Genomic footprints of repeated evolution of CAM photosynthesis in a Neotropical species radiation.

The adaptive radiation of Bromeliaceae (pineapple family) is one of the most diverse among Neotropical flowering plants. Diversification in this group was facilitated by shifts in several adaptive traits or "key innovations" including the transition from C3 to CAM photosynthesis associated with xeric (heat/drought) adaptation. We used phylogenomic approaches, complemented by differential gene expression (RNA-seq) and targeted metabolite profiling, to address the mechanisms of C3 /CAM evolution in the extremely species-rich bromeliad genus, Tillandsia, and related taxa. Evolutionary analyses of whole-genome sequencing and RNA-seq data suggest that evolution of CAM is associated with coincident changes to different pathways mediating xeric adaptation in this group. At the molecular level, C3 /CAM shifts were accompanied by gene expansion of XAP5 CIRCADIAN TIMEKEEPER homologs, a regulator involved in sugar- and light-dependent regulation of growth and development. Our analyses also support the re-programming of abscisic acid-related gene expression via differential expression of ABF2/ABF3 transcription factor homologs, and adaptive sequence evolution of an ENO2/LOS2 enolase homolog, effectively tying carbohydrate flux to abscisic acid-mediated abiotic stress response. By pinpointing different regulators of overlapping molecular responses, our results suggest plausible mechanistic explanations for the repeated evolution of correlated adaptive traits seen in a textbook example of an adaptive radiation.

Small RNA sequencing revealed various microRNAs involved in ethylene-triggered flowering process in Aechmea fasciata.

Ethylene-triggered flowering is a common phenomenon in plants of the family Bromeliaceae, but its molecular mechanism remains unclear. As a classical group of small RNAs, microRNAs play an essential role in the regulation of flowering. In this study, we found that various miRNAs participate in the ethylene-triggered flowering process in Aechmea fasciata via small RNA sequencing using juvenile and adult plants treated with ethylene for 24 hours. Finally, 63 known miRNAs, 52 novel miRNAs and 1721 target genes were identified or predicted. Expression changes of specific miRNAs were validated by qRT-PCR and northern blotting. Some predicted targets, including SPL, GAMYB and ARF, were verified in RLM-RACE experiments. Gene Ontology (GO) and KEGG analysis showed that numerous developmental and RNA-related processes were enriched. Integrated analysis of the transcriptomic data with small RNA sequencing revealed that numerous miRNAs and targets involved in ethylene-triggered flowering in A. fasciata. Our study is helpful for illuminating the molecular basis of the ethylene-triggered flowering phenomenon in Bromeliaceae.

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.

Bromeliaceae subfamilies show divergent trends of genome size evolution.

Genome size is known to vary widely across plants. Yet, the evolutionary drivers and consequences of genome size variation across organisms are far from understood. We investigated genome size variation and evolution in two major subfamilies of the Neotropical family Bromeliaceae by determining new genome size values for 83 species, testing phylogenetic signal in genome size variation, and assessing the fit to different evolutionary models. For a subset of epiphytic bromeliad species, we also evaluated the relationship of genome size with thermal traits and relative growth rate (RGR), respectively. Genome size variation in Bromelioideae appears to be evolutionary conserved, while genome size among Tillandsioideae varies considerably, not just due to polyploidy but arguably also due to environmental factors. The subfamilies show fundamental differences in genome size and RGR: Bromelioideae have, on average, lower genome sizes than Tillandsioideae and at the same time exhibit higher RGR. We attribute this to different resource use strategies in the subfamilies. Analyses among subfamilies, however, revealed unexpected positive relationships between RGR and genome size, which might be explained by the nutrient regime during cultivation. Future research should test whether there is indeed a trade-off between genome size and growth efficiency as a function of nutrient supply.

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.

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.

Adaptive variation in vein placement underpins diversity in a major Neotropical plant radiation.

Vein placement has been hypothesised to control leaf hydraulic properties, but the ecophysiological significance of variation in vein placement in the angiosperms has remained poorly understood. The highly diverse Neotropical Bromeliaceae offers an excellent system for exploring understudied relationships between leaf vein placement, physiological functions, and species ecology. To test key hypotheses regarding the links between vein placement, functional type divergences, and ecological diversity in the Bromeliaceae, I characterised the ratio of interveinal distance (IVD) to vein-epidermis distance (VED) in 376 species, representing all major functional types and 10% of the species diversity in the family, as well as bioclimatic properties and key leaf traits for subsets of species. There were significant differences in vein placement parameters in species of contrasting functional type, habitat association, and bioclimatic distribution. In many C3 tank-epiphytes, a greater ratio between interveinal distance and the depth of veins within the mesophyll reflects optimisation for resource foraging in shady, humid habitats. In succulent terrestrials, overinvestment in veins probably facilitates rapid recharge of water storage tissue, as well as restricting water loss. These results highlight how divergences in vein placement relate to distinctive ecophysiological strategies between and within bromeliad functional types, and provide timely insights into how structural-functional innovation has impacted the evolution of ecological diversity in a major radiation of tropical herbaceous angiosperms.

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.

