TRPA5 encodes a thermosensitive ankyrin ion channel receptor in a triatomine insect.

As ectotherms, insects need heat-sensitive receptors to monitor environmental temperatures and facilitate thermoregulation. We show that TRPA5, a class of ankyrin transient receptor potential (TRP) channels absent in dipteran genomes, may function as insect heat receptors. In the triatomine bug Rhodnius prolixus (order: Hemiptera), a vector of Chagas disease, the channel RpTRPA5B displays a uniquely high thermosensitivity, with biophysical determinants including a large channel activation enthalpy change (72 kcal/mol), a high temperature coefficient (Q10 = 25), and in vitro temperature-induced currents from 53°C to 68°C (T0.5 = 58.6°C), similar to noxious TRPV receptors in mammals. Monomeric and tetrameric ion channel structure predictions show reliable parallels with fruit fly dTRPA1, with structural uniqueness in ankyrin repeat domains, the channel selectivity filter, and potential TRP functional modulator regions. Overall, the finding of a member of TRPA5 as a temperature-activated receptor illustrates the diversity of insect molecular heat detectors.

Comprehensive phylogeny of Pieridae butterflies reveals strong correlation between diversification and temperature.

Temperature is thought to be a key factor influencing global species richness patterns. We investigate the link between temperature and diversification in the butterfly family Pieridae by combining next generation DNA sequences and published molecular data with fine-grained distribution data. We sampled nearly 600 pierid butterfly species to infer the most comprehensive molecular phylogeny of the family and curated a distribution dataset of more than 800,000 occurrences. We found strong evidence that species in environments with more stable daily temperatures or cooler maximum temperatures in the warm seasons have higher speciation rates. Furthermore, speciation and extinction rates decreased in tandem with global temperatures through geological time, resulting in a constant net diversification.

To and fro in the archipelago: Repeated inter-island dispersal and New Guinea's orogeny affect diversification of Delias, the world's largest butterfly genus.

The world's largest butterfly genus Delias, commonly known as Jezebels, comprises ca. 251 species found throughout Asia, Australia, and Melanesia. Most species are endemic to islands in the Indo-Australian Archipelago or to New Guinea and nearby islands in Melanesia, and many species are restricted to montane habitats over 1200 m. We inferred an extensively sampled and well-supported molecular phylogeny of the group to better understand the spatial and temporal dimensions of its diversification. The remarkable diversity of Delias evolved in just ca. 15-16 Myr (crown age). The most recent common ancestor of a clade with most of the species dispersed out of New Guinea ca. 14 Mya, but at least six subsequently diverging lineages dispersed back to the island. Diversification was associated with frequent dispersal of lineages among the islands of the Indo-Australian Archipelago, and the divergence of sister taxa on a single landmass was rare and occurred only on the largest islands, most notably on New Guinea. We conclude that frequent inter-island dispersal during the Neogene-likely facilitated by frequent sea level change-sparked much diversification during that period. Many extant New Guinea lineages started diversifying 5 Mya, suggesting that orogeny facilitated their diversification. Our results largely agree with the most recently proposed species group classification system, and we use our large taxon sample to extend this system to all described species. Finally, we summarize recent insights to speculate how wing pattern evolution, mimicry, and sexual selection might also contribute to these butterflies' rapid speciation and diversification.

Gut microbiomes of cycad-feeding insects tolerant to ß-methylamino-L-alanine (BMAA) are rich in siderophore biosynthesis.

Ingestion of the cycad toxins ß-methylamino-L-alanine (BMAA) and azoxyglycosides is harmful to diverse organisms. However, some insects are specialized to feed on toxin-rich cycads with apparent immunity. Some cycad-feeding insects possess a common set of gut bacteria, which might play a role in detoxifying cycad toxins. Here, we investigated the composition of gut microbiota from a worldwide sample of cycadivorous insects and characterized the biosynthetic potential of selected bacteria. Cycadivorous insects shared a core gut microbiome consisting of six bacterial taxa, mainly belonging to the Proteobacteria, which we were able to isolate. To further investigate selected taxa from diverging lineages, we performed shotgun metagenomic sequencing of co-cultured bacterial sub-communities. We characterized the biosynthetic potential of four bacteria from Serratia, Pantoea, and two different Stenotrophomonas lineages, and discovered a suite of biosynthetic gene clusters notably rich in siderophores. Siderophore semi-untargeted metabolomics revealed a broad range of chemically related yet diverse iron-chelating metabolites, including desferrioxamine B, suggesting the occurrence of an unprecedented desferrioxamine-like biosynthetic pathway that remains to be identified. These results provide a foundation for future investigations into how cycadivorous insects tolerate diets rich in azoxyglycosides, BMAA, and other cycad toxins, including a possible role for bacterial siderophores.

