Metabarcoding of the phytotelmata of Pseudalcantarea grandis (Bromeliaceae) from an arid zone.

BACKGROUND: Pseudalcantarea grandis (Schltdl.) Pinzón & Barfuss is a tank bromeliad that grows on cliffs in the southernmost portion of the Chihuahuan desert. Phytotelmata are water bodies formed by plants that function as micro-ecosystems where bacteria, algae, protists, insects, fungi, and some vertebrates can develop. We hypothesized that the bacterial diversity contained in the phytotelma formed in a bromeliad from an arid zone would differ in sites with and without surrounding vegetation. Our study aimed to characterize the bacterial composition and putative metabolic functions in P. grandis phytotelmata collected in vegetated and non-vegetated sites. METHODS: Water from 10 individuals was sampled. Five individuals had abundant surrounding vegetation, and five had little or no vegetation. We extracted DNA and amplified seven hypervariable regions of the 16S gene (V2, V4, V8, V3-6, 7-9). Metabarcoding sequencing was performed on the Ion Torrent PGM platform. Taxonomic identity was assigned by the binning reads and coverage between hit and query from the reference database of at least 90%. Putative metabolic functions of the bacterial families were assigned mainly using the FAPROTAX database. The dominance patterns in each site were visualized with rank/abundance curves using the number of Operational Taxonomic Units (OTUs) per family. A percentage similarity analysis (SIMPER) was used to estimate dissimilarity between the sites. Relationships among bacterial families (identified by the dominance analysis and SIMPER), sites, and their respective putative functions were analyzed with shade plots. RESULTS: A total of 1.5 million useful bacterial sequences were obtained. Sequences were clustered into OTUs, and taxonomic assignment was conducted using BLAST in the Greengenes databases. Bacterial diversity was 23 phyla, 52 classes, 98 orders, 218 families, and 297 genera. Proteobacteria (37%), Actinobacteria (19%), and Firmicutes (15%) comprised the highest percentage (71%). There was a 68.3% similarity between the two sites at family level, with 149 families shared. Aerobic chemoheterotrophy and fermentation were the main metabolic functions in both sites, followed by ureolysis, nitrate reduction, aromatic compound degradation, and nitrogen fixation. The dominant bacteria shared most of the metabolic functions between sites. Some functions were recorded for one site only and were related to families with the lowest OTUs richness. Bacterial diversity in the P. grandis tanks included dominant phyla and families present at low percentage that could be considered part of a rare biosphere. A rare biosphere can form genetic reservoirs, the local abundance of which depends on external abiotic and biotic factors, while their interactions could favor micro-ecosystem resilience and resistance.

Description of the immature stages, biology and DNA-barcoding of Quichuana pogonosa (Diptera: Syrphidae) collected in Bromeliaceae in Paraná, Brazil

Quichuana Knab, 1913 is a Neotropical genus of flower flies (Diptera: Syrphidae) with 50 valid species. Adults of this genus are flower visitors and their larvae are usually associated with the phytotelmata of bromeliads and heliconias, actively participating in the recycling of nutrients in forest environments. Despite their importance in ecosystem dynamics, Quichuana larvae are poorly known. Herein we describe the immature stages of Quichuana pogonosa Fluke, 1937 from samples collected from the phytotelmata of two terrestrial bromeliad species in the state of Paraná, Brazil. We provide illustrations of the egg, third instar larva and puparium, as well as information on the life cycle of the species. Additionally, we describe and illustrate the male genitalia and present the DNA-barcoding based on larva and adult specimens.(AU)

Description of the immature stages, biology and DNA-barcoding of Quichuana pogonosa (Diptera: Syrphidae) collected in Bromeliaceae in Paraná, Brazil

Quichuana Knab, 1913 is a Neotropical genus of flower flies (Diptera: Syrphidae) with 50 valid species. Adults of this genus are flower visitors and their larvae are usually associated with the phytotelmata of bromeliads and heliconias, actively participating in the recycling of nutrients in forest environments. Despite their importance in ecosystem dynamics, Quichuana larvae are poorly known. Herein we describe the immature stages of Quichuana pogonosa Fluke, 1937 from samples collected from the phytotelmata of two terrestrial bromeliad species in the state of Paraná, Brazil. We provide illustrations of the egg, third instar larva and puparium, as well as information on the life cycle of the species. Additionally, we describe and illustrate the male genitalia and present the DNA-barcoding based on larva and adult specimens.

