Transcript and metabolite changes during the early phase of abscisic acid-mediated induction of crassulacean acid metabolism in Talinum triangulare.

Crassulacean acid metabolism (CAM) has evolved as a water-saving strategy, and its engineering into crops offers an opportunity to improve their water use efficiency. This requires a comprehensive understanding of the regulation of the CAM pathway. Here, we use the facultative CAM species Talinum triangulare as a model in which CAM can be induced rapidly by exogenous abscisic acid. RNA sequencing and metabolite measurements were employed to analyse the changes underlying CAM induction and identify potential CAM regulators. Non-negative matrix factorization followed by k-means clustering identified an early CAM-specific cluster and a late one, which was specific for the early light phase. Enrichment analysis revealed abscisic acid metabolism, WRKY-regulated transcription, sugar and nutrient transport, and protein degradation in these clusters. Activation of the CAM pathway was supported by up-regulation of phosphoenolpyruvate carboxylase, cytosolic and chloroplastic malic enzymes, and several transport proteins, as well as by increased end-of-night titratable acidity and malate accumulation. The transcription factors HSFA2, NF-YA9, and JMJ27 were identified as candidate regulators of CAM induction. With this study we promote the model species T. triangulare, in which CAM can be induced in a controlled way, enabling further deciphering of CAM regulation.

Comparative cytogenetics of the ACPT clade (Anacampserotaceae, Cactaceae, Portulacaceae, and Talinaceae): a very diverse group of the suborder Cactineae, Caryophyllales.

The clade ACPT (Anacampserotaceae, Cactaceae, Portulacaceae, and Talinaceae) is the most diverse lineage of the subordem Cactineae. The relationships between these families are still uncertain, with different topologies suggested by phylogenetic analyses with several combinations of markers. Different basic numbers (x) have been suggested for each family and for the subord, often in a contestable way. Comparative cytogenetic has helped to understand the evolutionary relationships of phylogenetically poorly resolved groups, as well as their mechanisms of karyotype evolution. The karyotype evolution in representatives of Cactineae was analyzed, focusing on the ACPT clade, through the analysis of chromosome number in a phylogenetic bias. The phylogeny obtained showed a well-resolved topology with support for the monophyly of the five families. Although a chromosomal number is known for less than 30% of the Cactineae species, the analyses revealed a high karyotype variability, from 2n = 8 to 2n = 110. The analysis of character reconstruction of the ancestral haploid numbers (p) suggested p = 12 for Cactineae, with distinct basic numbers for the clade family ACPT: Cactaceae and Montiaceae (p = 11), Talinaceae (p = 12), and Anacampserotaceae and Portulacaceae (p = 9). Talinaceae, Anacampserotaceae, and Cactaceae were stable, while Portulaca and Montiaceae were karyotypically variable. The chromosome evolution of this group was mainly due to events of descending disploidy and poliploidy. Our data confirm that the low phylogenetic resolution among the families of the ACPT clade is due to a divergence of this clade in a short period of time. However, each of these families can be characterized by basic chromosome numbers and unique karyotype evolution events.

Next-generation lineage discovery: A case study of tuberous Claytonia L.

PREMISE OF THE STUDY: Species formation is an intuitive endpoint of reproductive isolation, but circumscribing taxa that arise during speciation can be difficult because of gene flow, morphological continuity, hybridization or polyploidization, and low sequence variation among newly diverged lineages. Nonetheless, species complexes are ubiquitous, and their classification is essential for understanding how diversity influences ecosystem function. METHODS: We used modern sequencing technology to identify lineages of perennial Claytonia L. and assessed correspondence between genetic lineages and morphological/ecological variation. Subsets of 18 taxa from 63 populations were used for (a) lineage discovery using network and coalescent analyses, (b) leaf shape analyses using elliptical Fourier analysis and ordination, and (c) ecological analyses (soil chemistry, climate) using ANOVA and ordination. KEY RESULTS: Samples mainly aggregated into groups representing each of the previously recognized species in each of the genetic data sets. Compared to the double-digest restriction-site-associated DNA sequencing data set, genome skimming data provided more resolution and further opportunity to probe into patterns of nuclear and chloroplast genome diversity. Morphological and ecological associations are significantly different (albeit intergrading) among the taxa investigated. A new species, Claytonia crawfordii, is described based on morphological data presented here. CONCLUSIONS: Genetic data presented in this study provide some of the first insights into phylogenetic relationships among recently diverged perennial Claytonia and are suggestive of past hybridization among caudicose and tuberous species. Given prior difficulties in understanding species boundaries among newly diverged plant lineages, this case study demonstrates the revolutionary breakthrough for systematics research that high throughput sequencing represents.

