Salt tolerance in a neotropical orchid in the absence of local adaptation to salt spray.

PREMISE: Salt tolerance has rarely been investigated regionally in the neotropics and even more rarely in Orchidaceae, one of the largest families. Therefore, investigating local adaptation to salt spray and its physiological basis in Epidendrum fulgens, a neotropical orchid species, brings important new insights. METHODS: We assessed the degree of salt tolerance in E. fulgens by testing whether coastal populations are more tolerant to salt, which could point to local adaptation. To understand the physiological basis of such salt tolerance, we exposed wild-collected individuals to salt spray for 60 days, then measured leaf expansion, osmotic potential, sodium leaf concentration, chlorophyll leaf index, chlorophyll fluorescence, relative growth rate, and pressure-volume curves. RESULTS: There is no local adaptation to salt spray since both inland and coastal plants have a high tolerance to salt stress. This tolerance is explained by the ability to tolerate high concentrations of salt in leaf tissues, which is related to the high succulence displayed by this species. CONCLUSIONS: We showed an unprecedented salt tolerance level for an orchid species, highlighting our limited knowledge of that trait beyond the traditional studied groups. Another interesting finding is that salt tolerance in E. fulgens is linked to succulence, is widespread, and is not the result of local adaptation. We suggest that E. fulgens and its allied species could be an interesting group to explore the evolution of important traits related to tolerance to salt stress, like succulence.

Ecological transcriptomics reveals stress response pathways of a ground-herb species in a waterlogging gradient of Amazonian riparian forests.

Environmental stress is a fundamental facet of life and a significant driver of natural selection in the wild. Gene expression diversity may facilitate adaptation to environmental changes, without necessary genetic change, but its role in adaptive divergence remains largely understudied in Neotropical systems. In Amazonian riparian forests, species distribution is predominantly influenced by species' waterlogging tolerance. The flooding gradient delineates distinct wetland forest types, shaping habitats and species characteristics. Here we investigated the molecular basis of environmental stress response in a tropical ground-herb species (Ischnosiphon puberulus) to environmental variation in Amazonian riparian forests. We compared environmental variables and gene expression profiles from individuals collected in two forest types: Igapó and Terra firme in the Amazonian riparian forests. Predictable seasonal flooding poses a significant challenge in Igapó compared to Terra firme environments, with the former presenting higher water column height and longer flooding duration. Our findings suggest that contrasting environmental conditions related to flooding regimes are important drivers of population genetic differentiation and differential gene expression in I. puberulus. Enriched gene ontology terms highlight associations with environmental stresses, such as defence response, water transport, phosphorylation, root development, response to auxin, salicylic acid and oxidative stress. By uncovering key environmental stress response pathways conserved across populations, I. puberulus offers novel genetic insights into the molecular basis of plant reactions to environmental constraints found in flooded areas of this highly biodiverse neotropical ecosystem.

Understanding desiccation tolerance and sex-specific responses in Bryum argenteum: insights from phenological phases and physiological analyses.

BACKGROUND AND AIMS: Desiccation tolerance (DT) is crucial for survival in arid environments, where organisms develop strategies in reproduction, maintenance and defence to cope with water scarcity. Therefore, investigating the relationship between reproduction and DT is essential to understand the ecology and adaptive strategies of species. This study explores the connection between the development of male and female gametangia in the moss Bryum argenteum and the decrease in DT during the progression of phenological phases in gametangia and protonema. METHODS: Samples collected from a dry tropical forest in Brazil were cultivated, cloned and subjected to desiccation. Subsequently, the physiological parameters of shoots and protonemata were analysed. Shoot and protonema regeneration were monitored for 28 d after the physiological analyses. Both phases were subjected to control and desiccation treatments. KEY RESULTS: Significant effects of desiccation and sex on the physiological parameters and regeneration capacity of shoots and protonemata were found. Male shoots generally exhibited lower values of Fv/Fm (quantum efficiency of photosystem II) and ϕPSII (effective quantum yield of photosystem II), while females demonstrated higher values and better recovery after desiccation. Protonemata also showed variation in Fv/Fm over time and with sex, with no significant differences in ϕPSII between them. Desiccated male shoots had higher mortality rates and produced fewer new shoots. For females, the regeneration patterns varied between the desiccation-exposed groups and the control, with decreased shoot production, and some protonemata growing into filaments without forming shoots. CONCLUSION: These findings improve our understanding of the ecological responses of bryophytes to desiccation stress and provide insights into their adaptive strategies in challenging environments, such as the possible rarity of males in dioicous moss populations.

