome number counts in the family. Despite the large number of counts made, they only represent approximately 16% of the species currently recognized. These counts are here presented in an indexed list with standardized nomenclature following a draft copy of the World Checklist of Cyperaceae. Additionally, I explore variation within genera where a significant number of counts have been made. Given the distributions of counts within genera there is evidence for both agmatoploid and polyploid chromosome number changes.

Basic helix-loop-helix transcription factor gene family phylogenetics and nomenclature

A phylogenetic analysis of the basic helix-loop-helix (bHLH) gene superfamily was performed using seven different species (human, mouse, rat, worm, fly, yeast, and plant Arabidopsis) and involving over 600 bHLH genes (Stevens et al., 2008). All bHLH genes were identified in the genomes of the various species, including expressed sequence tags, and the entire coding sequence was used in the analysis. Nearly 15% of the gene family has been updated or added since the original publication. A super-tree involving six clades and all structural relationships was established and is now presented for four of the species. The wealth of functional data available for members of the bHLH gene superfamily provides us with the opportunity to use this exhaustive phylogenetic tree to predict potential functions of uncharacterized members of the family. This phylogenetic and genomic analysis of the bHLH gene family has revealed unique elements of the evolution and functional relationships of the different genes in the bHLH gene family.

Phylogenetic Relationships in the Salicornioideae / Suaedoideae / Salsoloideae s.l. (Chenopodiaceae) Clade and a Clarification of the Phylogenetic Position of Bienertia and Alexandra Using Multiple DNA Sequence Datasets

Abstract The Chenopodiaceae includes taxa with both C3 and C4 photosynthesis with diverse kinds of Kranz anatomy and single-celled C4 species without Kranz anatomy; thus, it is of key importance for understanding evolution of C4 photosynthesis. All of the C4 genera except Atriplex, which belongs to Chenopodioideae, are in the Salicornioideae / Suaedoideae / Salsoloideae s.l. (including Camphorosmeae and Sclerolaeneae) clade. Our study focused on the relationships of the main lineages within this clade with an emphasis on the placement of the single cell functioning C4 genus Bienertia using maximum parsimony, maximum likelihood, and Bayesian inference phylogenetic analyses of the nuclear ribosomal ITS and five chloroplast DNA regions (atpB-rbcL, matK, psbB-psbH, rbcL, and trnL-trnF). Further we provide a detailed phylogeny of Alexandra and Suaeda based on ITS, atpB-rbcL, and psbB-psbH. Our molecular data provide strong statistical support for the monophyly of: (1) a Salicornioideae / Suaedoideae / Salsoloideae s.l. clade; (2) a Salicornioideae / Suaedoideae clade; (3) the subfamilies Salicornioideae, Suaedoideae (including Bienertia) and Salsoloideae s.l.; (4) the tribes Suaedeae, Salsoleae, and Camphorosmeae; (5) the Salicornieae if Halopeplideae is included; and (6) Suaeda if Alexandra is included. Alexandra lehmannii is therefore reclassified as Suaeda lehmannii and a new section of Suaeda is created, section Alexandra. There are four independent origins of C4 photosynthesis within the Suaedoideae including two parallel origins of Kranz C4 anatomy (in Suaeda sections Salsina s.l. and Schoberia) and two independent origins of C4 systems without Kranz anatomy (in Bienertia and in Suaeda section Borszczowia).