Endophytic ancestors of modern leaf miners may have evolved in the Late Carboniferous.

Endophytic feeding behaviors, including stem borings and galling, have been observed in the fossil record from as early as the Devonian and involve the consumption of a variety of plant (and fungal) tissues. Historically, the exploitation of internal stem tissues through galling has been well documented as emerging during the Pennsylvanian (c. 323-299 million years ago (Ma)), replaced during the Permian by galling of foliar tissues. However, leaf mining, a foliar endophytic behavior that today is exhibited exclusively by members of the four hyperdiverse holometabolous insect orders, has been more sparsely documented, with confirmed examples dating back only to the Early Triassic (c. 252-250 Ma). Here, we describe a trace fossil on seed-fern foliage from the Rhode Island Formation of Massachusetts, USA, representing the earliest indication of a general, endophytic type of feeding damage and dating from the Middle Pennsylvanian (c. 312 Ma). Although lacking the full features of Mesozoic leaf mines, this specimen provides evidence of how endophytic mining behavior may have originated. It sheds light on the evolutionary transition to true foliar endophagy, contributes to our understanding of the behaviors of early holometabolous insects, and enhances our knowledge of macroevolutionary patterns of plant-insect interactions.

Carnivorous Nepenthes Pitchers with Less Acidic Fluid House Nitrogen-Fixing Bacteria.

Carnivorous pitcher plants are uniquely adapted to nitrogen limitation, using pitfall traps to acquire nutrients from insect prey. Pitcher plants in the genus Sarracenia may also use nitrogen fixed by bacteria inhabiting the aquatic microcosms of their pitchers. Here, we investigated whether species of a convergently evolved pitcher plant genus, Nepenthes, might also use bacterial nitrogen fixation as an alternative strategy for nitrogen capture. First, we constructed predicted metagenomes of pitcher organisms from three species of Singaporean Nepenthes using 16S rRNA sequence data and correlated predicted nifH abundances with metadata. Second, we used gene-specific primers to amplify and quantify the presence or absence of nifH directly from 102 environmental samples and identified potential diazotrophs with significant differential abundance in samples that also had positive nifH PCR tests. Third, we analyzed nifH in eight shotgun metagenomes from four additional Bornean Nepenthes species. Finally, we conducted an acetylene reduction assay using greenhouse-grown Nepenthes pitcher fluids to confirm nitrogen fixation is indeed possible within the pitcher habitat. Results show active acetylene reduction can occur in Nepenthes pitcher fluid. Variation in nifH from wild samples correlates with Nepenthes host species identity and pitcher fluid acidity. Nitrogen-fixing bacteria are associated with more neutral fluid pH, while endogenous Nepenthes digestive enzymes are most active at low fluid pH. We hypothesize Nepenthes species experience a trade-off in nitrogen acquisition; when fluids are acidic, nitrogen is primarily acquired via plant enzymatic degradation of insects, but when fluids are neutral, Nepenthes plants take up more nitrogen via bacterial nitrogen fixation. IMPORTANCE Plants use different strategies to obtain the nutrients that they need to grow. Some plants access their nitrogen directly from the soil, while others rely on microbes to access the nitrogen for them. Carnivorous pitcher plants generally trap and digest insect prey, using plant-derived enzymes to break down insect proteins and generate a large portion of the nitrogen that they subsequently absorb. In this study, we present results suggesting that bacteria living in the fluids formed by Nepenthes pitcher plants can fix nitrogen directly from the atmosphere, providing an alternative pathway for plants to access nitrogen. These nitrogen-fixing bacteria are only likely to be present when pitcher plant fluids are not strongly acidic. Interestingly, the plant's enzymes are known to be more active under strongly acidic conditions. We propose a potential trade-off where pitcher plants sometimes access nitrogen using their own enzymes to digest prey and at other times take advantage of bacterial nitrogen fixation.

Rapid radiation of ant parasitic butterflies during the Miocene aridification of Africa.