Metabolomic analysis reveals stress tolerance mechanisms in common bean (Phaseolus vulgaris L.) related to treatment with a biostimulant obtained from Corynebacterium glutamicum.

Microbial biostimulants have emerged as a sustainable alternative to increase the productivity and quality of important crops. Despite this, the effects of the treatment on plant metabolism are poorly understood. Thus, this study investigated the metabolic response of common bean (Phaseolus vulgaris) related to the treatment with a biostimulant obtained from the extract of Corynebacterium glutamicum that showed positive effects on the development, growth, and yield of crops previously. By untargeted metabolomic analysis using UHPLC-MS/MS, plants and seeds were subjected to treatment with the biostimulant. Under ideal growth conditions, the plants treated exhibited higher concentration levels of glutamic acid, nicotiflorin and glycosylated lipids derived from linolenic acid. The foliar application of the biostimulant under water stress conditions increased the chlorophyll content by 17% and induced the accumulation of flavonols, mainly quercetin derivatives. Also, germination seed assays exhibited longer radicle lengths for seeds treated compared to the untreated control even in the absence of light (13-18% increase, p-value <0.05). Metabolomic analysis of the seeds indicated changes in concentration levels of amino acids (tryptophan, phenylalanine, tyrosine, glutamine, and arginine) and their derivatives. The results point out the enhancement of abiotic stress tolerance and the metabolic processes triggered in this crop associated with the treatment with the biostimulant, giving the first insights into stress tolerance mechanisms in P. vulgaris.

The spatial arrangement of sexes is related to reproductive allocation in mosses: a comparative study of reproductive allocation in three different monoicous sexual systems.

BACKGROUND AND AIMS: We examined the relationship between reproductive allocation and vegetative growth in three monoicous sexual systems of bryophytes. The sexual systems show a gradient of increasing distance between the sexes, from gonioautoicous to cladautoicous to rhizautoicous. Here, we investigated the following two hypotheses: (1) reproductive allocation differs between sexes and sexual systems, and male reproductive allocation increases with increasing distance between male and female gametangia; and (2) reproductive allocation is negatively related to vegetative growth. METHODS: We sampled the three sexual systems, represented by three moss species of the genus Fissidens in the Atlantic Forest of Southeastern Brazil. Ramets were washed in the laboratory; the reproductive structures were detached from the vegetative ramets and sorted regarding sex and individual, dried at 70 °C for 72 h, and weighed in an ultramicrobalance. We calculated the mean reproductive and vegetative mass and reproductive allocation and used generalized linear models to test our predictions. KEY RESULTS: Reproductive allocation differed between species and sexes. It was higher in the rhizautoicous than in the cladautoicous and gonioautoicous species. Mean reproductive allocation was greater in males than in females of the rhizautoicous species, greater in females than males of the cladautoicous species, and did not differ between the sexes in the gonioautoicous species. Estimates of reproductive and vegetative mass were positively related in females of the rhizautoicous species. Vegetative mass was not related to reproductive allocation in the gonioautoicous species, but negatively related to reproductive allocation in the male and female branchlets of the cladautoicous species and in the female ramets of the rhizautoicous species. CONCLUSIONS: The reproductive allocation patterns differ between the rhizautoicous species and the 'truly' monoicous species, with shorter intersexual distances, which implies that our hypotheses were supported only in part. We suggest that the hypotheses should be reformulated and tested further by comparing 'truly' monoicous species with dioicous species and by including other genera.

Life-history traits and density dependence in metapopulations of a tropical moss: a monoicous species that is almost dioicous.

