Deciduous and evergreen oaks show contrasting adaptive responses in leaf mass per area across environments.

Increases in leaf mass per area (LMA) are commonly observed in response to environmental stresses and are achieved through increases in leaf thickness and/or leaf density. Here, we investigated how the two underlying components of LMA differ in relation to species native climates and phylogeny, across deciduous and evergreen species. Using a phylogenetic approach, we quantified anatomical, compositional and climatic variables from 40 deciduous and 45 evergreen Quercus species from across the Northern Hemisphere growing in a common garden. Deciduous species from shorter growing seasons tended to have leaves with lower LMA and leaf thickness than those from longer growing seasons, while the opposite pattern was found for evergreens. For both habits, LMA and thickness increased in arid environments. However, this shift was associated with increased leaf density in evergreens but reduced density in deciduous species. Deciduous and evergreen oaks showed fundamental leaf morphological differences that revealed a diverse adaptive response. While LMA in deciduous species may have diversified in tight coordination with thickness mainly modulated by aridity, diversification of LMA within evergreens appears to be dependent on the infrageneric group, with diversification in leaf thickness modulated by both aridity and cold, while diversification in leaf density is only modulated by aridity.

Thallus Growth Stage and Geographic Origin Shape Microalgal Diversity in Ramalina farinacea Lichen Holobionts.

Lichen symbioses are microecosystems hosting many other living organisms besides the two major lichen symbionts (i.e., lichenized fungi [the mycobiont] and green microalgae or cyanobacteria [the photobiont]). Recent investigations evidenced that other fungi, non-photosynthetic bacteria, and microalgae co-inhabit within the lichen thalli, but their diversity and their roles are still underinvestigated. Here we present an ad hoc stratified sampling design and in-depth Illumina paired-end metabarcoding approach to explore microalgal diversity in lichen thalli of the model species Ramalina farinacea from different ecologies. Lichen thalli were surveyed according to three different sizes, and different thallus parts were considered for molecular, bioinformatics, and community diversity analyses. The results revealed that microalgal diversity strongly depends on the growth stage of the thalli, the geographic area, and the habitat type. The results also show that microalgal diversity does not vary along the thallus branches (lacinias)-that is, it does not correlate with the apical growth and founder effects-and that there is no balanced co-presence of two main photobionts as previously established in R. farinacea. The sampling design performed here minimizes bias in the assessment of photobiont diversity in lichens and is proposed to be reliable and applicable to further study microalgal diversity in lichen symbioses.

Exploring molecular evolution of Rubisco in C3 and CAM Orchidaceae and Bromeliaceae.

BACKGROUND: The CO2-concentrating mechanism associated to Crassulacean acid metabolism (CAM) alters the catalytic context for Rubisco by increasing CO2 availability and provides an advantage in particular ecological conditions. We hypothesized about the existence of molecular changes linked to these particular adaptations in CAM Rubisco. We investigated molecular evolution of the Rubisco large (L-) subunit in 78 orchids and 144 bromeliads with C3 and CAM photosynthetic pathways. The sequence analyses were complemented with measurements of Rubisco kinetics in some species with contrasting photosynthetic mechanism and differing in the L-subunit sequence. RESULTS: We identified potential positively selected sites and residues with signatures of co-adaptation. The implementation of a decision tree model related Rubisco specific variable sites to the leaf carbon isotopic composition of the species. Differences in the Rubisco catalytic traits found among C3 orchids and between strong CAM and C3 bromeliads suggested Rubisco had evolved in response to differing CO2 concentration. CONCLUSIONS: The results revealed that the variability in the Rubisco L-subunit sequence in orchids and bromeliads is composed of coevolving sites under potential positive adaptive signal. The sequence variability was related to δ13C in orchids and bromeliads, however it could not be linked to the variability found in the kinetic properties of the studied species.

The chloroplast genome of the lichen-symbiont microalga Trebouxia sp. Tr9 (Trebouxiophyceae, Chlorophyta) shows short inverted repeats with a single gene and loss of the rps4 gene, which is encoded by the nucleus.

