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.

Geoclimatic drivers of diversification in the largest arid and semi-arid environment of the Neotropics: Perspectives from phylogeography.

The South American Dry Diagonal, also called the Diagonal of Open Formations, is a large region of seasonally dry vegetation extending from northeastern Brazil to northern Argentina, comprising the Caatinga, Cerrado, and Chaco subregions. A growing body of phylogeography literature has determined that a complex history of climatic changes coupled with more ancient geological events has produced a diverse and endemic-rich Dry Diagonal biota. However, the exact drivers are still under investigation, and their relative strengths and effects are controversial. Pleistocene climatic fluctuations structured lineages via vegetation shifts, refugium formation, and corridors between the Amazon and Atlantic forests. In some taxa, older geological events, such as the reconfiguration of the São Francisco River, uplift of the Central Brazilian Plateau, or the Miocene inundation of the Chaco by marine incursions, were more important. Here, we review the Dry Diagonal phylogeography literature, discussing each hypothesized driver of diversification and assessing degree of support. Few studies statistically test these hypotheses, with most support drawn from associating encountered phylogeographic patterns such as population structure with the timing of ancient geoclimatic events. Across statistical studies, most hypotheses are well supported, with the exception of the Pleistocene Arc Hypothesis. However, taxonomic and regional biases persist, such as a proportional overabundance of herpetofauna studies, and the under-representation of Chaco studies. Overall, both Pleistocene climate change and Neogene geological events shaped the evolution of the Dry Diagonal biota, though the precise effects are regionally and taxonomically varied. We encourage further use of model-based analyses to test evolutionary scenarios, as well as interdisciplinary collaborations to progress the field beyond its current focus on the traditional set of geoclimatic hypotheses.

Tree species occurring in Amazonian wetland forests consistently show broader range sizes and niche breadths than trees in upland forests.

Generally, species with broad niches also show large range sizes. We investigated the relationship between hydrological niche breadth and geographic range size for Amazonian tree species seeking to understand the role of habitat specialization to Amazonian wetlands and upland forests on the current distribution of tree species. We obtained 571,092 valid occurrence points from GBIF and SpeciesLink to estimate the range size and the niche breadth of 76% of all known Amazonian tree species (5150 tree species). Hydrological niche breadth was measured on different unidimensional axes defined by (1) total annual precipitation; (2) precipitation seasonality; (3) actual evapotranspiration; and (4) water table depth. Geographic range sizes were estimated using alpha-hull adjustments. General linear models were used to relate niche breadth to range size while contrasting tree species occurring and not occurring in wetlands. The hydrological niche breadth of Amazonian tree species varied mostly along the water table depth axis. The average range size for an Amazonian tree species was 751,000 km2 (median of 154,000 km2 and standard deviation of 1,550,000 km2). Niche breadth-range size relationships for Amazonian tree species were positive for all models, and the explanatory power of the models improved when including whether a species occurred in wetlands or in terrestrial uplands. Wetland species had steeper positive slopes for the niche breadth-range size relationship, and consistently larger range sizes for a given niche breadth. Amazonian tree species varied strongly in hydrological niche breadth and range size, but most species had narrow niche breadths and range sizes. Our results suggest that the South American riverscape may have been acting as a corridor for species dispersal in the Neotropical lowlands.

Comparative phylogeography reveals the demographic patterns of neotropical ancient mountain species.

Mountains are renowned for their bountiful biodiversity. Explanations on the origin of such abundant life are usually regarded to their orogenic history. However, ancient mountain systems with geological stability also exhibit astounding levels of number of species and endemism, as illustrated by the Brazilian Quartzitic Mountains (BQM) in Eastern South America. Thus, cycles of climatic changes over the last couple million years are usually assumed to play an important role in the origin of mountainous biota. These climatic oscillations potentially isolated and reconnected adjacent populations, a phenomenon known as flickering connectivity, accelerating speciation events due to range fragmentation, dispersion, secondary contact, and hybridization. To evaluate the role of the climatic fluctuations on the diversification of the BQM biota, we estimated the ancient demography of distinct endemic species of animals and plants using hierarchical approximate Bayesian computation analysis and Ecological Niche Modelling. Additionally, we evaluated if climatic oscillations have driven a genetic spatial congruence in the genetic structure of codistributed species from the Espinhaço Range, one of the main BQM areas. Our results show that the majority of plant lineages underwent a synchronous expansion over the Last Glacial Maximum (LGM, c. 21 thousand years ago), although we could not obtain a clear demographic pattern for the animal lineages. We also obtained a signal of a congruent phylogeographic break between lineages endemic to the Espinhaço Range, suggesting how ancient climatic oscillations might have driven the evolutionary history of the Espinhaço's biota.