Life-history traits, such as reproductive allocation, sexual expression, sex ratio, and reproductive success, are central aspects of a species' ecology and evolution. For example, bias in male and female sex expression may play a large role in determining the viability of populations in the face of environmental pressures, such as population fragmentation, climate change and habitat occupancy. Thus, in this study, we investigated reproductive traits in 10 meta-populations of Fissidens flaccidus Mitt. From each meta-population, 30 patches were randomly selected, and 1 cm2 samples were collected form each patch. A total of 20,173 ramets were analyzed and classified into male, non-sporophytic female, sporophytic female, and non-sex expressing. In addition, population density in each patch was quantified. Our results showed that relative reproductive allocation in perigonia and sporophytes is greater than perichaetia. Trade-off between sexual relative reproductive allocation and asexual gemma production was observed, suggesting an important role of female ramets in asexual reproduction. The number of male ramets does not influence the reproductive success observed in each patch, and ramet density may induce male sex expression. Thus, we concluded that reproductive allocation in male function is efficient, since fewer male ramets can assure a considerable reproductive success. Furthermore, our results suggest that there may be a habitat preference between the sexes, since male ramets are found in patches with high density and mostly below female ramets, suggesting an avoidance of direct sunlight by male ramets.

Phylogenomics and plastome evolution of a Brazilian mycoheterotrophic orchid, Pogoniopsis schenckii.

PREMISE: Pogoniopsis likely represents an independent photosynthesis loss in orchids. We use phylogenomic data to better identify the phylogenetic placement of this fully mycoheterotrophic taxon, and investigate its molecular evolution. METHODS: We performed likelihood analysis of plastid and mitochondrial phylogenomic data to localize the position of Pogoniopsis schenckii in orchid phylogeny, and investigated the evolution of its plastid genome. RESULTS: All analyses place Pogoniopsis in subfamily Epidendroideae, with strongest support from mitochondrial data, which also place it near tribe Sobralieae with moderately strong support. Extreme rate elevation in Pogoniopsis plastid genes broadly depresses branch support; in contrast, mitochondrial genes are only mildly rate elevated and display very modest and localized reductions in bootstrap support. Despite considerable genome reduction, including loss of photosynthesis genes and multiple translation apparatus genes, gene order in Pogoniopsis plastomes is identical to related autotrophs, apart from moderately shifted inverted repeat (IR) boundaries. All cis-spliced introns have been lost in retained genes. Two plastid genes (accD, rpl2) show significant strengthening of purifying selection. A retained plastid tRNA gene (trnE-UUC) of Pogoniopsis lacks an anticodon; we predict that it no longer functions in translation but retains a secondary role in heme biosynthesis. CONCLUSIONS: Slowly evolving mitochondrial genes clarify the placement of Pogoniopsis in orchid phylogeny, a strong contrast with analysis of rate-elevated plastome data. We documented the effects of the novel loss of photosynthesis: for example, despite massive gene loss, its plastome is fully colinear with other orchids, and it displays only moderate shifts in selective pressure in retained genes.

Sex-specific differences in reproductive life-history traits of the moss Weissia jamaicensis.

PREMISE: We investigated sex-specific differences in the life-history traits of a metapopulation of the dioicous moss Weissia jamaicensis. Field observations revealed high rates of fertilization, which is uncommon for most dioicous bryophytes. We raised four hypotheses associated with the way the reproductive traits are related to the fertilization rate in this metapopulation. METHODS: We sampled 10 patches of the metapopulation and quantified sexual expression, sex ratio, reproductive success, and reproductive allocation. The ramets were classified as male, non-sporophytic female, sporophytic female, or non-sex-expressing. Thirty ramets from each of the categories expressing sex were placed for regeneration to test the effect of reproductive allocation on this trait. RESULTS: We found greater expression of the female function in all patches, implying a female bias in the metapopulation. The number of male ramets was variable in each patch and did not affect reproductive success. At the prezygotic level, the allocation of resources to the male function was higher. However, the large allocation of resources to sporophyte development in sporophytic females, which exceeded allocations at prezygotic levels, was related to the higher mortality rate of these ramets, suggesting reproductive cost. CONCLUSIONS: The prezygotic ramets that allocated the greatest amount of resources to reproduction expressed sex less frequently, biasing the sex ratio toward the sex that allocated the least amount of resources to reproduction. Overall, the ramets that allocated the greatest amount of resources to reproduction had the lowest regeneration rate, suggesting reproductive cost.