Africa has undergone a progressive aridification during the last 20 My that presumably impacted organisms and fostered the evolution of life history adaptations. We test the hypothesis that shift to living in ant nests and feeding on ant brood by larvae of phyto-predaceous Lepidochrysops butterflies was an adaptive response to the aridification of Africa that facilitated the subsequent radiation of butterflies in this genus. Using anchored hybrid enrichment we constructed a time-calibrated phylogeny for Lepidochrysops and its closest, non-parasitic relatives in the Euchrysops section (Poloyommatini). We estimated ancestral areas across the phylogeny with process-based biogeographical models and diversification rates relying on time-variable and clade-heterogeneous birth-death models. The Euchrysops section originated with the emerging Miombo woodlands about 22 million years ago (Mya) and spread to drier biomes as they became available in the late Miocene. The diversification of the non-parasitic lineages decreased as aridification intensified around 10 Mya, culminating in diversity decline. In contrast, the diversification of the phyto-predaceous Lepidochrysops lineage proceeded rapidly from about 6.5 Mya when this unusual life history likely first evolved. The Miombo woodlands were the cradle for diversification of the Euchrysops section, and our findings are consistent with the hypothesis that aridification during the Miocene selected for a phyto-predaceous life history in species of Lepidochrysops, with ant nests likely providing caterpillars a safe refuge from fire and a source of food when vegetation was scarce.

The Afrotropical breeding grounds of the Palearctic-African migratory painted lady butterflies (Vanessa cardui).

Migratory insects are key players in ecosystem functioning and services, but their spatiotemporal distributions are typically poorly known. Ecological niche modeling (ENM) may be used to predict species seasonal distributions, but the resulting hypotheses should eventually be validated by field data. The painted lady butterfly (Vanessa cardui) performs multigenerational migrations between Europe and Africa and has become a model species for insect movement ecology. While the annual migration cycle of this species is well understood for Europe and northernmost Africa, it is still unknown where most individuals spend the winter. Through ENM, we previously predicted suitable breeding grounds in the subhumid regions near the tropics between November and February. In this work, we assess the suitability of these predictions through i) extensive field surveys and ii) two-year monitoring in six countries: a large-scale monitoring scheme to study butterfly migration in Africa. We document new breeding locations, year-round phenological information, and hostplant use. Field observations were nearly always predicted with high probability by the previous ENM, and monitoring demonstrated the influence of the precipitation seasonality regime on migratory phenology. Using the updated dataset, we built a refined ENM for the Palearctic-African range of V. cardui. We confirm the relevance of the Afrotropical region and document the missing natural history pieces of the longest migratory cycle described in butterflies.

Cycad phylogeny predicts host plant use of Eumaeus butterflies.

Eumaeus butterflies are obligate herbivores of Zamia, the most diverse neotropical genus of cycads. Eumaeus-Zamia interactions have been characterized mainly for species distributed in North and Central America. However, larval host plant use by the southern Eumaeus clade remains largely unknown, precluding a comprehensive study of co-evolution between the genera. Here, we combine fieldwork with museum and literature surveys to expand herbivory records for Eumaeus from 21 to 38 Zamia species. We inferred a time-calibrated phylogeny of Eumaeus to test for distinct macroevolutionary scenarios of larval host plant conservatism and co-evolution. We found a remarkable coincidence between Eumaeus and Zamia diversification, with the butterfly stem group diverging at the same time as the most recent radiation of Zamia in the Miocene. Cophylogenetic reconciliation analyses show a strong cophylogenetic signal between cycads and their butterfly herbivores. Bipartite model-based approaches indicate that this is because closely related Zamia species are used by the same Eumaeus species, suggesting larval host plant resource tracking by the butterfly herbivores. Our results highlight a case of tight evolution between Eumaeus butterflies and cycads, pointing to the generality of correlated evolution and phylogenetic tracking in plant-herbivore interactions across seed plants.

A hypothesis for robust polarization vision: an example from the Australian imperial blue butterfly, Jalmenus evagoras.