Multilocus Data Analysis Reveal the Diversity of Cryptic Species in the Tillandsia ionantha (Bromeliaceae: Tillansiodeae) Complex.

Independent evolutionary lineages or species that lack phenotypic variation as an operative criterion for their delimitation are known as cryptic species. However, these have been delimited using other data sources and analysis. The aims of this study are: (1) to evaluate the divergence of the populations of the T. ionantha complex; and (2) to delimit the species using multilocus data, phylogenetic analysis and the coalescent model. Phylogenetic analyses, genetic diversity and population structure, and isolation by distance analysis were performed. A multispecies coalescent analysis to delimit the species was conducted. Phylogenetic analysis showed that T. ionantha is polyphyletic composed of eight evolutionary lineages. Haplotype distribution and genetic differentiation analysis detected strong population structure and high values of genetic differentiation among populations. The positive correlation between genetic differences with geographic distance indicate that the populations are evolving under the model of isolation by distance. The coalescent multispecies analysis performed with starBEAST supports the recognition of eight lineages as different species. Only three out of the eight species have morphological characters good enough to recognize them as different species, while five of them are cryptic species. Tillandsia scaposa and T. vanhyningii are corroborated as independent lineages, and T. ionantha var. stricta changed status to the species level.

Speciation with gene flow between two Neotropical sympatric species (Pitcairnia spp.: Bromeliaceae).

The study of mechanisms that generate new species is considered fundamental for broad areas of ecology and evolution. Speciation is a continuous process in which reproductive isolation is established, and it is of fundamental importance to understand the origins of the adaptations that contribute to this process. Hybrid zones are considered natural laboratories for the study of speciation and represent ideal systems for such studies. Here, we investigated genomic differentiation between hybridizing Neotropical species Pitcairnia staminea (G. Lodd.) and P. albiflos (Herb.). Using thousands of SNPs genotyped through RAD-seq, we estimate effective population sizes, interspecific gene flow, as well as time of divergence between these two sister species and identify candidate genomic regions for positive selection that may be related to reproductive isolation. We selected different scenarios of speciation and tested them by using approximate Bayesian computation (ABC); we found evidence of divergence with gradual reduction in gene flow between these species over time, compatible with the hypothesis of speciation with gene flow between these Pitcairnia species. The parameter estimates obtained through ABC suggested that the effective population size of P. albiflos was around three times larger than that of P. staminea. Our divergence date estimates showed that these two species diverged during the Pliocene (4.7 Mya; CI = 1.3-8.5 Mya), which has likely allowed this species to accumulate genome-wide differences. We also detected a total of 17 of 4165 loci which showed signatures of selection with high genetic differentiation (F ST > 0.85), 12 of these loci were annotated in de novo assembled transcriptomes of both species, and 4 candidate genes were identified to be putatively involved in reproductive isolation. These four candidate genes were previously associated with the function of pollen development, pollen tube germination and orientation, abiotic stress, and flower scent in plants, suggesting an interplay between pre- and postpollination barriers in the evolution of reproductive isolation between such species.

Deforestation is the turning point for the spreading of a weedy epiphyte: an IBM approach.