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.

Genetic diversity and karyotype of Pitcairnia azouryi: an endangered species of Bromeliaceae endemic to Atlantic Forest inselbergs.

Plant species of various families, such as those of Bromeliaceae, occur on inselbergs where they are subject to geographic isolation and environmental conditions that can lead to genetic erosion. This, in turn, can result in the loss of natural populations due to homozygosis, or changes in ploidy that may lead to reproductive isolation. The genetic diversity of five natural populations of Pitcairnia azouryi was measured using nine microsatellite markers transferred from P. albiflos and P. geyskesii. Chromosome numbers and nuclear DNA content were also evaluated. The results indicated moderate genetic differentiation among populations (FST = 0.188), and significant gene flow (Nm = 1.073) in four of the five populations. P. azouryi has, predominantly, 2n = 50 chromosomes and DNA content of 2C = 1.16 pg, but the tetraploid condition was found (2n = 100 and 2C = 2.32 pg) in seedlings of an individual of the most geographically isolated population. The moderate level of genetic structuring observed for P. azouryi seems to be related to its disjoint geographical distribution and the locally aggregated spatial structure of the populations, which are isolated from each other, hindering the inter and intrapopulational gene flow. This interpretation was also evidenced by the mantel test (r = 0.777, P < 0.05). The occurrence of diploid individuals with tetraploid seedlings is indicative of events of eupolyploidization, possibly due to the environmental conditions of this geographically isolated population.

Transcriptional foliar profile of the C3-CAM bromeliad Guzmania monostachia.

Guzmania monostachia is an epiphytic tank bromeliad that displays the inducible CAM photosynthesis under stressful conditions and had the highest stomata density in the leaf apex, while the base portion has the highest density of trichomes, which are specialized structures used to acquire water and nutrients from the tank solution. In order to correlate the genetic factors behind these morpho-physiological characteristics along the leaf blade of G. monostachia, a comparative transcriptome analysis was performed to identify the functional enriched pathways and unigenes that could play a role in the apical, middle and basal leaf portions. A total of 653 million reads were used for de novo transcriptome assembly, resulting in 48,051 annotated unigenes. Analysis of differentially expressed genes (DEGs) among distinct leaf regions revealed that 806 DEGs were upregulated in the apex compared to the middle portion, while 9685 DEGs were upregulated in the apex and 9784 DEGs were upregulated in the middle portions compared to the base. Our outcomes correlated some DEGs and identified unigenes with their physiological functions, mainly suggesting that the leaf apex was related to the regulation of stomatal movement, production of chlorophyll, cellular response to stress, and H2O2 catabolic process. In contrast, the middle portion showed DEGs associated with the transport of amino acids. Furthermore, DEGs from the leaf base were mainly correlated with responses to nutrients and nitrogen compounds, regulation of potassium ion import, response to water deprivation, and trichome branching, indicating that, at least in part, this leaf portion can replace some of the root functions of terrestrial plants. Therefore, possibly candidate unigenes and enriched pathways presented here could be prospected in future experimental work, opening new possibilities to bioengineer non-inducible CAM plants and/or improve the fertilization use efficiency by increasing leaf nutrient acquisition of crop plants.

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.

Involvement of aquaporins on nitrogen-acquisition strategies of juvenile and adult plants of an epiphytic tank-forming bromeliad.

