Selective Bacterial Community Enrichment between the Pitcher Plants Sarracenia minor and Sarracenia flava.

The interconnected and overlapping habitats present in natural ecosystems remain a challenge in determining the forces driving microbial community composition. The cuplike leaf structures of some carnivorous plants, including those of the family Sarraceniaceae, are self-contained ecological habitats that represent systems for exploring such microbial ecology questions. We investigated whether Sarracenia minor and Sarracenia flava cultivate distinct bacterial communities when sampled at the same geographic location and time. This sampling strategy eliminates many abiotic environmental variables present in other studies that compare samples harvested over time, and it could reveal biotic factors driving the selection of microbes. DNA extracted from the decomposing detritus trapped in each Sarracenia leaf pitcher was profiled using 16S rRNA amplicon sequencing. We identified a surprising amount of bacterial diversity within each pitcher, but we also discovered bacteria whose abundance was specifically enriched in one of the two Sarracenia species. These differences in bacterial community representation suggest some biotic influence of the Sarracenia plant on the bacterial composition of their pitchers. Overall, our results suggest that bacterial selection due to factors other than geographic location, weather, or prey availability is occurring within the pitchers of these two closely related plant species. This indicates that specific characteristics of S. minor and S. flava may play a role in fostering distinct bacterial communities. These confined, naturally occurring microbial ecosystems within Sarracenia pitchers may provide model systems to answer important questions about the drivers of microbial community composition, succession, and response to environmental perturbations. IMPORTANCE This study uses amplicon sequencing to compare the bacterial communities of environmental samples from the detritus of the leaf cavities of Sarracenia minor and Sarracenia flava pitcher plants. We sampled the detritus at the same time and in the same geographic location, eliminating many environmental variables present in other comparative studies. This study revealed that different species of Sarracenia contain distinct bacterial members within their pitchers, suggesting that these communities are not randomly established based on environmental factors and the prey pool but are potentially enriched for by the plants' chemical or physical environment. This study of these naturally occurring, confined microbial ecosystems will help further establish carnivorous pitcher plants as a model system for answering important questions about the development and succession of microbial communities.

Phylogeny and biogeography of South American marsh pitcher plant genus Heliamphora (Sarraceniaceae) endemic to the Guiana Highlands.

Heliamphora is a genus of carnivorous pitcher plants endemic to the Guiana Highlands with fragmented distributions. We present a well resolved, time-calibrated, and comprehensive Heliamphora phylogeny estimated using Bayesian inference and maximum likelihood based on nuclear genes (26S, ITS, and PHYC) and secondary calibration. We used stochastic mapping to infer ancestral states of morphological characters and ecological traits. Our ancestral state estimations revealed that the pitcher drainage structures characteristic of the genus transformed from a hole to a slit in single clade, while other features (scape pubescence and hammock-like growth) have been gained and lost multiple times. Habitat was similarly labile in Heliamphora, with multiple transitions from the ancestral highland habitats into the lowlands. Using a Mantel test, we found closely related species tend to be geographically closely distributed. Placing our phylogeny in a historical context, major clades likely emerged through both vicariance and dispersal during the Miocene with more recent diversification driven by vertical displacement during the Pleistocene glacial-interglacial thermal oscillations. Despite the dynamic climatic history experienced by Heliamphora, the temperature changes brought by global warming pose a significant threat, particularly for those species at the highest elevations.

Metabolite profiling of the carnivorous pitcher plants Darlingtonia and Sarracenia.

Sarraceniaceae is a New World carnivorous plant family comprising three genera: Darlingtonia, Heliamphora, and Sarracenia. The plants occur in nutrient-poor environments and have developed insectivorous capability in order to supplement their nutrient uptake. Sarracenia flava contains the alkaloid coniine, otherwise only found in Conium maculatum, in which its biosynthesis has been studied, and several Aloe species. Its ecological role and biosynthetic origin in S. flava is speculative. The aim of the current research was to investigate the occurrence of coniine in Sarracenia and Darlingtonia and to identify common constituents of both genera, unique compounds for individual variants and floral scent chemicals. In this comprehensive metabolic profiling study, we looked for compound patterns that are associated with the taxonomy of Sarracenia species. In total, 57 different Sarracenia and D. californica accessions were used for metabolite content screening by gas chromatography-mass spectrometry. The resulting high-dimensional data were studied using a data mining approach. The two genera are characterized by a large number of metabolites and huge chemical diversity between different species. By applying feature selection for clustering and by integrating new biochemical data with existing phylogenetic data, we were able to demonstrate that the chemical composition of the species can be explained by their known classification. Although transcriptome analysis did not reveal a candidate gene for coniine biosynthesis, the use of a sensitive selected ion monitoring method enabled the detection of coniine in eight Sarracenia species, showing that it is more widespread in this genus than previously believed.

Purple pitcher plant (Sarracenia rosea) Dieback and partial community disassembly following experimental storm surge in a coastal pitcher plant bog.