The rapid spread of many weeds into intensely disturbed landscapes is boosted by clonal growth and self-fertilization strategies, which conversely increases the genetic structure of populations. Here, we use empirical and modeling approaches to evaluate the spreading dynamics of Tillandsia recurvata (L.) L. populations, a common epiphytic weed with self-reproduction and clonal growth widespread in dry forests and deforested landscapes in the American continent. We introduce the TRec model, an individual-based approach to simulate the spreading of T. recurvata over time and across landscapes subjected to abrupt changes in tree density with the parameters adjusted according to the empirical genetic data based on microsatellites genotypes. Simulations with this model showed that the strong spatial genetic structure observed from empirical data in T. recurvata can be explained by a rapid increase in abundance and gene flow followed by stabilization after ca. 25 years. TRec model's results also indicate that deforestation is a turning point for the rapid increase in both individual abundance and gene flow among T. recurvata subpopulations occurring in formerly dense forests. Active reforestation can, in turn, reverse such a scenario, although with a milder intensity. The genetic-based study suggests that anthropogenic changes in landscapes may strongly affect the population dynamics of species with 'weedy' traits.

Global Commitments to Conserving and Monitoring Genetic Diversity Are Now Necessary and Feasible.

Global conservation policy and action have largely neglected protecting and monitoring genetic diversity-one of the three main pillars of biodiversity. Genetic diversity (diversity within species) underlies species' adaptation and survival, ecosystem resilience, and societal innovation. The low priority given to genetic diversity has largely been due to knowledge gaps in key areas, including the importance of genetic diversity and the trends in genetic diversity change; the perceived high expense and low availability and the scattered nature of genetic data; and complicated concepts and information that are inaccessible to policymakers. However, numerous recent advances in knowledge, technology, databases, practice, and capacity have now set the stage for better integration of genetic diversity in policy instruments and conservation efforts. We review these developments and explore how they can support improved consideration of genetic diversity in global conservation policy commitments and enable countries to monitor, report on, and take action to maintain or restore genetic diversity.

Evidence of local adaptation despite strong drift in a Neotropical patchily distributed bromeliad.

Both genetic drift and divergent selection are predicted to be drivers of population differentiation across patchy habitats, but the extent to which these forces act on natural populations to shape traits is strongly affected by species' ecological features. In this study, we infer the genomic structure of Pitcairnia lanuginosa, a widespread herbaceous perennial plant with a patchy distribution. We sampled populations in the Brazilian Cerrado and the Central Andean Yungas and discovered and genotyped SNP markers using double-digest restriction-site associated DNA sequencing. In addition, we analyzed ecophysiological traits obtained from a common garden experiment and compared patterns of phenotypic and genetic divergence (PST-FST comparisons) in a subset of populations from the Cerrado. Our results from molecular analyses pointed to extremely low genetic diversity and a remarkable population differentiation, supporting a major role of genetic drift. Approximately 0.3% of genotyped SNPs were flagged as differentiation outliers by at least two distinct methods, and Bayesian generalized linear mixed models revealed a signature of isolation by environment in addition to isolation by distance for high-differentiation outlier SNPs among the Cerrado populations. PST-FST comparisons suggested divergent selection on two ecophysiological traits linked to drought tolerance. We showed that these traits vary among populations, although without any particular macro-spatial pattern, suggesting local adaptation to differences in micro-habitats. Our study shows that selection might be a relevant force, particularly for traits involved in drought stress, even for populations experiencing strong drift, which improves our knowledge on eco-evolutionary processes acting on non-continuously distributed species.

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.

Phenotypic expression of floral traits in hybrid zones provides insights into their genetic architecture.

Information on the genetic architecture of phenotypic traits is helpful for constructing and testing models of the ecoevolutionary dynamics of natural populations. For plant groups with long life cycles there is a lack of line cross experiments that can unravel the genetic architecture of loci underlying quantitative traits. To fill this gap, we propose the use of variation for phenotypic traits expressed in natural hybrid zones as an alternative approach. We used data from orchid hybrid zones and compared expected and observed patterns of phenotypic trait expression in different early-generation hybrid classes identified by molecular genetic markers. We found evidence of additivity, dominance, and epistatic interactions for different phenotypic traits. We discuss the potential of this approach along with its limitations and suggest that it may represent a realistic way to gain an initial insight into the heritability and genomic architecture of traits in organismal groups with complex life history, such as orchids and many others.

Ecological niche modeling and a lack of phylogeographic structure in Vriesea incurvata suggest historically stable areas in the southern Atlantic Forest.