Ecological and phylogenetic constraints determine the stage of anthetic ovule development in orchids.

PREMISE: Unlike most flowering plants, orchid flowers have under-developed ovules that complete development only after pollination. Classical studies reported variation in the stage in which ovule development is arrested, but the extent of this variation and its evolutionary and ecological significance are unclear. METHODS: Here, we used light microscopy to observe ovule development at anthesis for 39 species not previously studied and surveyed the literature gaining information on 94 orchid species. Tropical and temperate members of all five orchid subfamilies as well as species with contrasting pollination strategies (rewarding versus deceptive) and life forms (epiphytic versus terrestrial) were represented. We analyzed the data using statistical comparisons and a phylogenetic generalized least square (PGLS) analysis. RESULTS: Apostasioideae, the sister to the rest of the orchids, have mature ovules similar to other Asparagales, while under-differentiated ovules are present in the other subfamilies. Ovule developmental stages showed high variation even among closely related groups. Ovules were more developed in terrestrial than in epiphytic, in temperate than in tropical, and in rewarding than in deceptive pollination orchid species. This latter comparison was also significant in the PGLS analysis. CONCLUSIONS: These results suggest that ovule developmental stage in orchids can be shaped by ecological factors, such as seasonality and pollination strategy, and can be selected for optimizing female reproductive investment.

Reproductive development and genetic structure of the mycoheterotrophic orchid Pogoniopsis schenckii Cogn.

BACKGROUND: Pogoniopsis schenckii Cogn. is a mycoheterotrophic orchid that can be used as a model to understand the influence of mycoheterotrophy at different stages of the reproductive cycle. We aimed to verify the presence of endophytic and epiphytic fungi at each stage of the reproductive process and investigated how the breeding system may relate to genetic structure and diversity of populations. In this study we performed anatomical and ultrastructural analyses of the reproductive organs, field tests to confirm the breeding system, and molecular analysis to assess genetic diversity and structure of populations. RESULTS: During the development of the pollen grain, embryo sac and embryogenesis, no fungal infestation was observed. The presence of endophytic fungal hyphae was observed just within floral stems and indehiscent fruit. Beyond assuring the presence of fungus that promote seed germination, specific fungi hyphae in the fruit may affect other process, such as fruit ripening. As other mycoheterotrophic orchids, P. schenckii is autogamous, which may explain the low genetic diversity and high genetic structure in populations. CONCLUSIONS: We discuss an interesting interaction: fungal hyphae in the indehiscent fruit. These fungal hyphae seem to play different roles inside fruit tissues, such as acting in the fruit maturation process and increasing the proximity between fungi and plant seeds even before dispersion occurs. As other mycoheterotrophic orchids, P. schenckii is autogamous, which may explain the low genetic diversity and high genetic structure in populations. Altogether, our findings provide important novel information about the mechanisms shaping ecology and evolution of fragmented populations of mycoheterotrophic plant.

Plastid phylogenomics resolves ambiguous relationships within the orchid family and provides a solid timeframe for biogeography and macroevolution.

Recent phylogenomic analyses based on the maternally inherited plastid organelle have enlightened evolutionary relationships between the subfamilies of Orchidaceae and most of the tribes. However, uncertainty remains within several subtribes and genera for which phylogenetic relationships have not ever been tested in a phylogenomic context. To address these knowledge-gaps, we here provide the most extensively sampled analysis of the orchid family to date, based on 78 plastid coding genes representing 264 species, 117 genera, 18 tribes and 28 subtribes. Divergence times are also provided as inferred from strict and relaxed molecular clocks and birth-death tree models. Our taxon sampling includes 51 newly sequenced plastid genomes produced by a genome skimming approach. We focus our sampling efforts on previously unplaced clades within tribes Cymbidieae and Epidendreae. Our results confirmed phylogenetic relationships in Orchidaceae as recovered in previous studies, most of which were recovered with maximum support (209 of the 262 tree branches). We provide for the first time a clear phylogenetic placement for Codonorchideae within subfamily Orchidoideae, and Podochilieae and Collabieae within subfamily Epidendroideae. We also identify relationships that have been persistently problematic across multiple studies, regardless of the different details of sampling and genomic datasets used for phylogenetic reconstructions. Our study provides an expanded, robust temporal phylogenomic framework of the Orchidaceae that paves the way for biogeographical and macroevolutionary studies.

