Abstract:
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.
Publisher's Version