The Trebouxiophyceae is the class of Chlorophyta algae from which the highest number of chloroplast genome (cpDNA) sequences has been obtained. Several species in this class participate in symbioses with fungi to form lichens. However, no cpDNA has been obtained from any Trebouxia lichen-symbiont microalgae, which are present in approximately half of all lichens. Here, we report the sequence of the completely assembled cpDNA from Trebouxia sp. TR9 and a comparative study with other Trebouxio-phyceae. The organization of the chloroplast genome of Trebouxia sp. TR9 has certain features that are unusual in the Trebouxiophyceae and other green algae. The most remarkable characteristics are the presence of long intergenic spacers, a quadripartite structure with short inverted repeated sequences (IRs), and the loss of the rps4 gene. The presence of long intergenic spacers accounts for a larger cpDNA size in comparison to other closely related Trebouxiophyceae. The IRs, which were thought to be lost in the Trebouxiales, are distinct from most of cpDNAs since they lack the rRNA operon and uniquely includes the rbcL gene. The functional transfer of the rps4 gene to the nuclear genome has been confirmed by sequencing and examination of the gene architecture, which includes three spliceosomal introns as well as the verification of the presence of the corresponding transcript. This is the first documented transfer of the rps4 gene from the chloroplast to the nucleus among Viridiplantae. Additionally, a fairly well-resolved phylogenetic reconstruction, including Trebouxia sp. TR9 along with other Trebouxiophyceae, was obtained based on a set of conserved chloroplast genes.

Leaf anatomy does not explain apparent short-term responses of mesophyll conductance to light and CO2 in tobacco.

Mesophyll conductance to CO2 (gm ), a key photosynthetic trait, is strongly constrained by leaf anatomy. Leaf anatomical parameters such as cell wall thickness and chloroplast area exposed to the mesophyll intercellular airspace have been demonstrated to determine gm in species with diverging phylogeny, leaf structure and ontogeny. However, the potential implication of leaf anatomy, especially chloroplast movement, on the short-term response of gm to rapid changes (i.e. seconds to minutes) under different environmental conditions (CO2 , light or temperature) has not been examined. The aim of this study was to determine whether the observed rapid variations of gm in response to variations of light and CO2 could be explained by changes in any leaf anatomical arrangements. When compared to high light and ambient CO2 , the values of gm estimated by chlorophyll fluorescence decreased under high CO2 and increased at low CO2 , while it decreased with decreasing light. Nevertheless, no changes in anatomical parameters, including chloroplast distribution, were found. Hence, the gm estimated by analytical models based on anatomical parameters was constant under varying light and CO2 . Considering this discrepancy between anatomy and chlorophyll fluorescence estimates, it is concluded that apparent fast gm variations should be due to artefacts in its estimation and/or to changes in the biochemical components acting on diffusional properties of the leaf (e.g. aquaporins and carbonic anhydrase).

Molecular and morphological diversity of Trebouxia microalgae in sphaerothallioid Circinaria spp. lichens1.

Three vagrant (Circinaria hispida, Circinaria gyrosa, and Circinaria sp. 'paramerae') and one crustose (semi-vagrant, Circinaria sp. 'oromediterranea') lichens growing in very continental areas in the Iberian Peninsula were selected to study the phycobiont diversity. Mycobiont identification was checked using nrITS DNA barcoding: Circinaria sp. 'oromediterranea' and Circinaria sp. 'paramerae' formed a new clade. Phycobiont diversity was analyzed in 50 thalli of Circinaria spp. using nrITS DNA and LSU rDNA, with microalgae coexistence being found in all the species analyzed by Sanger sequencing. The survey of phycobiont diversity showed up to four different Trebouxia spp. as the primary phycobiont in 20 thalli of C. hispida, in comparison with the remaining Circinaria spp., where only one Trebouxia was the primary microalga. In lichen species showing coexistence, some complementary approaches are needed (454 pyrosequencing and/or ultrastructural analyses). Five specimens were selected for high-throughput screening (HTS) analyses: 22 Trebouxia OTUs were detected, 10 of them not previously known. TEM analyses showed three different cell morphotypes (Trebouxia sp. OTU A12, OTU S51, and T. cretacea) whose ultrastructure is described here in detail for the first time. HTS revealed a different microalgae pool in each species studied, and we cannot assume a specific pattern between these pools and the ecological and/or morphological characteristics. The mechanisms involved in the selection of the primary phycobiont and the other microalgae by the mycobiont are unknown, and require complex experimental designs. The systematics of the genus Circinaria is not yet well resolved, and more analyses are needed to establish a precise delimitation of the species.

