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. 2023 Jul 26;89(7):e0081223.
doi: 10.1128/aem.00812-23. Epub 2023 Jun 20.

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

Affiliations

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

Leonora S Bittleston et al. Appl Environ Microbiol. .

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. 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.

Keywords: carnivorous plant; convergent evolution; microbial ecology; nitrogen fixation; symbiosis; trade-off.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

FIG 1
FIG 1
The acidity (pH) of pitcher fluid and nifH presence in Nepenthes species, with representative photographs of each species. (A) Pitcher fluid pH in the following three Singaporean species: N. gracilis, N. rafflesiana, and N. ampullaria. (B) PICRUSt-predicted relative abundance of nifH from amplicon sequencing of Singaporean species. (C) Proportion of samples with nifH as assayed by PCR in Singaporean species. The number of samples with nifH over total number of samples is shown as text above each bar. (D) Pitcher fluid pH in the following four Bornean species: N. stenophylla, N. veitchii, N. reinwardtiana, and N. hirsuta. (E) Normalized nifH relative abundance from the metagenomes of the Bornean species. Photographs by L.S. Bittleston.
FIG 2
FIG 2
The presence of nifH strongly correlates with pitcher fluid pH across our three methods. (A) PICRUSt-predicted relative abundance of nifH from amplicon sequencing of Singaporean species by pitcher fluid pH. (B) Effect plot from model lme2 of data from panel A. (C) Counts of samples from Singaporean species with nifH as assessed by PCR, organized by pH level. (D) Effect plot from model glmer2 using PCR data. (E) Normalized nifH relative abundance from Bornean Nepenthes species by pitcher fluid pH. (F) Effect plot from model lm1 using data from panel E.
FIG 3
FIG 3
Bacterial taxa associated with increased nifH abundance. (A) A stacked bar plot showing the relative abundance of the top 12 bacterial families, organized from left to right by increasing predicted nifH abundance. Legend is shown below. (B) Top 5 bacterial ASVs with higher relative abundance in samples with positive nifH PCR results. These ASVs are significantly differentially abundant as measured by ANCOM-II. Their taxonomic assignments are listed in the legend at the genus and family levels. Family names followed by stars are known to contain nitrogen-fixing bacteria.

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