Predicting photosynthetic pathway from anatomy using machine learning.

Plants with Crassulacean acid metabolism (CAM) have long been associated with a specialized anatomy, including succulence and thick photosynthetic tissues. Firm, quantitative boundaries between non-CAM and CAM plants have yet to be established - if they indeed exist. Using novel computer vision software to measure anatomy, we combined new measurements with published data across flowering plants. We then used machine learning and phylogenetic comparative methods to investigate relationships between CAM and anatomy. We found significant differences in photosynthetic tissue anatomy between plants with differing CAM phenotypes. Machine learning-based classification was over 95% accurate in differentiating CAM from non-CAM anatomy, and had over 70% recall of distinct CAM phenotypes. Phylogenetic least squares regression and threshold analyses revealed that CAM evolution was significantly correlated with increased mesophyll cell size, thicker leaves, and decreased intercellular airspace. Our findings suggest that machine learning may be used to aid the discovery of new CAM species and that the evolutionary trajectory from non-CAM to strong, obligate CAM requires continual anatomical specialization.

Targeted Enrichment of Large Gene Families for Phylogenetic Inference: Phylogeny and Molecular Evolution of Photosynthesis Genes in the Portullugo Clade (Caryophyllales).

Hybrid enrichment is an increasingly popular approach for obtaining hundreds of loci for phylogenetic analysis across many taxa quickly and cheaply. The genes targeted for sequencing are typically single-copy loci, which facilitate a more straightforward sequence assembly and homology assignment process. However, this approach limits the inclusion of most genes of functional interest, which often belong to multi-gene families. Here, we demonstrate the feasibility of including large gene families in hybrid enrichment protocols for phylogeny reconstruction and subsequent analyses of molecular evolution, using a new set of bait sequences designed for the "portullugo" (Caryophyllales), a moderately sized lineage of flowering plants (~ 2200 species) that includes the cacti and harbors many evolutionary transitions to C$_{\mathrm{4}}$ and CAM photosynthesis. Including multi-gene families allowed us to simultaneously infer a robust phylogeny and construct a dense sampling of sequences for a major enzyme of C$_{\mathrm{4}}$ and CAM photosynthesis, which revealed the accumulation of adaptive amino acid substitutions associated with C$_{\mathrm{4}}$ and CAM origins in particular paralogs. Our final set of matrices for phylogenetic analyses included 75-218 loci across 74 taxa, with ~ 50% matrix completeness across data sets. Phylogenetic resolution was greatly improved across the tree, at both shallow and deep levels. Concatenation and coalescent-based approaches both resolve the sister lineage of the cacti with strong support: Anacampserotaceae $+$ Portulacaceae, two lineages of mostly diminutive succulent herbs of warm, arid regions. In spite of this congruence, BUCKy concordance analyses demonstrated strong and conflicting signals across gene trees. Our results add to the growing number of examples illustrating the complexity of phylogenetic signals in genomic-scale data.

Molecular evolution of key metabolic genes during transitions to C4 and CAM photosynthesis.

PREMISE OF THE STUDY: Next-generation sequencing facilitates rapid production of well-sampled phylogenies built from very large genetic data sets, which can then be subsequently exploited to examine the molecular evolution of the genes themselves. We present an evolutionary analysis of 83 gene families (19 containing carbon-concentrating mechanism (CCM) genes, 64 containing non-CCM genes) in the portullugo clade (Caryophyllales), a diverse lineage of mostly arid-adapted plants that contains multiple evolutionary origins of all known photosynthesis types in land plants (C3 , C4 , CAM, C4 -CAM, and various intermediates). METHODS: We inferred a phylogeny of 197 individuals from 167 taxa using coalescent-based approaches and individual gene family trees using maximum likelihood. Positive selection analyses were conducted on individual gene family trees with a mixed effects model of evolution (MEME). We devised new indices to compare levels of convergence and prevalence of particular residues between CCM and non-CCM genes and between species with different photosynthetic pathways. KEY RESULTS: Contrary to expectations, there were no significant differences in the levels of positive selection detected in CCM versus non-CCM genes. However, we documented a significantly higher level of convergent amino acid substitutions in CCM genes, especially in C4 taxa. CONCLUSIONS: Our analyses reveal a new suite of amino acid residues putatively important for C4 and CAM function. We discuss both the advantages and challenges of using targeted enrichment sequence data for exploratory studies of molecular evolution.