Weak coordination between leaf structure and function among closely related tomato species.

Theory predicts that natural selection should favor coordination between leaf physiology, biochemistry and anatomical structure along a functional trait spectrum from fast, resource-acquisitive syndromes to slow, resource-conservative syndromes. However, the coordination hypothesis has rarely been tested at a phylogenetic scale most relevant for understanding rapid adaptation in the recent past or for the prediction of evolutionary trajectories in response to climate change. We used a common garden to examine genetically based coordination between leaf traits across 19 wild and cultivated tomato taxa. We found weak integration between leaf structure (e.g. leaf mass per area) and physiological function (photosynthetic rate, biochemical capacity and CO2 diffusion), even though all were arrayed in the predicted direction along a 'fast-slow' spectrum. This suggests considerable scope for unique trait combinations to evolve in response to new environments or in crop breeding. In particular, we found that partially independent variation in stomatal and mesophyll conductance may allow a plant to improve water-use efficiency without necessarily sacrificing maximum photosynthetic rates. Our study does not imply that functional trait spectra, such as the leaf economics spectrum, are unimportant, but that many important axes of variation within a taxonomic group may be unique and not generalizable to other taxa.

Coordination between leaf CO2 diffusion and Rubisco properties allows maximizing photosynthetic efficiency in Limonium species.

High photosynthetic efficiency intrinsically demands tight coordination between traits related to CO2 diffusion capacity and leaf biochemistry. Although this coordination constitutes the basis of existing mathematical models of leaf photosynthesis, it has been barely explored among closely related species, which could reveal rapid adaptation clues in the recent past. With this aim, we characterized the photosynthetic capacity of 12 species of Limonium, possessing contrasting Rubisco catalytic properties, grown under optimal (WW) and extreme drought conditions (WD). The availability of CO2 at the site of carboxylation (Cc ) determined the photosynthetic capacity of Limonium under WD, while both diffusional and biochemical components governed the photosynthetic performance under WW. The variation in the in vivo caboxylation efficiency correlated with both the concentration of active Rubisco sites and the in vitro-based properties of Rubisco, such as the maximum carboxylase turnover rate (kcatc ) and the Michaelis-Menten constant for CO2 (Kc ). Notably, the results confirmed the hypothesis of coordination between the CO2 offer and demand functions of photosynthesis: those Limonium species with high total leaf conductance to CO2 have evolved towards increased velocity (i.e. higher kcatc ), at the penalty of lower affinity for CO2 (i.e. lower specificity factor, Sc/o ).

Environmentally driven evolution of Rubisco and improved photosynthesis and growth within the C3 genus Limonium (Plumbaginaceae).