Differential gene expression reveals mechanisms related to habitat divergence between hybridizing orchids from the Neotropical coastal plains.

BACKGROUND: Closely related hybridizing species are ideal systems for identifying genomic regions underlying adaptive divergence. Although gene expression plays a central role in determining ecologically-based phenotypic differences, few studies have inferred the role of gene expression for adaptive divergence in Neotropical systems. In this study, we conduct genome-wide expression analysis alongside soil elemental analysis in sympatric and allopatric populations of Epidendrum fulgens and E. puniceoluteum (Orchidaceae), which occur in contrasting adjacent habitats in the Neotropical coastal plains. RESULTS: These species were highly differentiated by their gene expression profiles, as determined by 18-21% of transcripts. Gene ontology (GO) terms associated with reproductive processes were enriched according to comparisons between species in both allopatric and sympatric populations. Species showed differential expression in genes linked to salt and waterlogging tolerance according to comparisons between species in sympatry, and biological processes related to environmental stimulus appeared as representative among those transcripts associated with edaphic characteristics in each sympatric zone. Hybrids, in their turn, were well differentiated from E. fulgens, but exhibited a similar gene expression profile to flooding-tolerant E. puniceolutem. When compared with parental species, hybrids showed no transcripts with additive pattern of expression and increased expression for almost all transgressive transcripts. CONCLUSIONS: This study sheds light on general mechanisms promoting ecological differentiation and assortative mating, and suggests candidate genes, such as those encoding catalase and calcium-dependent protein kinase, underling adaptation to harsh edaphic conditions in the Neotropical coastal plains. Moreover, it demonstrates that differential gene expression plays a central role in determining ecologically-based phenotypic differences among co-occurring species and their hybrids.

Correction: From micro- to macroevolution: insights from a Neotropical bromeliad with high population genetic structure adapted to rock outcrops.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

From micro- to macroevolution: insights from a Neotropical bromeliad with high population genetic structure adapted to rock outcrops.

Geographic isolation and reduced population sizes can lead to local extinction, low efficacy of selection and decreased speciation. However, population differentiation is an essential step of biological diversification. In allopatric speciation, geographically isolated populations differentiate and persist until the evolution of reproductive isolation and ecological divergence completes the speciation process. Pitcairnia flammea allows us to study the evolutionary consequences of habitat fragmentation on naturally disjoint rock-outcrop species from the Brazilian Atlantic Rainforest (BAF). Our main results showed low-to-moderate genetic diversity within populations, and deep population structuring caused by limited gene flow, low connectivity, genetic drift and inbreeding of long-term isolation and persistence of rock-outcrop populations throughout Quaternary climatic oscillations. Bayesian phylogenetic and model-based clustering analyses found no clear northern and southern phylogeographic structure commonly reported for many BAF organisms. Although we found two main lineages diverging by ~2 Mya during the early Pleistocene, species' delimitation analysis assigned most of the populations as independent evolving entities, suggesting an important role of disjoint rock outcrops in promoting high endemism in this rich biome. Lastly, we detected limited gene flow in sympatric populations although some hybridization and introgression were observed, suggesting a continuous speciation process in this species complex. Our data not only inform us about the extensive differentiation and limited gene flow found among Pitcairnia flammea species complex, but they also contain information about the mechanisms that shape the genetic architecture of small and fragmented populations of isolated rock outcrop of recently radiated plants.