Phylogeny, evolution, and biogeographic history of Calandrinia (Montiaceae).

PREMISE OF THE STUDY: Calandrinia are small, succulent herbs that vary broadly in habitat, morphology, life history, and photosynthetic metabolism. The lineage is placed within the Montiaceae, which in turn is sister to the rest of the Portulacineae (Caryophyllales). Calandrinia occupy two distinct biogeographic regions, one in the Americas (~14 species), and one in Australia (~74 species). Past analyses of the Montiaceae present conflicting hypotheses for the phylogenetic placement and monophyly of Calandrinia, and to date, there has been no molecular phylogenetic analysis of the Australian species. METHODS: Using a targeted gene enrichment approach, we sequenced 297 loci from multiple gene families across the Montiaceae, including all named and 16 putative new species of Australian Calandrinia, and the enigmatic monotypic genus Rumicastrum. KEY RESULTS: All data sets and analyses reject the monophyly of Calandrinia, with Australian and New World Calandrinia each comprising distinct and well-supported clades, and Rumicastrum nested within Australian Calandrinia. We provide the first well-supported phylogeny for Australian Calandrinia, which includes all named species and several phrase-named taxa. CONCLUSIONS: This study brings much needed clarity to relationships within Montiaceae and confirms that New World and Australian Calandrinia do not form a clade. Australian Calandrinia is a longtime resident of the continent, having diverged from its sister lineage ~30 Ma, concurrent with separation of Australia from Antarctica. Most diversification occurred during the middle Miocene, with lowered speciation and/or higher extinction rates coincident with the establishment of severe aridity by the late Miocene.

Facultative CAM photosynthesis (crassulacean acid metabolism) in four species of Calandrinia, ephemeral succulents of arid Australia.

Crassulacean acid metabolism (CAM) was demonstrated in four small endemic Australian terrestrial succulents from the genus Calandrinia (Montiaceae) viz. C. creethiae, C. pentavalvis, C. quadrivalvis and C. reticulata. CAM was substantiated by measurements of CO2 gas-exchange and nocturnal acidification. In all species, the expression of CAM was overwhelmingly facultative in that nocturnal H+ accumulation was greatest in droughted plants and zero, or close to zero, in plants that were well-watered, including plants that had been droughted and were subsequently rewatered, i.e. the inducible component was proven to be reversible. Gas-exchange measurements complemented the determinations of acidity. In all species, net CO2 uptake was restricted to the light in well-watered plants, and cessation of watering was followed by a progressive reduction of CO2 uptake in the light and a reduction in nocturnal CO2 efflux. In C. creethiae, C. pentavalvis and C. reticulata net CO2 assimilation was eventually observed in the dark, whereas in C. quadrivalvis nocturnal CO2 exchange approached the compensation point but did not transition to net CO2 gain. Following rewatering, all species returned to their original well-watered CO2 exchange pattern of net CO2 uptake restricted solely to the light. In addition to facultative CAM, C. quadrivalvis and C. reticulata exhibited an extremely small constitutive CAM component as demonstrated by the nocturnal accumulation in well-watered plants of small amounts of acidity and by the curved pattern of the nocturnal course of CO2 efflux. It is suggested that low-level CAM and facultative CAM are more common within the Australian succulent flora, and perhaps the world succulent flora, than has been previously assumed.

CAM photosynthesis in desert blooming Cistanthe of the Atacama, Chile.

When plants of the Atacama desert undergo episodic blooms, among the most prominent are succulent-leaved Cistanthe (Montiaceae). We demonstrate that two Cistanthe species, the perennial Cistanthe sp. aff. crassifolia and the annual/biannual Cistanthe sp. aff. longiscapa, can exhibit net CO2 uptake and leaf acidification patterns typical of crassulacean acid metabolism (CAM). In C. sp. aff. crassifolia leaves, CAM expression was facultative. CAM-type nocturnal net CO2 uptake and acid accumulation occurred in drought-stressed but not in well-watered plants. By contrast, CAM expression in C. sp. aff. longiscapa was largely constitutive. Nocturnal acid accumulation was present in leaves of well-watered and in droughted plants. Following water-deficit stress, net nocturnal CO2 uptake was induced and the level of acid accumulated increased. Neither nocturnal CO2 uptake nor acid accumulation was reduced when the plants were re-watered. δ13C values of a further nine field-collected Cistanthe species are consistent with a contribution of CAM to their carbon pools. In the Portulacinae, a suborder with eight CAM-containing families, Cistanthe becomes the sixth genus with CAM within the family Montiaceae, and it is likely that the ancestor of all Portulacineae also possessed CAM photosynthesis. In the stochastic rainfall landscape of the Atacama, carbon uptake in the dark is a water-use efficient mechanism that increases the carbon pool available for seed production or dormancy. The next rain event may be years away.