The Australian lycaenid butterfly Jalmenus evagoras has iridescent wings that are sexually dimorphic, spectrally and in their degree of polarization, suggesting that these properties are likely to be important in mate recognition. We first describe the results of a field experiment showing that free-flying individuals of J. evagoras discriminate between visual stimuli that vary in polarization content in blue wavelengths but not in others. We then present detailed reflectance spectrophotometry measurements of the polarization content of male and female wings, showing that female wings exhibit blue-shifted reflectance, with a lower degree of polarization relative to male wings. Finally, we describe a novel method for measuring alignment of ommatidial arrays: by measuring variation of depolarized eyeshine intensity from patches of ommatidia as a function of eye rotation, we show that (a) individual rhabdoms contain mutually perpendicular microvilli; (b) many rhabdoms in the array have their microvilli misaligned with respect to neighboring rhabdoms by as much as 45 deg; and (c) the misaligned ommatidia are useful for robust polarization detection. By mapping the distribution of the ommatidial misalignments in eye patches of J. evagoras, we show that males and females exhibit differences in the extent to which ommatidia are aligned. Both the number of misaligned ommatidia suitable for robust polarization detection and the number of aligned ommatidia suitable for edge detection vary with respect to both sex and eye patch elevation. Thus, J. evagoras exhibits finely tuned ommatidial arrays suitable for perception of polarized signals, likely to match sex-specific life history differences in the utility of polarized signals.

Complex hemolymph circulation patterns in grasshopper wings.

An insect's living systems-circulation, respiration, and a branching nervous system-extend from the body into the wing. Wing hemolymph circulation is critical for hydrating tissues and supplying nutrients to living systems such as sensory organs across the wing. Despite the critical role of hemolymph circulation in maintaining healthy wing function, wings are often considered "lifeless" cuticle, and flows remain largely unquantified. High-speed fluorescent microscopy and particle tracking of hemolymph in the wings and body of the grasshopper Schistocerca americana revealed dynamic flow in every vein of the fore- and hindwings. The global system forms a circuit, but local flow behavior is complex, exhibiting three distinct types: pulsatile, aperiodic, and "leaky" flow. Thoracic wing hearts pull hemolymph from the wing at slower frequencies than the dorsal vessel; however, the velocity of returning hemolymph (in the hindwing) is faster than in that of the dorsal vessel. To characterize the wing's internal flow mechanics, we mapped dimensionless flow parameters across the wings, revealing viscous flow regimes. Wings sustain ecologically important insect behaviors such as pollination and migration. Analysis of the wing circulatory system provides a template for future studies investigating the critical hemodynamics necessary to sustaining wing health and insect flight.

Herbarium specimens reveal herbivory patterns across the genus Cucurbita.

PREMISE: Quantifying how closely related plant species differ in susceptibility to insect herbivory is important for understanding the variation in evolutionary pressures on plant functional traits. However, empirically measuring in situ variation in herbivory spanning the geographic range of a plant-insect complex is logistically difficult. Recently, new methods have been developed using herbarium specimens to investigate patterns in plant-insect symbioses across large geographic scales. Such investigations provide insights into how accelerated anthropogenic changes may impact plant-insect interactions that are of ecological or agricultural importance. METHODS: Here, we analyze 274 pressed herbarium samples to investigate variation in herbivory damage in 13 different species of the economically important plant genus Cucurbita (Cucurbitaceae). This collection is composed of specimens of wild, undomesticated Cucurbita that were collected from across their native range, and Cucurbita cultivars collected from both within their native range and from locations where they have been introduced for agriculture in temperate North America. RESULTS: Herbivory is common on individuals of all Cucurbita species collected throughout their geographic ranges. However, estimates of herbivory varied considerably among individuals, with mesophytic species accruing more insect damage than xerophytic species, and wild specimens having more herbivory than specimens collected from human-managed habitats. CONCLUSIONS: Our study suggests that long-term evolutionary changes in habitat from xeric to mesic climates and wild to human-managed habitats may mediate the levels of herbivory pressure from coevolved herbivores. Future investigations into the potential factors that contribute to herbivory may inform the management of domesticated crop plants and their insect herbivores.

Fine-scale genome-wide signature of Pleistocene glaciation in Thitarodes moths (Lepidoptera: Hepialidae), host of Ophiocordyceps fungus in the Hengduan Mountains.

