Carnivorous 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 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, , 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 using 16S rRNA sequence data and correlated predicted abundances with metadata. Second, we used gene-specific primers to amplify and quantify the presence or absence of directly from 102 environmental samples and identified potential diazotrophs with significant differential abundance in samples that also had positive PCR tests. Third, we analyzed in eight shotgun metagenomes from four additional Bornean species. Finally, we conducted an acetylene reduction assay using greenhouse-grown pitcher fluids to confirm nitrogen fixation is indeed possible within the pitcher habitat. Results show active acetylene reduction can occur in pitcher fluid. Variation in from wild samples correlates with host species identity and pitcher fluid acidity. Nitrogen-fixing bacteria are associated with more neutral fluid pH, while endogenous digestive enzymes are most active at low fluid pH. We hypothesize 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, plants take up more nitrogen via bacterial nitrogen fixation. 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 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.