First Record of Ategmic Ovules in Orchidaceae Offers New Insights Into Mycoheterotrophic Plants.

The number of integuments found in angiosperm ovules is variable. In orchids, most species show bitegmic ovules, except for some mycoheterotrophic species that show ovules with only one integument. Analysis of ovules and the development of the seed coat provide important information regarding functional aspects such as dispersal and seed germination. This study aimed to analyze the origin and development of the seed coat of the mycoheterotrophic orchid Pogoniopsis schenckii and to compare this development with that of other photosynthetic species of the family. Flowers and fruits at different stages of development were collected, and the usual methodology for performing anatomical studies, scanning microscopy, and transmission microscopy following established protocols. P. schenckii have ategmic ovules, while the other species are bitegmic. No evidence of integument formation at any stage of development was found through anatomical studies. The reduction of integuments found in the ovules could facilitate fertilization in this species. The seeds of P. schenckii, Vanilla planifolia, and V. palmarum have hard seed coats, while the other species have seed coats formed by the testa alone, making them thin and transparent. P. schenckii, in contrast to the other species analyzed, has a seed coat that originates from the nucellar epidermis, while in other species, the seed coat originates from the outer integument.

Hybridization and geographic distribution shapes the spatial genetic structure of two co-occurring orchid species.

Multiple ecological and life-history traits shape the fine-scale spatial genetic structure (FSGS) of a given population. The occurrence in core versus peripheral populations, levels of outcrossing, pollen and seed dispersal, and hybridization are important biological properties that influence the kinship of individuals within populations. We examined spatial genetic structure within 15 populations of Epidendrum fulgens and E. puniceoluteum distributed along a linear gradient of Brazilian coastal vegetation, including both allopatric and sympatric populations where the two orchid species hybridize. We analyzed 581 mapped specimens using nine simple sequence repeat loci, aiming to investigate how geographic distribution and hybridization shape within-population FSGS. A significant increase in FSGS was found towards peripheral populations, compared to core populations. Analysis of short-distance and long-distance components of FSGS identified biparental inbreeding and higher levels of FSGS at peripheral populations, when compared to core populations. In contrast, the relatively high density of reproductive adults in core populations potentially leads to highly overlapping seed and pollen movement, decreasing FSGS. Hybridization was an important factor shaping within-population spatial genetic structure at sympatric sites, decreasing the FSGS observed in parental species. Our results indicate that different ecological forces act in concert to create a gradient of FSGS along species distribution ranges, shaped by extensive levels of intraspecific and interspecific gene exchange.

Unidirectional transitions in nectar gain and loss suggest food deception is a stable evolutionary strategy in Epidendrum (Orchidaceae): insights from anatomical and molecular evidence.

BACKGROUND: Nectar gain and loss are important flower transitions observed in angiosperms, and are particularly common in orchids. To understand such transitions, the availability of detailed anatomical data and species-level phylogenies are crucial. We investigated the evolution of food deception in Epidendrum, one of the largest orchid genera, using genus phylogeny to map transitions between nectar gain and loss among different clades. Associations between anatomical and histochemical changes and nectar gain and loss were examined using fresh material available from 27 species. The evolution of nectar presence/absence in Epidendrum species was investigated in a phylogenetic framework of 47 species, using one nuclear and five plastid DNA regions available from GenBank and sequenced in this study. RESULTS: The presence or absence of nectar was strongly associated with changes in the inner epidermal tissues of nectaries. Nectar-secreting species have unornamented epidermal tissue, in contrast to the unicellular trichomes found on the epidermis of food deceptive species. Bayesian tests confirmed that transitions occurred preferentially from nectar presence to nectar absence across the Epidendrum phylogeny. In addition, independent nectar loss events were found across the phylogeny, suggesting a lack of constraint for these transitions. CONCLUSIONS: Ornamented nectaries may play an important role in the deceptive pollination strategy by secreting volatile organic compounds and providing tactile stimuli to pollinators. The recurrent and apparently irreversible pattern of nectar loss in Epidendrum suggests that food deception may constitute an alternative evolutionarily stable strategy, as observed in other orchid groups.