PREMISE: Despite the efforts to understand the processes that shape neotropical biodiversity, the complexity of certain biomes, such as the Atlantic Forest (AF), prevents the generalization of patterns. Initially, ecological niche modeling (ENM), with phylogeographic studies, identified past stable areas in the central and northern portions of the AF, while the southern portion was thought to be highly fragmented. Here, we examined the phylogeography, historical patterns, genetic diversity, and population structure of Vriesea incurvata, an endemic species of the southern portion of the AF. METHODS: We evaluated 149 individuals using two plastid DNA regions (cpDNA) and 13 nuclear microsatellite markers (nuSSRs) to access the historical patterns, genetic diversity, and structure of V. incurvata populations. We also conducted historical demography and ENM analyses. RESULTS: We found moderate to high genetic diversity and low population structure for both genomes. The cpDNA network revealed high haplotype sharing. The ENM suggested no drastic changes in suitable areas for V. incurvata occurrence, corroborating the finding of no phylogeographic structure. CONCLUSIONS: Contrary to some studies, our results indicate that the southern AF was a historically stable climate region for V. incurvata occupation after southward colonization by the species. Past climatic changes probably did not cause structuring among its populations.

Dispersal and local persistence shape the genetic structure of a widespread Neotropical plant species with a patchy distribution.

BACKGROUND AND AIMS: Isolated populations constitute an ideal laboratory to study the consequences of intraspecific divergence, because intrinsic incompatibilities are more likely to accumulate under reduced gene flow. Here, we use a widespread bromeliad with a patchy distribution, Pitcairnia lanuginosa, as a model to infer processes driving Neotropical diversification and, thus, to improve our understanding of the origin and evolutionary dynamics of biodiversity in this highly speciose region. METHODS: We assessed the timing of lineage divergence, genetic structural patterns and historical demography of P. lanuginosa, based on microsatellites, and plastid and nuclear sequence data sets using coalescent analyses and an Approximate Bayesian Computation framework. Additionally, we used species distribution models (SDMs) to independently estimate potential changes in habitat suitability. KEY RESULTS: Despite morphological uniformity, plastid and nuclear DNA data revealed two distinct P. lanuginosa lineages that probably diverged through dispersal from the Cerrado to the Central Andean Yungas, following the final uplift of the Andes, and passed through long-term isolation with no evidence of migration. Microsatellite data indicate low genetic diversity and high levels of inbreeding within populations, and restricted gene flow among populations, which are likely to be a consequence of bottlenecks (or founder events), and high selfing rates promoting population persistence in isolation. SDMs showed a slight expansion of the suitable range for P. lanuginosa lineages during the Last Glacial Maximum, although molecular data revealed a signature of older divergence. Pleistocene climatic oscillations thus seem to have played only a minor role in the diversification of P. lanuginosa, which probably persisted through adverse conditions in riparian forests. CONCLUSIONS: Our results imply drift as a major force shaping the evolution of P. lanuginosa, and suggest that dispersal events have a prominent role in connecting Neotropical open and forest biomes.

Insights into the evolutionary dynamics of Neotropical biomes from the phylogeography and paleodistribution modeling of Bromelia balansae.

PREMISE OF THE STUDY: Historical abiotic and biotic factors have strongly affected species diversification and speciation. Although pre-Pleistocene events have been linked to the divergence of several Neotropical organisms, studies have highlighted a more prominent role of Pleistocene climatic oscillations in shaping current patterns of genetic variation of plants. METHODS: We performed phylogeographic analyses based on plastidial markers and modeled the current distribution and paleodistribution of Bromelia balansae (Bromeliaceae), an herbaceous species with a wide geographical distribution in South America, to infer the processes underlying its evolutionary history. KEY RESULTS: Combined molecular and paleodistributional modeling analyses indicated retraction during the Last Glacial Maximum followed by interglacial expansion. Populations occurring in the semideciduous Atlantic Forest and the Cerrado formed two distinct genetic clusters, which have been historically or ecologically isolated since late Pliocene to early Pleistocene. Populations located in the transition zone had higher levels of genetic diversity, as expected by the long-term climatic stability in the region detected in our ecological niche models. CONCLUSIONS: Our study adds important information on how herbaceous species have been affected by past climate in Central and Southeast Brazil, helping to disentangle the complex processes that have triggered the evolution of Neotropical biota.