Carbon assimilation by most ecosystems requires ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Its kinetic parameters are likely to have evolved in parallel with intracellular CO2 availability, with the result that faster forms of Rubisco occur in species with CO2 -concentrating mechanisms. The Rubisco catalytic properties were determined and evaluated in relation to growth and carbon assimilation capacity in Mediterranean Limonium species, inhabiting severe stress environments. Significant kinetic differences between closely related species depended on two amino acid substitutions at functionally important residues 309 and 328 within the Rubisco large subunit. The Rubisco of species facing the largest CO2 restrictions during drought had relatively high affinity for CO2 (low Michaelis-Menten constant for CO2 Kc) but low maximum rates of carboxylation (kcatc), while the opposite was found for species that maintained higher CO2 concentrations under similar conditions. Rubisco kinetic characteristics were correlated with photosynthetic rate in both well-watered and drought-stressed plants. Moreover, the drought-mediated decrease in plant biomass accumulation was consistently lower in species with higher Rubisco carboxylase catalytic efficiency (kcatc/Kc). The present study is the first demonstration of Rubisco adaptation during species diversification within closely related C3 plants, revealing a direct relationship between Rubisco molecular evolution and the biomass accumulation of closely related species subjected to unfavourable conditions.

Improving the estimation of mesophyll conductance to CO2: on the role of electron transport rate correction and respiration.

Mesophyll conductance (gm) can markedly limit photosynthetic CO2 assimilation and is required to estimate the parameters of the Farquhar-von Caemmerer-Berry (FvCB) model properly. The variable J (electron transport rate) is the most frequently used method for estimating gm, and the correct determination of J is one of its requirements. Recent evidence has shown that calibrating J can lead to some errors in estimating gm, but to what extent the parameterization of the FvCB model is affected by calibrations is not well known. In addition to determining the FvCB parameters, variants of the J calibration method were tested to address whether varying CO2 or light levels, possible alternative electron sinks, or contrasting leaf structural properties might play a role in determining differences in αß, the product of the leaf absorptance (α) and the photosystem II optical cross-section (ß). It was shown that differences in αß were mainly attributed to the use of A/C(i) or A/PPFD curves to calibrate J. The different αß values greatly influenced g(m), leading to a high number of unrealistic values in addition to affecting the estimates of the FvCB model parameters. A new approach was devised to retrieve leaf respiration in the light from combined A/C(i) and A/C(c) curves and a framework to understand the high variation in observed gm values. Overall, a background is provided to decrease the noise in gm, facilitating data reporting and allowing better retrieval of the information presented in A/C(i) and A/C(c) curves.

Photosynthesis limitations in three fern species.

Maximum photosynthesis rates in ferns are generally lower than those of seed plants, but little is known about the limiting factors, which are crucial to understand the evolution of photosynthesis in land plants. To address this issue, a gas exchange/chlorophyll fluorescence analysis was performed in three fern species spanning high phylogenetic range within Polypodiopsida (Osmunda regalis, Blechnum gibbum and Nephrolepis exaltata) to determine their maximum net photosynthesis (AN ), stomatal (gs ) and mesophyll (gm ) conductances to CO2 , and the maximum velocity of carboxylation (Vc,max ). The in vitro Rubisco specificity factor (SC /O ) was also determined. All three species had values for SC /O similar to those typical of seed plants, but values of AN , gs , gm and Vc,max were within the lowest range of those observed in seed plants. In addition, gs was unresponsive to light and CO2 , as already described in other fern species. On the contrary, gm varied with changes CO2 . A quantitative photosynthesis limitation analysis suggested that early land plants (ferns) presented not only stomatal limitations-which were less adjustable to the environment-but also restricted gm and Vc,max , resulting in limited maximum photosynthesis rates.

The Tripartite Lichen Ricasolia virens: Involvement of Cyanobacteria and Bacteria in Its Morphogenesis.

Ricasolia virens is an epiphytic lichen-forming fungus mainly distributed in Western Europe and Macaronesia in well-structured forests with ecological continuity that lack eutrophication. It is considered to be threatened or extinct in many territories in Europe (IUCN). Despite its biological and ecological relevance, studies on this taxon are scarce. The thalli are tripartite, and the mycobiont has a simultaneous symbiotic relationship with cyanobacteria and green microalgae, which represent interesting models to analyse the strategies and adaptations resulting from the interactions of lichen symbionts. The present study was designed to contribute to a better understanding of this taxon, which has shown a clear decline over the last century. The symbionts were identified by molecular analysis. The phycobiont is Symbiochloris reticulata, and the cyanobionts (Nostoc) are embedded in internal cephalodia. Light, transmission electron and low-temperature scanning microscopy techniques were used to investigate the thallus anatomy, ultrastructure of microalgae and ontogeny of pycnidia and cephalodia. The thalli are very similar to its closest relative, Ricasolia quercizans. The cellular ultrastructure of S. reticulata by TEM is provided. Non-photosynthetic bacteria located outside the upper cortex are introduced through migratory channels into the subcortical zone by the splitting of fungal hyphae. Cephalodia were very abundant, but never as external photosymbiodemes.