The Hengduan Mountains region is a biodiversity hotspot known for its topologically complex, deep valleys and high mountains. While landscape and glacial refugia have been evoked to explain patterns of interspecies divergence, the accumulation of intra-species (i.e., population level) genetic divergence across the mountain-valley landscape in this region has received less attention. We used genome-wide restriction site-associated DNA sequencing (RADseq) to reveal signatures of Pleistocene glaciation in populations of Thitarodes shambalaensis (Lepidoptera: Hepialidae), the host moth of parasitic Ophiocordyceps sinensis (Hypocreales: Ophiocordycipitaceae) or "caterpillar fungus" endemic to the glacier of eastern Mt. Gongga. We used moraine history along the glacier valleys to model the distribution and environmental barriers to gene flow across populations of T. shambalaensis. We found that moth populations separated by less than 10 km exhibited valley-based population genetic clustering and isolation-by-distance (IBD), while gene flow among populations was best explained by models using information about their distributions at the local last glacial maximum (LGML , 58 kya), not their contemporary distribution. Maximum likelihood lineage history among populations, and among subpopulations as little as 500 m apart, recapitulated glaciation history across the landscape. We also found signals of isolated population expansion following the retreat of LGML glaciers. These results reveal the fine-scale, long-term historical influence of landscape and glaciation on the genetic structuring of populations of an endangered and economically important insect species. Similar mechanisms, given enough time and continued isolation, could explain the contribution of glacier refugia to the generation of species diversity among the Hengduan Mountains.

Migratory behaviour is positively associated with genetic diversity in butterflies.

Migration is typically associated with risk and uncertainty at the population level, but little is known about its cost-benefit trade-offs at the species level. Migratory insects in particular often exhibit strong demographic fluctuations due to local bottlenecks and outbreaks. Here, we use genomic data to investigate levels of heterozygosity and long-term population size dynamics in migratory insects, as an alternative to classical local and short-term approaches such as regional field monitoring. We analyse whole-genome sequences from 97 Lepidoptera species and show that individuals of migratory species have significantly higher levels of genome-wide heterozygosity, a proxy for effective population size, than do nonmigratory species. Also, we contribute whole-genome data for one of the most emblematic insect migratory species, the painted lady butterfly (Vanessa cardui), sampled across its worldwide distributional range. This species exhibits one of the highest levels of genomic heterozygosity described in Lepidoptera (2.95 ± 0.15%). Coalescent modelling (PSMC) shows historical demographic stability in V. cardui, and high effective population size estimates of 2-20 million individuals 10,000 years ago. The study reveals that the high risks associated with migration and local environmental fluctuations do not seem to decrease overall genetic diversity and demographic stability in migratory Lepidoptera. We propose a "compensatory" demographic model for migratory r-strategist organisms in which local bottlenecks are counterbalanced by reproductive success elsewhere within their typically large distributional ranges. Our findings highlight that the boundaries of populations are substantially different for sedentary and migratory insects, and that, in the latter, local and even regional field monitoring results may not reflect whole population dynamics. Genomic diversity patterns may elucidate key aspects of an insect's migratory nature and population dynamics at large spatiotemporal scales.

A high-throughput multispectral imaging system for museum specimens.

We present an economical imaging system with integrated hardware and software to capture multispectral images of Lepidoptera with high efficiency. This method facilitates the comparison of colors and shapes among species at fine and broad taxonomic scales and may be adapted for other insect orders with greater three-dimensionality. Our system can image both the dorsal and ventral sides of pinned specimens. Together with our processing pipeline, the descriptive data can be used to systematically investigate multispectral colors and shapes based on full-wing reconstruction and a universally applicable ground plan that objectively quantifies wing patterns for species with different wing shapes (including tails) and venation systems. Basic morphological measurements, such as body length, thorax width, and antenna size are automatically generated. This system can increase exponentially the amount and quality of trait data extracted from museum specimens.

Molecular advances to study the function, evolution and spectral tuning of arthropod visual opsins.

Visual opsins of vertebrates and invertebrates diversified independently and converged to detect ultraviolet to long wavelengths (LW) of green or red light. In both groups, colour vision largely derives from opsin number, expression patterns and changes in amino acids interacting with the chromophore. Functional insights regarding invertebrate opsin evolution have lagged behind those for vertebrates because of the disparity in genomic resources and the lack of robust in vitro systems to characterize spectral sensitivities. Here, we review bioinformatic approaches to identify and model functional variation in opsins as well as recently developed assays to measure spectral phenotypes. In particular, we discuss how transgenic lines, cAMP-spectroscopy and sensitive heterologous expression platforms are starting to decouple genotype-phenotype relationships of LW opsins to complement the classical physiological-behavioural-phylogenetic toolbox of invertebrate visual sensory studies. We illustrate the use of one heterologous method by characterizing novel LW Gq opsins from 10 species, including diurnal and nocturnal Lepidoptera, a terrestrial dragonfly and an aquatic crustacean, expressing them in HEK293T cells, and showing that their maximum absorbance spectra (λmax) range from 518 to 611 nm. We discuss the advantages of molecular approaches for arthropods with complications such as restricted availability, lateral filters, specialized photochemistry and/or electrophysiological constraints. This article is part of the theme issue 'Understanding colour vision: molecular, physiological, neuronal and behavioural studies in arthropods'.