MAIN CONCLUSION: Depending on the N source and plant ontogenetic state, the epiphytic tank-forming bromeliad Vriesea gigantea can modulate aquaporin expression to maximize the absorption of the most available nitrogen source. Epiphytic bromeliads frequently present a structure formed by the overlapping of leaf bases where water and nutrients can be accumulated and absorbed, called tank. However, this structure is not present during the juvenile ontogenetic phase, leading to differences in nutrient acquisition strategies. Recent studies have shown a high capacity of the bromeliad Vriesea gigantea, an epiphytic tank-forming bromeliad, to absorb urea by their leaves. Since plant aquaporins can facilitate the diffusion of urea through the membranes, we cloned three foliar aquaporin genes, VgPIP1;1, VgPIP1;2 and VgTIP2;1 from V. gigantea plants. Through functional studies, we observed that besides water, VgTIP2;1 was capable of transporting urea while VgPIP1;2 may facilitate ammonium/ammonia diffusion. Moreover, aiming at identifying urea and ammonium-induced changes in aquaporin expression in leaves of juvenile and adult-tank plants, we showed that VgPIP1;1 and VgPIP1;2 transcripts were up-regulated in response to either urea or ammonium only in juvenile plants, while VgTIP2;1 was up-regulated in response to urea only in adult-tank plants. Thereby, an ontogenetic shift from juvenile to adult-tank-forming-plant appears to occur with metabolic changes regarding nitrogen metabolism regulation. Investigating urea metabolism in wild species that naturally cope with organic N sources, such as V. gigantea, may provide the knowledge to modify nitrogen use efficiency of crop plants.

De novo assembly of Vriesea carinata leaf transcriptome to identify candidate cysteine-proteases.

Vriesea carinata is an endemic bromeliad from the Brazilian Atlantic Forest. It has trichome and tank system in their leaves which allows to absorb water and nutrients. It belongs to Bromeliaceae family, which includes several species highly enriched of cysteine-proteases (CysPs). These proteolytic enzymes regulate processes as senescence, cell differentiation, pathogen-linked programmed cell death and mobilization of proteins. Although, their biological importance, there are not genomic resources in V. carinata that can help to identify and understand their molecular mechanisms involved in different biological processes. Thus high-throughput transcriptome sequencing of V. carinata is necessary to generate sequences for the purpose of gene discovery and functional genomic studies. In the present study, we sequenced and assembled the V. carinata transcriptome to the identification of CysPs. A total of 43,232 contigs were assembled for the leaf tissue. BLAST analysis indicated that 23,803 contigs exhibited similarity to non-redundant Viridiplantae proteins. 28.24% of the contigs were classified into the COG database, and gene ontology categorized them into 61 functional groups. A metabolic pathway analysis with KEGG revealed 9679 contigs assigned to 31 metabolic pathways. Among 16 full-length CysPs identified, 11 were evaluated in respect to their expression patterns in the leaf apex, base and inflorescence tissues. The results showed differential expression levels of legumain, metacaspase, pyroglutamyl and papain-like CysPs depending of the leaf region. These results provide a global overview of V. carinata gene functions and expression activities of CysPs in those tissues.

The role of hybridization and introgression in maintaining species integrity and cohesion in naturally isolated inselberg bromeliad populations.

Hybridization is a widespread phenomenon present in numerous lineages across the tree of life. Its evolutionary consequences range from effects on the origin and maintenance, to the loss of biodiversity. We studied genetic diversity and intra- and interspecific gene flow between two sympatric populations of closely-related species, Pitcairnia flammea and P. corcovadensis (Bromeliaceae), which are adapted to naturally fragmented Neotropical inselbergs, based on nuclear and plastidial DNA. Our main results indicate a strong reproductive isolation barrier, although low levels of interspecific gene flow were observed in both sympatric populations. The low rates of intraspecific gene flow observed for both P. corcovadensis and P. flammea populations corroborate the increasing body of evidence that inselberg bromeliad species are maintained as discrete evolutionary units despite the presence of low genetic connectivity. Nuclear patterns of genetic diversity and gene flow revealed that hybridization and introgression might not cause species extinction via genetic assimilation of the rare P. corcovadensis. In the face of reduced intraspecific gene exchange, hybridization and introgression may be important aspects of the Pitcairnia diversification process, with a positive evolutionary impact at the bromeliad community level, and thus contribute to increasing and maintaining genetic diversity in local isolated inselberg populations.

Pervasive migration across rainforest and sandy coastal plain Aechmea nudicaulis (Bromeliaceae) populations despite contrasting environmental conditions.