Patterns of genetic variability and habitat occupancy in Crepis triasii (Asteraceae) at different spatial scales: insights on evolutionary processes leading to diversification in continental islands.

BACKGROUND AND AIMS: Archipelagos are unique systems for studying evolutionary processes promoting diversification and speciation. The islands of the Mediterranean basin are major areas of plant richness, including a high proportion of narrow endemics. Many endemic plants are currently found in rocky habitats, showing varying patterns of habitat occupancy at different spatial scales throughout their range. The aim of the present study was to understand the impact of varying patterns of population distribution on genetic diversity and structure to shed light on demographic and evolutionary processes leading to population diversification in Crepis triasii, an endemic plant from the eastern Balearic Islands. METHODS: Using allozyme and chloroplast markers, we related patterns of genetic structure and diversity to those of habitat occupancy at a regional (between islands and among populations within islands) and landscape (population size and connectivity) scale. KEY RESULTS: Genetic diversity was highly structured both at the regional and at the landscape level, and was positively correlated with population connectivity in the landscape. Populations located in small isolated mountains and coastal areas, with restricted patterns of regional occupancy, were genetically less diverse and much more differentiated. In addition, more isolated populations had stronger fine-scale genetic structure than well-connected ones. Changes in habitat availability and quality arising from marine transgressions during the Quaternary, as well as progressive fragmentation associated with the aridification of the climate since the last glaciation, are the most plausible factors leading to the observed patterns of genetic diversity and structure. CONCLUSIONS: Our results emphasize the importance of gene flow in preventing genetic erosion and maintaining the evolutionary potential of populations. They also agree with recent studies highlighting the importance of restricted gene flow and genetic drift as drivers of plant evolution in Mediterranean continental islands.

Trebouxia lynnae sp. nov. (Former Trebouxia sp. TR9): Biology and Biogeography of an Epitome Lichen Symbiotic Microalga

Two microalgal species, Trebouxia jamesii and Trebouxia sp. TR9, were detected as the main photobionts coexisting in the thalli of the lichen Ramalina farinacea. Trebouxia sp. TR9 emerged as a new taxon in lichen symbioses and was successfully isolated and propagated in in vitro culture and thoroughly investigated. Several years of research have confirmed the taxon Trebouxia sp. TR9 to be a model/reference organism for studying mycobiont−photobiont association patterns in lichen symbioses. Trebouxia sp. TR9 is the first symbiotic, lichen-forming microalga for which an exhaustive characterization of cellular ultrastructure, physiological traits, genetic and genomic diversity is available. The cellular ultrastructure was studied by light, electron and confocal microscopy; physiological traits were studied as responses to different abiotic stresses. The genetic diversity was previously analyzed at both the nuclear and organelle levels by using chloroplast, mitochondrial, and nuclear genome data, and a multiplicity of phylogenetic analyses were carried out to study its intraspecific diversity at a biogeographical level and its specificity association patterns with the mycobiont. Here, Trebouxia sp. TR9 is formally described by applying an integrative taxonomic approach and is presented to science as Trebouxia lynnae, in honor of Lynn Margulis, who was the primary modern proponent for the significance of symbiosis in evolution. The complete set of analyses that were carried out for its characterization is provided.

Biodiversity Patterns and Ecological Preferences of the Photobionts Associated With the Lichen-Forming Genus Parmelia.