Molecular phylogenetics of the Ronnbergia Alliance (Bromeliaceae, Bromelioideae) and insights into their morphological evolution.

The tank-epiphytic clade of berry-fruited bromeliads, also known as the Core Bromelioideae, represents a remarkable event of adaptive radiation within the Bromeliaceae; however, the details of this radiation have been difficult to study because this lineage is plagued with generic delimitation problems. In this study, we used a phylogenetic approach to investigate a well supported, albeit poorly understood, lineage nested within the Core Bromelioideae, here called the "Ronnbergia Alliance". In order to assess the monophyly and phylogenetic relationships of this group, we used three plastid and three nuclear DNA sequence markers combined with a broad sampling across three taxonomic groups and allied species of Aechmea expected to comprise the Ronnbergia Alliance. We combined the datasets to produce a well-supported and resolved phylogenetic hypothesis. Our main results indicated that the Ronnbergia Alliance was a well-supported monophyletic group, sister to the remaining Core Bromelioideae, and it was composed by species of the polyphyletic genera Aechmea, Hohenbergia and Ronnbergia. We identified two major internal lineages with high geographic structure within the Ronnbergia Alliance. The first of these lineages, called the Pacific Clade, contained species of Aechmea and Ronnbergia that occur exclusively from southern Central America to northwestern South America. The second clade, called the Atlantic Clade, contained species of Aechmea, Hohenbergia and Ronnbergia mostly limited to the Atlantic Forest and the Caribbean. We also explored the diagnostic and evolutionary importance of 13 selected characters using ancestral character reconstructions on the phylogenetic hypothesis. We found that the combination of tubular corollas apically spreading and unappendaged ovules had diagnostic value for the Ronnbergia Alliance, whereas flower size, length of the corolla tube, and petal pigmentation and apex were important characters to differentiate the Pacific and Atlantic clades. This study opens new perspectives for future taxonomic reorganizations and provides a framework for evolutionary and biogeographic studies.

AfAP2-1, An Age-Dependent Gene of Aechmea fasciata, Responds to Exogenous Ethylene Treatment.

The Bromeliaceae family is one of the most morphologically diverse families with a pantropical distribution. To schedule an appropriate flowering time for bromeliads, ethylene is commonly used to initiate flower development in adult plants. However, the mechanism by which ethylene induces flowering in adult bromeliads remains unknown. Here, we identified an APETALA2 (AP2)-like gene, AfAP2-1, in Aechmea fasciata. AfAP2-1 contains two AP2 domains and is a nuclear-localized protein. It functions as a transcriptional activator, and the activation domain is located in the C-terminal region. The expression level of AfAP2-1 is higher in juvenile plants than in adult plants, and the AfAP2-1 transcript level was rapidly and transiently reduced in plants treated with exogenous ethylene. Overexpression of AfAP2-1 in Arabidopsis thaliana results in an extremely delayed flowering phenotype. These results suggested that AfAP2-1 responds to ethylene and is a putative age-dependent flowering regulator in A. fasciata.

Adaptive radiation, correlated and contingent evolution, and net species diversification in Bromeliaceae.

We present an integrative model predicting associations among epiphytism, the tank habit, entangling seeds, C3 vs. CAM photosynthesis, avian pollinators, life in fertile, moist montane habitats, and net rates of species diversification in the monocot family Bromeliaceae. We test these predictions by relating evolutionary shifts in form, physiology, and ecology to time and ancestral distributions, quantifying patterns of correlated and contingent evolution among pairs of traits and analyzing the apparent impact of individual traits on rates of net species diversification and geographic expansion beyond the ancestral Guayana Shield. All predicted patterns of correlated evolution were significant, and the temporal and spatial associations of phenotypic shifts with orogenies generally accorded with predictions. Net rates of species diversification were most closely coupled to life in fertile, moist, geographically extensive cordilleras, with additional significant ties to epiphytism, avian pollination, and the tank habit. The highest rates of net diversification were seen in the bromelioid tank-epiphytic clade (D(crown) = 1.05 My⁻¹), associated primarily with the Serra do Mar and nearby ranges of coastal Brazil, and in the core tillandsioids (D(crown) = 0.67 My⁻¹), associated primarily with the Andes and Central America. Six large-scale adaptive radiations and accompanying pulses of speciation account for 86% of total species richness in the family. This study is among the first to test a priori hypotheses about the relationships among phylogeny, phenotypic evolution, geographic spread, and net species diversification, and to argue for causality to flow from functional diversity to spatial expansion to species diversity.

Karyotype characterization and nuclear DNA content measurement in Bromeliaceae: state of the art and future perspectives.

