Replication Data and Code for: "Carnivorous Nepenthes pitchers with less acidic fluid house nitrogen-fixing bacteria"

Abstract: Background. Carnivorous pitcher plants are well known for a unique adaptation to nitrogen limitation: pitchers are pitfall traps used to capture and digest insect prey, and acquire nutrients. Pitcher plants in the genus Sarracenia appear to also use nitrogen fixed by bacteria inhabiting the aquatic microcosms within pitchers. To investigate whether species of a convergently evolved pitcher plant genus, Nepenthes, also use this alternative strategy for nitrogen capture, we measured the presence and relative abundance of an essential nitrogen fixation gene, nifH, in Nepenthes microbiome samples taken from pitcher fluids, and measured correlations between nifH abundance and pitcher characteristics. Methods. We used four approaches to investigate nitrogen fixation in pitcher fluid samples. First, we used 16S rRNA sequence data from the fluids of three species of Singaporean Nepenthes to construct a PICRUST-predicted metagenome of the pitcher plant microbes and correlate predicted nifH abundances with sample metadata. Second, we used gene-specific primers to amplify and quantify the presence or absence of nifH directly from our environmental samples. Third, we complemented our results from Singapore by analyzing nifH relative abundances in eight shotgun metagenomes from four additional Nepenthes species from Malaysian Borneo. Finally, we conducted an acetylene reduction assay using the pitcher fluids of Nepenthes housed in a greenhouse to prove nitrogen fixation is possible within the Nepenthes pitcher habitat. Results. The gene nifH frequently appears in the microbial communities of Nepenthes pitchers, and variation in nifH presence correlates with host plant species identity and particularly strongly with pitcher fluid acidity. The genes are more commonly found in more neutral fluids. We also present evidence for active acetylene reduction in greenhouse Nepenthes pitcher fluid. Discussion. In the aggregate, data suggest Nepenthes pitcher plants have the potential to access nitrogen via bacterial nitrogen fixation in addition to accessing nitrogen from the digestion of insect prey. Moreover, the proliferation of nitrogen-fixing bacteria appears to be associated with physiological features of the pitchers, and particularly fluid pH. Endogenous Nepenthes digestive enzymes are most active at low pH levels. Thus, we hypothesize Nepenthes species experience a tradeoff in nitrogen acquisition: when fluids are acidic, nitrogen is primarily acquired via plant enzymatic degradation of insects, but when fluids are neutral, Nepenthes plants may take up more nitrogen via bacterial nitrogen fixation in pitcher microcosms.