The worldwide, ecologically relevant lichen-forming genus Parmelia currently includes 41 accepted species, of which the Parmelia sulcata group (PSULgp) and the Parmelia saxatilis group (PSAXgp) have received considerable attention over recent decades; however, phycobiont diversity is poorly known in Parmelia s. lat. Here, we studied the diversity of Trebouxia microalgae associated with 159 thalli collected from 30 locations, including nine Parmelia spp.: P. barrenoae, P. encryptata, P. ernstiae, P. mayi, P. omphalodes, P. saxatilis, P. serrana, P. submontana, and P. sulcata. The mycobionts were studied by carrying out phylogenetic analyses of the nrITS. Microalgae genetic diversity was examined by using both nrITS and LSU rDNA markers. To evaluate putative species boundaries, three DNA species delimitation analyses were performed on Trebouxia and Parmelia. All analyses clustered the mycobionts into two main groups: PSULgp and PSAXgp. Species delimitation identified 13 fungal and 15 algal species-level lineages. To identify patterns in specificity and selectivity, the diversity and abundance of the phycobionts were identified for each Parmelia species. High specificity of each Parmelia group for a given Trebouxia clade was observed; PSULgp associated only with clade I and PSAXgp with clade S. However, the degree of specificity is different within each group, since the PSAXgp mycobionts were less specific and associated with 12 Trebouxia spp., meanwhile those of PSULgp interacted only with three Trebouxia spp. Variation-partitioning analyses were conducted to detect the relative contributions of climate, geography, and symbiotic partner to phycobiont and mycobiont distribution patterns. Both analyses explained unexpectedly high portions of variability (99 and 98%) and revealed strong correlations between the fungal and algal diversity. Network analysis discriminated seven ecological clusters. Even though climatic conditions explained the largest proportion of the variation among these clusters, they seemed to show indifference relative to climatic parameters. However, the cluster formed by P. saxatilis A/P. saxatilis B/Trebouxia sp. 2/Trebouxia sp. S02/Trebouxia sp. 3A was identified to prefer cold-temperate as well as humid summer environments.

Symbiotic microalgal diversity within lichenicolous lichens and crustose hosts on Iberian Peninsula gypsum biocrusts.

This study analyses the interactions among crustose and lichenicolous lichens growing on gypsum biocrusts. The selected community was composed of Acarospora nodulosa, Acarospora placodiiformis, Diploschistes diacapsis, Rhizocarpon malenconianum and Diplotomma rivas-martinezii. These species represent an optimal system for investigating the strategies used to share phycobionts because Acarospora spp. are parasites of D. diacapsis during their first growth stages, while in mature stages, they can develop independently. R. malenconianum is an obligate lichenicolous lichen on D. diacapsis, and D. rivas-martinezii occurs physically close to D. diacapsis. Microalgal diversity was studied by Sanger sequencing and 454-pyrosequencing of the nrITS region, and the microalgae were characterized ultrastructurally. Mycobionts were studied by performing phylogenetic analyses. Mineralogical and macro- and micro-element patterns were analysed to evaluate their influence on the microalgal pool available in the substrate. The intrathalline coexistence of various microalgal lineages was confirmed in all mycobionts. D. diacapsis was confirmed as an algal donor, and the associated lichenicolous lichens acquired their phycobionts in two ways: maintenance of the hosts' microalgae and algal switching. Fe and Sr were the most abundant microelements in the substrates but no significant relationship was found with the microalgal diversity. The range of associated phycobionts are influenced by thallus morphology.

Dynamic evolution of mitochondrial genomes in Trebouxiophyceae, including the first completely assembled mtDNA from a lichen-symbiont microalga (Trebouxia sp. TR9).