Sea-level rise and frequent intense hurricanes associated with climate change will result in recurrent flooding of inland systems such as Gulf Coastal pitcher plant bogs by storm surges. These surges can transport salt water and sediment to freshwater bogs, greatly affecting their biological integrity. Purple pitcher plants (Sarracenia rosea) are Gulf Coast pitcher plant bog inhabitants that could be at a disadvantage under this scenario because their pitcher morphology may leave them prone to collection of saline water and sediment after a surge. We investigated the effects of storm surge water salinity and sediment type on S. rosea vitality, plant community structure, and bog soil-water conductivity. Plots (containing ≥1 ramet of S. rosea) were experimentally flooded with fresh or saline water crossed with one of three sediment types (local, foreign, or no sediment). There were no treatment effects on soil-water conductivity; nevertheless, direct exposure to saline water resulted in significantly lower S. rosea cover until the following season when a prescribed fire and regional drought contributed to the decline of all the S. rosea to near zero percent cover. There were also significant differences in plant community structure between treatments over time, reflecting how numerous species increased in abundance and a few species decreased in abundance. However, in contrast to S. rosea, most of the other species in the community appeared resilient to the effects of storm surge. Thus, although the community may be somewhat affected by storm surge, those few species that are particularly sensitive to the storm surge disturbance will likely drop out of the community and be replaced by more resilient species. Depending on the longevity of these biological legacies, Gulf Coastal pitcher plant bogs may be incapable of fully recovering if they become exposed to storm surge more frequently due to climate change.

Resolving phylogenetic relationships of the recently radiated carnivorous plant genus Sarracenia using target enrichment.

The North American carnivorous pitcher plant genus Sarracenia (Sarraceniaceae) is a relatively young clade (<3 million years ago) displaying a wide range of morphological diversity in complex trapping structures. This recently radiated group is a promising system to examine the structural evolution and diversification of carnivorous plants; however, little is known regarding evolutionary relationships within the genus. Previous attempts at resolving the phylogeny have been unsuccessful, most likely due to few parsimony-informative sites compounded by incomplete lineage sorting. Here, we applied a target enrichment approach using multiple accessions to assess the relationships of Sarracenia species. This resulted in 199 nuclear genes from 75 accessions covering the putative 8-11 species and 8 subspecies/varieties. In addition, we recovered 42kb of plastome sequence from each accession to estimate a cpDNA-derived phylogeny. Unsurprisingly, the cpDNA had few parsimony-informative sites (0.5%) and provided little information on species relationships. In contrast, use of the targeted nuclear loci in concatenation and coalescent frameworks elucidated many relationships within Sarracenia even with high heterogeneity among gene trees. Results were largely consistent for both concatenation and coalescent approaches. The only major disagreement was with the placement of the purpurea complex. Moreover, results suggest an Appalachian massif biogeographic origin of the genus. Overall, this study highlights the utility of target enrichment using multiple accessions to resolve relationships in recently radiated taxa.

Photodynamic activity of plant extracts from Sarawak, Borneo.

Photodynamic therapy (PDT) is a medical treatment that involves the irradiation of an administered photosensitizing drug with light of a particular wavelength to activate the photosensitizer to kill abnormal cells. To date, only a small number of photosensitizers have been clinically approved for PDT, and researchers continue to look for new molecules that have more desirable properties for clinical applications. Natural products have long been important sources of pharmaceuticals, and there is a great potential for discovery of novel chemotypes from under-explored biodiversities in the world. The objective of this study is to mine the terrestrial plants in Sarawak, Borneo Island, for new photosensitizers for PDT. In a screening program from 2004 to 2008, we prepared and studied 2,400 extracts from 888 plants for their photosensitizing activities. This report details the bioprospecting process, preparation and testing of extracts, analysis of the active samples, fractionation of four samples, and isolation and characterization of photosensitizers.

Phylogeny and biogeography of the carnivorous plant family Sarraceniaceae.