LepTraits 1.0 A globally comprehensive dataset of butterfly traits.

Here, we present the largest, global dataset of Lepidopteran traits, focusing initially on butterflies (ca. 12,500 species records). These traits are derived from field guides, taxonomic treatments, and other literature resources. We present traits on wing size, phenology,voltinism, diapause/overwintering stage, hostplant associations, and habitat affinities (canopy, edge, moisture, and disturbance). This dataset will facilitate comparative research on butterfly ecology and evolution and our goal is to inspire future research collaboration and the continued development of this dataset.

Characterization and Comparison of Convergence Among Cephalotus follicularis Pitcher Plant-Associated Communities With Those of Nepenthes and Sarracenia Found Worldwide.

The Albany pitcher plant, Cephalotus follicularis, has evolved cup-shaped leaves and a carnivorous habit completely independently from other lineages of pitcher plants. It is the only species in the family Cephalotaceae and is restricted to a small region of Western Australia. Here, we used metabarcoding to characterize the bacterial and eukaryotic communities living in C. follicularis pitchers at two different sites. Bacterial and eukaryotic communities were correlated in both richness and composition; however, the factors associated with richness were not the same across bacteria and eukaryotes, with bacterial richness differing with fluid color, and eukaryotic richness differing with the concentration of DNA extracted from the fluid, a measure roughly related to biomass. For turnover in composition, the variation in both bacterial and eukaryotic communities primarily differed with fluid acidity, fluid color, and sampling site. We compared C. follicularis-associated community diversity with that of Australian Nepenthes mirabilis, as well as a global comparison of Southeast Asian Nepenthes and North American Sarracenia. Our results showed similarity in richness with communities from other pitcher plants, and specific bacterial taxa shared among all three independent lineages of pitcher plants. Overall, we saw convergence in richness and particular clades colonizing pitcher plants around the world, suggesting that these highly specialized habitats select for certain numbers and types of inhabitants.

Profiling, monitoring and conserving caterpillar fungus in the Himalayan region using anchored hybrid enrichment markers.

The collection of caterpillar fungus accounts for 50-70% of the household income of thousands of Himalayan communities and has an estimated market value of $5-11 billion across Asia. However, Himalayan collectors are at multiple economic disadvantages compared with collectors on the Tibetan Plateau because their product is not legally recognized. Using a customized hybrid-enrichment probe set and market-grade caterpillar fungus (with samples up to 30 years old) from 94 production zones across Asia, we uncovered clear geography-based signatures of historical dispersal and significant isolation-by-distance among caterpillar fungus hosts. This high-throughput approach can readily distinguish samples from major production zones with definitive geographical resolution, especially for samples from the Himalayan region that form monophyletic clades in our analysis. Based on these results, we propose a two-step procedure to help local communities authenticate their produce and improve this multi-national trade-route without creating opportunities for illegal exports and other forms of economic exploitation. We argue that policymakers and conservation practitioners must encourage the fair trade of caterpillar fungus in addition to sustainable harvesting to support a trans-boundary conservation effort that is much needed for this natural commodity in the Himalayan region.

Sarracenia pitcher plant-associated microbial communities differ primarily by host species across a longitudinal gradient.

Plant-associated microbial communities can profoundly affect plant health and success, and research is still uncovering factors driving the assembly of these communities. Here, we examine how geography versus host species affects microbial community structure and differential abundances of individual taxa. We use metabarcoding to characterize the bacteria and eukaryotes associated with five, often co-occurring species of Sarracenia pitcher plants (Sarraceniaceae) and three natural hybrids along the longitudinal gradient of the U.S. Gulf Coast, as well as samples from S. purpurea in Massachusetts. To tease apart the effects of geography versus host species, we focus first on sites with co-occurring species and then on species located across different sites. Our analyses show that bacterial and eukaryotic community structures are clearly and consistently influenced by host species identity, with geographic factors also playing a role. Naturally occurring hybrids appear to also host unique communities, which are in some ways intermediate between their parent species. We see significant effects of geography (site and longitude), but these generally explain less of the variation among pitcher communities. Overall, in Sarracenia pitchers, host plant phenotype significantly affects the pitcher microbiomes and other associated organisms.