Understanding the colonization of extreme marginal habitats and the relative roles of space and environment in maintaining peripheral populations remains challenging. Here, we leverage a system of pairs of rainforest and sandy coastal plain communities that allow us to decouple spatial and environmental effects in the population structure and migration rates of the bromeliad Aechmea nudicaulis. Structure and gene flow between populations were estimated from Bayesian clustering and coalescent-based migration models applied to chloroplast sequence and nuclear microsatellite data. Contrary to our initial expectation, the sharp environmental gradient between rainforest and sandy plains does not seem to have affected the colonization and migration dynamics in A. nudicaulis. Our analyses uncover pervasive gene flow between neighbouring habitats in both chloroplast and nuclear data despite the striking differences in environmental conditions. This result is consistent with a scenario of repeated colonization of the sandy coastal plains from forest populations through seed dispersal, as well as the maintenance of gene flow between habitats through pollination. We also recovered a broad north/south population structure that has been found in other Atlantic rainforest groups and possibly reflects older phylogeographic dynamics.

Ammonium intensifies CAM photosynthesis and counteracts drought effects by increasing malate transport and antioxidant capacity in Guzmania monostachia.

Guzmania monostachia (Bromeliaceae) is a tropical epiphyte capable of up-regulating crassulacean acid metabolism (CAM) in its photosynthetic tissues in response to changing nutrient and water availability. Previous studies have shown that under drought there is a gradient of increasing CAM expression from the basal (youngest) to the apical (oldest) portion of the leaves, and additionally that nitrogen deficiency can further increase CAM intensity in the leaf apex of this bromeliad. The present study investigated the inter-relationships between nitrogen source (nitrate and/or ammonium) and water deficit in regulating CAM expression in G. monostachia leaves. The highest CAM activity was observed under ammonium nutrition in combination with water deficit. This was associated with enhanced activity of the key enzyme phosphoenolpyruvate carboxylase, elevated rates of ATP- and PPi-dependent proton transport at the vacuolar membrane in the presence of malate, and increased transcript levels of the vacuolar malate channel-encoding gene, ALMT. Water deficit was consistently associated with higher levels of total soluble sugars, which were maximal under ammonium nutrition, as were the activities of several antioxidant enzymes (superoxide dismutase, catalase, ascorbate peroxidase, and glutathione reductase). Thus, ammonium nutrition, whilst associated with the highest degree of CAM induction in G. monostachia, also mitigates the effects of water deficit by osmotic adjustment and can limit oxidative damage in the leaves of this bromeliad under conditions that may be typical of its epiphytic habitat.

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.

Cross-amplification and characterization of microsatellite markers in Alcantarea patriae Versieux & Wand.

The Alcantarea patriae is a Bromeliaceae endemic to the inselbergs of the Atlantic Forest. This taxon, described in the year of 2007 by Versieux & Wanderley, presents restricted and fragmented distribution outside conservation units. Studies to evaluate the genetic structure of its populations can contribute to the conservation and management strategies for the species. In this study, 31 microsatellite markers, descriptive to six different Bromeliaceae species, were evaluated by cross-amplification tests in 20 individuals of A. patriae. The individuals were collected in the district of Vila Cruzeiro, in the municipality of Jerônimo Monteiro. Twelve markers were polymorphic and 10 monomorphic, with an amplification success rate of 71%. The displayed polymorphism information content was considered high, indicating that the selected markers are informative. The values found for the fixation index were positive and indicated the occurrence of inbreeding. The mean number of alleles was 4.66 (3-6), the mean expected and observed heterozygosities were 0.6605 and 0.4618, respectively. The detection of polymorphic markers was important for future studies of diversity and genetic structuring of natural populations and for germplasm bank creation aiming to contribute to in situ and ex situ conservations of A. patriae.

Population genetic structure of the rock outcrop species Encholirium spectabile (Bromeliaceae): The role of pollination vs. seed dispersal and evolutionary implications.