The carnivorous plant family Sarraceniaceae comprises three genera of wetland-inhabiting pitcher plants: Darlingtonia in the northwestern United States, Sarracenia in eastern North America, and Heliamphora in northern South America. Hypotheses concerning the biogeographic history leading to this unusual disjunct distribution are controversial, in part because genus- and species-level phylogenies have not been clearly resolved. Here, we present a robust, species-rich phylogeny of Sarraceniaceae based on seven mitochondrial, nuclear, and plastid loci, which we use to illuminate this family's phylogenetic and biogeographic history. The family and genera are monophyletic: Darlingtonia is sister to a clade consisting of Heliamphora+Sarracenia. Within Sarracenia, two clades were strongly supported: one consisting of S. purpurea, its subspecies, and S. rosea; the other consisting of nine species endemic to the southeastern United States. Divergence time estimates revealed that stem group Sarraceniaceae likely originated in South America 44-53 million years ago (Mya) (highest posterior density [HPD] estimate = 47 Mya). By 25-44 (HPD = 35) Mya, crown-group Sarraceniaceae appears to have been widespread across North and South America, and Darlingtonia (western North America) had diverged from Heliamphora+Sarracenia (eastern North America+South America). This disjunction and apparent range contraction is consistent with late Eocene cooling and aridification, which may have severed the continuity of Sarraceniaceae across much of North America. Sarracenia and Heliamphora subsequently diverged in the late Oligocene, 14-32 (HPD = 23) Mya, perhaps when direct overland continuity between North and South America became reduced. Initial diversification of South American Heliamphora began at least 8 Mya, but diversification of Sarracenia was more recent (2-7, HPD = 4 Mya); the bulk of southeastern United States Sarracenia originated co-incident with Pleistocene glaciation, <3 Mya. Overall, these results suggest climatic change at different temporal and spatial scales in part shaped the distribution and diversity of this carnivorous plant clade.

Deep phylogeographic structure and environmental differentiation in the carnivorous plant Sarracenia alata.

We collected ~29 kb of sequence data using Roche 454 pyrosequencing in order to estimate the timing and pattern of diversification in the carnivorous pitcher plant Sarracenia alata. Utilizing modified protocols for reduced representation library construction, we generated sequence data from 86 individuals across 10 populations from throughout the range of the species. We identified 76 high-quality and high-coverage loci (containing over 500 SNPs) using the bioinformatics pipeline PRGmatic. Results from a Bayesian clustering analysis indicate that populations are highly structured, and are similar in pattern to the topology of a population tree estimated using *BEAST. The pattern of diversification within Sarracenia alata implies that riverine barriers are the primary factor promoting population diversification, with divergence across the Mississippi River occurring more than 60,000 generations before present. Further, significant patterns of niche divergence and the identification of several outlier loci suggest that selection may contribute to population divergence. Our results demonstrate the feasibility of using next-generation sequencing to investigate intraspecific genetic variation in nonmodel species.

The microbial Phyllogeography of the carnivorous plant Sarracenia alata.

Carnivorous pitcher plants host diverse microbial communities. This plant-microbe association provides a unique opportunity to investigate the evolutionary processes that influence the spatial diversity of microbial communities. Using next-generation sequencing of environmental samples, we surveyed microbial communities from 29 pitcher plants (Sarracenia alata) and compare community composition with plant genetic diversity in order to explore the influence of historical processes on the population structure of each lineage. Analyses reveal that there is a core S. alata microbiome, and that it is similar in composition to animal gut microfaunas. The spatial structure of community composition in S. alata (phyllogeography) is congruent at the deepest level with the dominant features of the landscape, including the Mississippi river and the discrete habitat boundaries that the plants occupy. Intriguingly, the microbial community structure reflects the phylogeographic structure of the host plant, suggesting that the phylogenetic structure of bacterial communities and population genetic structure of their host plant are influenced by similar historical processes.

Development of microalgae communities in the Phytotelmata of allochthonous populations of Sarracenia purpurea (Sarraceniaceae).

The phytotelmata of the North American pitcher plant Sarracenia purpurea are colonised by a great variety of aquatic organisms and, thus, provide an ideal model to study trophic interactions in small freshwater ecosystems. Although algae are discussed as a potential food source for predators, little is known about the structure of algae coenoses in pitchers of S. purpurea. This study aims to elucidate temporal shifts in the algae community structure in pitchers of an allochthonous population of S. purpurea in Saxony, Germany. A total of 78 algae taxa was found in the pitchers. Mean algae abundances in new and old pitchers were similar (2.6 x 10(5) and 2.3 x 10(5) algae ml(-1), respectively). Taxa from the orders Chlamydomonadales, Chlorococcales, and Ochromonadales were the primary colonisers. With increasing age of the pitchers the filamentous green algae from the order Klebsormidiales became more abundant. In contrast, pennate diatoms dominated the algae coenoses in the fen. Algae community structure in vase-shaped 50 ml Greiner tubes was similar to those of natural pitchers. Differences in the temporal patterns of algae coenoses in individual pitchers suggested a colonisation of the pitchers by algae via trapped insects, air and rain water rather than via the surrounding fen. Biomass of algae approximated 0.3 mg C ml(-1), which corresponds to 82.8 % of the living biomass (bacteria, heterotrophic nanoflagellates, algae, protozoans and rotifers). Rotifers were abundant in new pitchers; nematodes and mites were seldom found in all pitchers. A similar qualitative and quantitative composition of the aquatic biocoenoses was observed in pitchers of another allochthonous S. purpurea population growing in Blekinge, Sweden. Biomass of algae represented nearly one quarter of the total organic matter content in the pitchers. Thus, nitrogen and phosphorus compounds present in the algae biomass might be used by the carnivorous S. purpurea plant as additional food source in allochthonous populations in Europe lacking top predators.