Trebouxiophyceae (Chlorophyta) is a species-rich class of green algae with a remarkable morphological and ecological diversity. Currently, there are a few completely sequenced mitochondrial genomes (mtDNA) from diverse Trebouxiophyceae but none from lichen symbionts. Here, we report the mitochondrial genome sequence of Trebouxia sp. TR9 as the first complete mtDNA sequence available for a lichen-symbiont microalga. A comparative study of the mitochondrial genome of Trebouxia sp. TR9 with other chlorophytes showed important organizational changes, even between closely related taxa. The most remarkable change is the enlargement of the genome in certain Trebouxiophyceae, which is principally due to larger intergenic spacers and seems to be related to a high number of large tandem repeats. Another noticeable change is the presence of a relatively large number of group II introns interrupting a variety of tRNA genes in a single group of Trebouxiophyceae, which includes Trebouxiales and Prasiolales. In addition, a fairly well-resolved phylogeny of Trebouxiophyceae, along with other Chlorophyta lineages, was obtained based on a set of seven well-conserved mitochondrial genes.

Unexpected associated microalgal diversity in the lichen Ramalina farinacea is uncovered by pyrosequencing analyses.

The current literature reveals that the intrathalline coexistence of multiple microalgal taxa in lichens is more common than previously thought, and additional complexity is supported by the coexistence of bacteria and basidiomycete yeasts in lichen thalli. This replaces the old paradigm that lichen symbiosis occurs between a fungus and a single photobiont. The lichen Ramalina farinacea has proven to be a suitable model to study the multiplicity of microalgae in lichen thalli due to the constant coexistence of Trebouxia sp. TR9 and T. jamesii in long-distance populations. To date, studies involving phycobiont diversity within entire thalli are based on Sanger sequencing, but this method seems to underestimate the diversity. Here, we aim to analyze both the microalgal diversity and its community structure in a single thallus of the lichen R. farinacea by applying a 454 pyrosequencing approach coupled with a careful ad hoc-performed protocol for lichen sample processing prior to DNA extraction. To ascertain the reliability of the pyrosequencing results and the applied bioinformatics pipeline results, the thalli were divided into three sections (apical, middle and basal zones), and a mock community sample was used. The developed methodology allowed 40448 filtered algal reads to be obtained from a single lichen thallus, which encompassed 31 OTUs representative of different microalgae genera. In addition to corroborating the coexistence of the two Trebouxia sp. TR9 and T. jamesii taxa in the same thallus, this study showed a much higher microalgal diversity associated with the lichen. Along the thallus ramifications, we also detected variations in phycobiont distribution that might correlate with different microenvironmental conditions. These results highlight R. farinacea as a suitable material for studying microalgal diversity and further strengthen the concept of lichens as multispecies microecosystems. Future analyses will be relevant to ecophysiological and evolutionary studies to understand the roles of the multiple photobionts in lichen symbioses.

Positively selected amino acid replacements within the RuBisCO enzyme of oak trees are associated with ecological adaptations.

Phylogenetic analysis by maximum likelihood (PAML) has become the standard approach to study positive selection at the molecular level, but other methods may provide complementary ways to identify amino acid replacements associated with particular conditions. Here, we compare results of the decision tree (DT) model method with ones of PAML using the key photosynthetic enzyme RuBisCO as a model system to study molecular adaptation to particular ecological conditions in oaks (Quercus). We sequenced the chloroplast rbcL gene encoding RuBisCO large subunit in 158 Quercus species, covering about a third of the global genus diversity. It has been hypothesized that RuBisCO has evolved differentially depending on the environmental conditions and leaf traits governing internal gas diffusion patterns. Here, we show, using PAML, that amino acid replacements at the residue positions 95, 145, 251, 262 and 328 of the RuBisCO large subunit have been the subject of positive selection along particular Quercus lineages associated with the leaf traits and climate characteristics. In parallel, the DT model identified amino acid replacements at sites 95, 219, 262 and 328 being associated with the leaf traits and climate characteristics, exhibiting partial overlap with the results obtained using PAML.

Correction: Positively selected amino acid replacements within the RuBisCO enzyme of oak trees are associated with ecological adaptations.

[This corrects the article DOI: 10.1371/journal.pone.0183970.].