In Bromeliaceae, cytogenetic and flow cytometry analyses have been performed to clarify systematic and evolutionary aspects. Karyotyping approaches have shown the relatively high chromosome number, similar morphology and small size of the chromosomes. These facts have prevented a correct chromosome counting and characterization. Authors have established a basic chromosome number of x = 25 for Bromeliaceae. Recently, one karyomorphological analysis revealed that x = 25 is no longer the basic chromosome number, whose genome may have a polyploid origin. Besides cytogenetic characterization, the 2C DNA content of bromeliads has been measured. Nuclear DNA content has varied from 2C = 0.60 to 2C = 3.34 picograms. Thus, in relation to most angiosperms, the 2C DNA content of Bromeliaceae species as well as their chromosome size can be considered relatively small. In spite of some advances, cytogenetic and flow cytometry data are extremely scarce in this group. In this context, this review reports the state of the art in karyotype characterization and nuclear DNA content measurement in Bromeliaceae, emphasizing the main problems and suggesting prospective solutions and ideas for future research.

Phylogeny, adaptive radiation, and historical biogeography in Bromeliaceae: insights from an eight-locus plastid phylogeny.

PREMISE: Bromeliaceae form a large, ecologically diverse family of angiosperms native to the New World. We use a bromeliad phylogeny based on eight plastid regions to analyze relationships within the family, test a new, eight-subfamily classification, infer the chronology of bromeliad evolution and invasion of different regions, and provide the basis for future analyses of trait evolution and rates of diversification. METHODS: We employed maximum-parsimony, maximum-likelihood, and Bayesian approaches to analyze 9341 aligned bases for four outgroups and 90 bromeliad species representing 46 of 58 described genera. We calibrate the resulting phylogeny against time using penalized likelihood applied to a monocot-wide tree based on plastid ndhF sequences and use it to analyze patterns of geographic spread using parsimony, Bayesian inference, and the program S-DIVA. RESULTS: Bromeliad subfamilies are related to each other as follows: (Brocchinioideae, (Lindmanioideae, (Tillandsioideae, (Hechtioideae, (Navioideae, (Pitcairnioideae, (Puyoideae, Bromelioideae))))))). Bromeliads arose in the Guayana Shield ca. 100 million years ago (Ma), spread centrifugally in the New World beginning ca. 16-13 Ma, and dispersed to West Africa ca. 9.3 Ma. Modern lineages began to diverge from each other roughly 19 Ma. CONCLUSIONS: Nearly two-thirds of extant bromeliads belong to two large radiations: the core tillandsioids, originating in the Andes ca. 14.2 Ma, and the Brazilian Shield bromelioids, originating in the Serro do Mar and adjacent regions ca. 9.1 Ma.

Phylogenetic estimation of the core Bromelioids with an emphasis on the genus Aechmea (Bromeliaceae).

We developed a phylogeny of the core Bromelioideae including Aechmea and related genera, with the specific goals of investigating the monophyly of Aechmea and its allied genera, redefining monophyletic lineages for taxonomic revision, and investigating the biogeographic history of the group. Chloroplast, nuclear ribosomal, and low copy nuclear DNA sequences from 150 species within the Bromelioideae were used to develop the phylogeny. Phylogenies constructed with the combined four gene dataset provided sufficient resolution for investigating evolutionary relationships among species. Many genera are nested within Aechmea, or are rendered para- or polyphyletic by inclusion of Aechmea species. Several genera and subgenera of Aechmea with species in disjunct geographic locations are found to be polyphyletic, divided into separate clades that reflect geographic distribution rather than morphological similarity. This suggests that certain morphological characteristics thought to be indicative of common ancestry have instead evolved multiple times in parallel (i.e. ecological conservatism), possibly indicative of local adaptations to an epiphytic habit across the range of the Bromelioideae. These apparently homoplastic morphological characters used to assign species to genera or subgenera may be useful taxonomically when geography is also taken into account.

Within-population spatial genetic structure in four naturally fragmented species of a neotropical inselberg radiation, Alcantarea imperialis, A. geniculata, A. glaziouana and A. regina (Bromeliaceae).

Studies of organisms on 'terrestrial islands' can improve our understanding of two unresolved issues in evolutionary genetics: the likely long-term effects of habitat fragmentation and the genetic underpinnings of continental species radiations in island-like terrestrial habitats. We have addressed both issues for four closely related plant species of the adaptive radiation Bromeliaceae, Alcantarea imperialis, A. geniculata, A. regina and A. glaziouana. All four are adapted to ancient, isolated inselberg rock outcrops in the Brazilian Atlantic rainforest and are thus long-term fragmented by nature. We used eight nuclear microsatellites to study within-population spatial genetic structure (SGS) and historical gene dispersal in nine populations of these species. Within-population SGS reflected known between-species differences in mating systems. The strongest SGS observed in A. glaziouana (Sp=0.947) was stronger than literature estimates available for plants. Analysis of short- and long-distance components of SGS identified biparental inbreeding, selfing and restricted seed dispersal as main determinants of SGS, with restricted pollen dispersal by bats contributing in some localities. The ability of Alcantarea spp. to colonize isolated inselbergs probably stems from their flexible mating systems and an ability to tolerate inbreeding. Short-ranging gene dispersal (average sigma=7-27 m) is consistent with a loss of dispersal power in terrestrial island habitats. Population subdivision associated with sympatric colour morphs in A. imperialis is accompanied by between-morph differences in pollen and seed dispersal. Our results indicate a high potential for divergence with gene flow in inselberg bromeliads and they provide base-line data about the long-term effects of fragmentation in plants.