PREMISE OF THE STUDY: Inselbergs are terrestrial, island-like rock outcrop environments that present a highly adapted flora. The epilithic bromeliad Encholirium spectabile is a dominant species on inselbergs in the Caatinga of northeastern Brazil. We conducted a population genetic analysis to test whether the substantial phenotypic diversity of E. spectabile could be explained by limited gene flow among populations and to assess the relative impact of pollen vs. seed dispersal on the genetic structure of the species. METHODS: Nuclear and chloroplast microsatellite markers were used to genotype E. spectabile individuals from 20 rock outcrop locations, representing four geographic regions: northern Espinhaço Range, Borborema Plateau, southwestern Caatinga and southeastern Caatinga. F-statistics, structure, and other tools were applied to evaluate the genetic makeup of populations. KEY RESULTS: Considerable levels of genetic diversity were revealed. Genetic structuring among populations was stronger on the plastid as compared with the nuclear level, indicating higher gene flow via bat pollination as compared with seed dispersal by wind. structure and AMOVA analyses of the nuclear data suggested a high genetic differentiation between two groups, one containing all populations from the southeastern Caatinga and the other one comprising all remaining samples. CONCLUSIONS: The strong genetic differentiation between southeastern Caatinga and the remaining regions may indicate the occurrence of a cryptic species in E. spectabile. The unique genetic composition of each inselberg population suggests in situ conservation as the most appropriate protection measure for this plant lineage.

Genetic diversity of Bromeliaceae species from the Atlantic Forest.

The Bromeliaceae family includes a range of species used for many purposes, including ornamental use and use as food, medicine, feed, and fiber. The state of Espírito Santo, Brazil is a center of diversity for this family in the Atlantic Forest. We evaluated the genetic diversity of five populations of the Bromeliaceae family, including specimens of the genera Aechmea, Billbergia (subfamily Bromelioideae), and Pitcairnia (subfamily Pitcairnioidea), all found in the Atlantic Forest and distributed in the state of Espírito Santo. The number of alleles per locus in populations ranged from two to six and the fixation index (F), estimated for some simple sequence repeats in bromeliad populations, was less than zero in all populations. All markers in the Pitcairnia flammea population were in Hardy-Weinberg equilibrium (P < 0.05). Moreover, significant deviations from Hardy-Weinberg equilibrium were observed at some loci in populations of the five bromeliad species. In most cases, this can be attributed to the presence of inbreeding or the Wahlund effect. The genetic diversity indices of five species showed greater allelic richness in P. flammea (3.55). Therefore, we provide useful information for the characterization of genetic diversity in natural populations of Aechmea ramosa, Aechmea nudicaulis, Billbergia horrid, Billbergia euphemia, and P. flammea in Atlantic Forest remnants in the south of Espírito Santo state.

Ecological range shift in the polyploid members of the South American genus Fosterella (Bromeliaceae).

Background and Aims: The distribution of polyploidy along a relatively steep Andean elevation and climatic gradient is studied using the genus Fosterella L.B. Sm. (Bromeliaceae) as a model system. Ecological differentiation of cytotypes and the link of polyploidy with historical biogeographic processes such as dispersal events and range shift are assessed. Methods: 4',6-Diamidino-2-phenylindole (DAPI) staining of nuclei and flow cytometry were used to estimate the ploidy levels of 159 plants from 22 species sampled throughout the distribution range of the genus. Ecological differentiation among ploidy levels was tested by comparing the sets of climatic variables. Ancestral chromosome number reconstruction was carried out on the basis of a previously generated phylogeographic framework. Key Results: This study represents the first assessment of intrageneric, intraspecific and partially intrapopulational cytotype diversity in a genus of the Bromeliaceae family. In Fosterella , the occurrence of polyploidy was limited to the phylogenetically isolated penduliflora and rusbyi groups. Cytotypes were found to be ecologically differentiated, showing that polyploids preferentially occupy colder habitats with high annual temperature variability (seasonality). The combined effects of biogeographic history and adaptive processes are presumed to have shaped the current cytotype distribution in the genus. Conclusions: The results provide indirect evidence for both adaptive ecological and non-adaptive historical processes that jointly influenced the cytotype distribution in the predominantly Andean genus Fosterella (Bromeliaceae). The results also exemplify the role of polyploidy as an important driver of speciation in a topographically highly structured and thus climatically diverse landscape.

Diversification of Bromelioideae (Bromeliaceae) in the Brazilian Atlantic rainforest: A case study in Aechmea subgenus Ortgiesia.