The Evolution of CAM Photosynthesis in Australian Calandrinia Reveals Lability in C3+CAM Phenotypes and a Possible Constraint to the Evolution of Strong CAM.

Australian Calandrinia has radiated across the Australian continent during the last 30 Ma, and today inhabits most Australian ecosystems. Given its biogeographic range and reports of facultative Crassulacean acid metabolism (CAM) photosynthesis in multiple species, we hypothesized (1) that CAM would be widespread across Australian Calandrinia and that species, especially those that live in arid regions, would engage in strong CAM, and (2) that Australian Calandrinia would be an important lineage for informing on the CAM evolutionary trajectory. We cultivated 22 Australian Calandrinia species for a drought experiment. Using physiological measurements and δ13C values we characterized photosynthetic mode across these species, mapped the resulting character states onto a phylogeny, and characterized the climatic envelopes of species in their native ranges. Most species primarily utilize C3 photosynthesis, with CAM operating secondarily, often upregulated following drought. Several phylogenetically nested species are C3, indicating evolutionary losses of CAM. No strong CAM was detected in any of the species. Results highlight the limitations of δ13C surveys in detecting C3+CAM phenotypes, and the evolutionary lability of C3+CAM phenotypes. We propose a model of CAM evolution that allows for lability and reversibility among C3+CAM phenotypes and C3 and suggest that an annual life-cycle may preclude the evolution of strong CAM.

Optional use of CAM photosynthesis in two C4 species, Portulaca cyclophylla and Portulaca digyna.

Low levels of crassulacean acid metabolism (CAM) are demonstrated in two species with C4 photosynthesis, Portulaca cyclophylla and P. digyna. The expression of CAM in P. cyclophylla and P. digyna is facultative, i.e. optional. Well-watered plants did not accumulate acid at night and exhibited gas-exchange patterns consistent with C4 photosynthesis. CAM-type nocturnal acidification was reversible in that it was induced following drought and lost when droughted plants were rewatered. In P. cyclophylla, droughting was accompanied by a small but discernible net uptake of CO2 during the dark, whereas in P. digyna, net CO2 exchange at night approached the CO2 compensation point but did not transition beyond it. This report brings the number of known C4 species with a capacity for expressing CAM to six. All are species of Portulaca. The observation of CAM in P. cyclophylla and P. digyna is the first for species in the opposite-leaved (OL) Portulacelloid-anatomy lineage of Portulaca and for the Australian clade therein. The other four species are within the alternate-leaved (AL) lineage, in the Atriploid-anatomy Oleracea and the Pilosoid-anatomy Pilosa clades. Studies of the evolutionary origins of C4 and CAM in Portulaca will benefit from a more wide-range survey of CAM across its species, particularly in the C3-C4 intermediate-containing Cryptopetala clade.

Australia lacks stem succulents but is it depauperate in plants with crassulacean acid metabolism (CAM)?

In the flora of Australia, the driest vegetated continent, crassulacean acid metabolism (CAM), the most water-use efficient form of photosynthesis, is documented in only 0.6% of native species. Most are epiphytes and only seven terrestrial. However, much of Australia is unsurveyed, and carbon isotope signature, commonly used to assess photosynthetic pathway diversity, does not distinguish between plants with low-levels of CAM and C3 plants. We provide the first census of CAM for the Australian flora and suggest that the real frequency of CAM in the flora is double that currently known, with the number of terrestrial CAM species probably 10-fold greater. Still unresolved is the question why the large stem-succulent life - form is absent from the native Australian flora even though exotic large cacti have successfully invaded and established in Australia.