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.

Natural hybridization and genetic and morphological variation between two epiphytic bromeliads.

Reproductive isolation is of fundamental importance for maintaining species boundaries in sympatry. Here, we examine the genetic and morphological differences between two closely related bromeliad species: Vriesea simplex and Vriesea scalaris. Furthermore, we examined the occurrence of natural hybridization and discuss the action of reproductive isolation barriers. Nuclear genomic admixture suggests hybridization in sympatric populations, although interspecific gene flow is low among species in all sympatric zones (Nem < 0.5). Thus, morphological and genetic divergence (10.99 %) between species can be maintained despite ongoing natural hybridization. Cross-evaluation of our genetic and morphological data suggests that species integrity is maintained by the simultaneous action of multiple barriers, such as divergent reproductive systems among species, differences in floral traits and low hybrid seed viability.

Incomplete lineage sorting and hybridization in the evolutionary history of closely related, endemic yellow-flowered Aechmea species of subgenus Ortgiesia (Bromeliaceae).

PREMISE OF THE STUDY: The yellow-flowered Aechmea subgenus Ortgiesia (yfAsO) (Bromeliaceae) is a group of seven morphologically similar bromeliads found mostly in the southern Brazilian Atlantic rainforest. The recent origin of this group probably contributes to its taxonomic complexity. The aims of this study were to investigate the levels of genetic diversity and structure at the population and species levels, to gain insight into the processes behind the diversification of the group, and to contribute to the establishment of species boundaries. METHODS: We sequenced two noncoding regions of the chloroplast genome (rpl32-trnL and rps16-trnK) and the nuclear phyC gene in 204 and 153 individuals, respectively, representing the seven species of the group. Phylogeographical and population genetics approaches were used. KEY RESULTS: Three of the seven yfAsO showed some degree of genetic differentiation among species. Divergence time for the group was dated to around 4 million years ago. Areas of conservation value were identified, and a scenario of multiple refugia in the southern Brazilian Atlantic rainforest during the Pleistocene climatic oscillations is suggested. CONCLUSIONS: We hypothesized that incomplete lineage sorting and localized hybridization events are responsible for the low levels of genetic differentiation and the taxonomic complexity observed among and within the seven yfAsO species. Further studies on Aechmea comata and Aechmea kertesziae will be necessary to clarify the boundary between these two species. Most of the populations sampled showed high genetic diversity and/or unique haplotypes; they should be prioritized for conservation purposes.

Strong but permeable barriers to gene exchange between sister species of Epidendrum.

PREMISE OF THE STUDY: The investigation of reproductive barriers between sister species can provide insights into how new lineages arise, and how species integrity is maintained in the face of interspecific gene flow. Different pre- and postzygotic barriers can limit interspecific gene exchange in sympatric populations, and different sources of evidence are often required to investigate the role of multiple reproductive isolation (RI) mechanisms. METHODS: We tested the hypothesis of hybridization and potential introgression between Epidendrum secundum and Epidendrum xanthinum, two Neotropical food-deceptive orchid species, using nuclear and plastid microsatellites, experimental crosses, pollen tube growth observations, and genome size estimates. KEY RESULTS: A large number of hybrids between E. secundum and E. xanthinum were detected, suggesting weak premating barriers. The low fertility of hybrid plants and the absence of haplotype sharing between parental species indicated strong postmating barriers, reducing interspecific gene exchange and the development of advanced generation hybrids. Despite the strength of reproductive barriers, fertile seeds were produced in some backcrossing experiments, and the existence of interspecific gene exchange could not be excluded. CONCLUSIONS: Strong but permeable barriers were found between E. secundum and E. xanthinum. Indeed, haplotype sharing was not detected between parental species, suggesting that introgression is limited by a combination of genic incompatibilities, including negative cytonuclear interactions. Most taxonomic uncertainties in this group were potentially influenced by incomplete RI barriers between species, which mainly occurred sympatrically.