Aechmea subgenus Ortgiesia comprises ca. 20 species distributed in Brazil, Argentina, Paraguay, and Uruguay, with a center of diversity in the Brazilian Atlantic rainforest. We examined interspecific relationships of Ortgiesia based on Amplified Fragment Length Polymorphisms (AFLP). Ninety-six accessions belonging to 14 species of Ortgiesia were sampled, and genotyped with 11 AFLP primer combinations. The neighbor joining (NJ) tree depicted two main genetic groups within Aechmea subgenus Ortgiesia, and four subgroups. The NJ tree showed short internal branches, indicating an overall shallow genetic divergence among Ortgiesia species as expected for the recently radiated subfamily Bromelioideae. Our results suggest that hybridization and/or incomplete lineage sorting may have hampered the reconstruction of interspecific relationships in Aechmea subgenus Ortgiesia. The mapping of petal color (yellow, blue, pink, or white), inflorescence type (simple or compound), and inflorescence shape (ellipsoid, subcylindric, cylindric, or pyramidal) against the NJ tree indicated that these characters are of limited taxonomic use in Aechmea subgenus Ortgiesia due to homoplasy. An analysis of the current distribution of Ortgiesia identified the southern region of the Brazilian Atlantic rainforest, between latitudes of 26° and 27°S, as the center of diversity for the subgenus.

De novo assembly and characterization of leaf and floral transcriptomes of the hybridizing bromeliad species (Pitcairnia spp.) adapted to Neotropical Inselbergs.

We present the leaf and floral transcriptomes of two hybridizing bromeliad species that differ in their major pollinator systems. Here we identified candidate genes responsible for pollinator attraction and reproductive isolation in these two species. We searched for candidate genes involved in floral traits, such as colour. Approximately 34 Gbp of cDNA sequence data were produced from both tissues and species, resulting in a total of 424 506 914 raw reads. The de novo-assembled transcriptomes consisted of a total of 263 955 contigs, further clustered into 110 977 unigenes. Over 58% of the unigenes were functionally annotated and assigned to one or more Gene Ontology terms. The transcriptomes revealed 144 unique transcripts that encode key enzymes in the flavonoid and anthocyanin biosynthesis pathways. The domain/family annotation and phylogenetic analysis allowed us to infer, by homology, potential functions of the genes encoding MYB, HD-ZIP and bZIP-HY5 transcription factors, as well as WD40 protein, which may be involved in anthocyanin and flavonoid regulation in these species. These candidate genes are associated with natural regulation in flower colour in other plant species and will facilitate future studies aimed at elucidating the molecular basis of adaptive differentiation and the evolution of mechanisms of pollinator-mediated reproductive isolation in these two bromeliads. In addition, we identified a total of 49 439 microsatellite loci. These resources will assist future research into adaptation and speciation events in bromeliad species, thus providing a starting point for investigation of the molecular mechanisms of the traits responsible for their reproductive isolation.

Genetic diversity in three natural populations of Pitcairnia flammea (l.) John (Bromeliaceae) estimated by ISSR markers.

Bromeliads are greatly represented in the Atlantic Forest, although many species are threatened with extinction owing to habitat fragmentation and intense extraction for ornamental purposes. Therefore, it is necessary to conduct studies generating knowledge about genetic diversity and the distribution of this diversity among and within natural populations to establish conservation strategies. These studies can be performed with the use of molecular markers. Molecular markers are advantageous for studies of natural populations, for conservation programs, and to aid in properly classifying plant species. This study aimed to evaluate the genetic diversity among and within natural populations of Pitcairnia flammea, occurring in three fragments of the Atlantic Forest in the southern State of Espírito Santo through the use of inter-simple sequence repeat (ISSR) markers. DNA samples from 55 individuals were amplified with 18 ISSR primers, generating 180 bands, 159 of which were polymorphic. The Shannon genetic diversity index ranged from 0.348 to 0.465, with an average of 0.412. The Bayesian approach for the molecular data indicated the existence of two genetic groups. Analysis of molecular variance indicated the existence of 90.3% diversity within the population and 9.74% among populations. The amount of genetic differentiation of populations was moderate (0.0974), indicating that gene flow rates may be enough to counteract the effects of genetic drift. Greater genetic variability found in population B indicates that this area is an important source of genetic variability.