An experimental test of local adaptation among cytotypes within a polyploid complex.

The geographic distributions of polyploids suggest they can have distinct and sometimes broader niches compared to diploids. However, relatively few field experiments have investigated whether range differences are associated with local adaptation or reflect other processes, such as dispersal limitation. In three years of transplants across the elevational ranges of five cytotypes in the Claytonia perfoliata complex, we found evidence for local adaptation. In at least one study year germination was higher within the natural range for each cytotype, and four of the five cytotypes attained larger biomass within their natural range. Fitness within and beyond range varied across years, with two instances of cytotypes showing higher fitness beyond the range, highlighting a potential role of temporal variability in cytotype differentiation. Polyploids as a group did not outperform diploids, but the cytotype with highest fitness across environments was a hexaploid reported to be invasive. Our results suggest that differences in geographic ranges within the C. perfoliata complex reflect local adaptation of cytotypes. Although we did not find a general polyploid advantage, our findings support the idea that occasional polyploid cytotypes exhibit high fitness relative to other cytotypes, and contribute to growing evidence supporting ecological differentiation of cytotypes within polyploid complexes.

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.

Cytogeography and genome size variation in the Claytonia perfoliata (Portulacaceae) polyploid complex.

BACKGROUND AND AIMS: Genome duplication is a central process in plant evolution and contributes to patterns of variation in genome size within and among lineages. Studies that combine cytogeography with genome size measurements contribute to our basic knowledge of cytotype distributions and their associations with variation in genome size. METHODS: Ploidy and genome size were assessed with direct chromosome counts and flow cytometry for 78 populations within the Claytonia perfoliata complex, comprised of three diploid taxa with numerous polyploids that range to the decaploid level. The relationship between genome size and temperature and precipitation was investigated within and across cytotypes to test for associations between environmental factors and nuclear DNA content. KEY RESULTS: A euploid series (n = 6) of diploids to octoploids was documented through chromosome counts, and decaploids were suggested by flow cytometry. Increased variation in genome size among populations was found at higher ploidy levels, potentially associated with differential contributions of diploid parental genomes, variation in rates of genomic loss or gain, or undetected hybridization. Several accessions were detected with atypical genome sizes, including a diploid population of C. parviflora ssp. grandiflora with an 18 % smaller genome than typical, and hexaploids of C. perfoliata and C. parviflora with genomes 30 % larger than typical. There was a slight but significant association of larger genome sizes with colder winter temperature across the C. perfoliata complex as a whole, and a strong association between lower winter temperatures and large genome size for tetraploid C. parviflora. CONCLUSIONS: The C. perfoliata complex is characterized by polyploids ranging from tetraploid to decaploid, with large magnitude variation in genome size at higher ploidy levels, associated in part with environmental variation in temperature.

Polyploidy associated with altered and broader ecological niches in the Claytonia perfoliata (Portulacaceae) species complex.

PREMISE OF THE STUDY: Polyploids are often hypothesized to have distinct and broader niches than their diploid progenitors. Differences in geographic distributions between diploid and polyploids are frequently used to infer niche differentiation and increased breadth, but they are seldom used to test these hypotheses explicitly. METHODS: Niche overlap and breadth were compared for diploids, tetraploids, and hexaploids of three taxa in the Claytonia perfoliata complex (C. parviflora, C. perfoliata, and C. rubra) with the use of species distribution models. Resampling and randomization approaches were used to test hypotheses of niche differentiation, breadth, and conservatism. KEY RESULTS: Niche differentiation was detected between polyploid and diploid cytotypes assigned to the same taxon (e.g., C. parviflora 2× vs. 4×) but not between hexaploids and tetraploids within a taxon (e.g., C. parviflora 4× vs. 6×). Individual hexaploid cytotypes had broader ecological niches than individual diploid cytotypes. However, as a group the three hexaploid taxa did not exceed the combined niche breadth of the three diploids, suggesting that polyploidy does not result in transgressive niche breadth for this group. Niche overlap was lowest among diploids and was highest among the three hexaploid cytotypes, consistent with introgression associated with polyploidy resulting in greater ecological similarity. Although cytotypes possessed nonidentical niches, after accounting for environmental differences among ranges, cytotypes were more similar than expected, suggesting niche conservatism and similar responses to environmental characteristics. CONCLUSIONS: These results suggest that polyploids occupy distinct and broader niches relative to diploids but that cytotypes also share fundamentally similar responses to environmental variation across ploidy levels.

Evolutionary physiology: the extent of C4 and CAM photosynthesis in the genera Anacampseros and Grahamia of the Portulacaceae.

The Portulacaceae is one of the few terrestrial plant families known to have both C(4) and Crassulacean acid metabolism (CAM) species. There may be multiple origins of the evolution of CAM within the Portulacaceae but the only clear evidence of C(4) photosynthesis is found in members of the genus Portulaca. In the Portulaca, CAM succulent tissue is overlaid with the C(4) tissue in a unique fashion where both pathways are operating simultaneously. Earlier reports have shown that the clade containing the genera Anacampseros and Grahamia may also contain C(4) photosynthetic species similar to the Portulaca, which would indicate multiple origins of C(4) photosynthesis within the family. The aim of the present study was to ascertain the true photosynthetic nature of these genera. An initial survey of the carbon isotope composition of the Anacampseros ranged from -12.6 per thousand to -24.0 per thousand, indicating very little CAM activity in some species, with other values close to the C(4) range. Anacampseros (=Grahamia) australiana which had been previously identified as a C(4) species had a carbon isotope composition value of -24.0 per thousand, which is more indicative of a C(3) species with a slight contribution of CAM activity. Other Anacampseros species with C(4)-like values have been shown to be CAM plants. The initial isotope analysis of the Grahamia species gave values in the range of -27.1 per thousand to -23.6 per thousand, placing the Grahamia species well towards the C(3) photosynthetic range. Further physiological studies indicated increased night-time CO(2) uptake with imposition of water stress, associated with a large diurnal acid fluctuation and a marked increased phosphoenolpyruvate carboxylase activity. This showed that the Grahamia species are actually facultative CAM plants despite their C(3)-like carbon isotope values. The results indicate that the Grahamia and Anacampseros species do not utilize the C(4) photosynthetic pathway. This is the first to identify that the Grahamia species are facultative CAM plants where CAM can be induced by water stress. This work supports earlier physiological work that indicates that this clade containing Anacampseros and Grahamia species comprises predominantly facultative CAM plants. This report suggests there may be only one clade which contains C(4) photosynthetic members with CAM-like characteristics.

Opposing natural selection from herbivores and pathogens may maintain floral-color variation in Claytonia virginica (Portulacaceae).

The maintenance of floral-color variation within natural populations is enigmatic because directional selection through pollinator preferences combined with random genetic drift should lead to the rapid loss of such variation. Fluctuating, balancing, and negative frequency-dependent selection mediated through pollinators have been identified as factors that may contribute to the maintenance of floral-color variation, and recently it has been suggested that indirect responses to selection on correlated characters through agents of selection other than pollinators may substantially shape the evolution of floral traits. Here, I provide empirical support for this latter view in Claytonia virginica (Portulacaceae) through a multiseason field study, a pollen supplementation study, and an artificial herbivory experiment. These studies show that most individuals fall into one of four discrete color classes, and suggest pollinator-mediated selection for increased floral redness in concurrent years. Floral color is also an indirect target of opposing directional selection via herbivores and pathogens that fluctuates through time. Taken together, these data suggest a novel mechanism by which floral-color variation may be maintained, and illustrate the importance of an inclusive, pluralistic view of selection when investigating the evolution of complex phenotypes.