We investigated how Nepenthes pitchers might act as ecological filters, especially via their manipulation of the abiotic properties of their fluid. In order for Nepenthes species to affect the ability of microbes to establish and persist in their pitchers, they must be able to alter their fluid properties. We demonstrated that the various species in our experiment do in fact alter their fluid properties, including pH modification and the production of viscous and/or colored fluid. The pitchers in our study were all filled with the same pH 6.5 water at the beginning of the experiment and yet after about two weeks of acclimation, they ended with a pH range spanning from ~1–6, and clear differences in fluid viscosity and color. We designate the differences in fluid properties as caused by both biotic (fluid viscosity, color) and abiotic factors (pH); however, the designation is not clear cut. The pH level, like viscosity or fluid color, is largely a function of pitcher physiology, and accordingly shows interspecific variation (Fig. 1A). This level of variation is striking: even with relatively low replication within species, it is clear that species differ in their characteristic pH range, with some species being more stable than others. For example, out of 12 pitchers of N. x “Bill Bailey”, 10 were pH 2 and out of 7 pitchers of N. fusca, 6 were pH 5 (Fig. 1A).

We first tested how Nepenthes species identity shapes variation in the community compositions of organisms housed within pitchers. At a first approximation, the magnitude of the effect of host species identity on community composition is similar to that of pH; however, a post hoc test reveals that with the number of species involved, the differences between any species pair taken in isolation lacks statistical significance. To further probe the role of species identity as a force in shaping microbial assembly, we performed the ANCOM test for the presence of significantly differentially abundant OTUs within N. ramispina (a species with a visibly separated cluster in the ordination for bacteria, Fig. 2) versus all other species. While N. ramispina did contain OTUs that were unique or differentially abundant with respect to all other species pooled, these were OTUs that did not differ across the other species that share a similar pH mean and range (low and narrow, Fig. 1A). Hence these OTUs were likely associated with N. ramispina’s particular pH regulatory properties rather than N. ramispina itself. The fact that physiologically similar Nepenthes species do not contain characteristic and significantly different OTUs further suggests that trait variation rather than species identity per se is the factor that acts as an ecological filter in this study. This supports pH as the primary factor of importance among the traits we measured for community assembly within Nepenthes pitchers.

The influence of pH on both bacteria and eukaryotes is strong, both in terms of community composition and in terms of the dynamics of individual OTUs (as seen in ANCOM results, Fig. 3). Most bacterial OTUs are less abundant in highly acidic fluid, and the overall alpha diversity is lower as well. This speaks to the harshness of low pH conditions, where only a few specialized acidophiles are able to thrive, such as the Acetobacteraceae. Interestingly, species in the Acetobacteraceae, especially of the genus Acidocella, appear to be common associates of Nepenthes, not only in this study, but in wild samples as well (Supplemental Discussion).

For eukaryotes, all OTUs with significant differential abundance at different pH levels were found to decrease with increasing pH. All of these OTUs were assigned as fungus gnats (Insecta: Diptera: Sciaridae) by BLAST. The high numbers of fungus gnat sequences at low pH levels may have multiple non-exclusive explanations: (1) prey capture has been recorded in other species to lower pH by inducing fluid acidification74,75; (2) pitchers belonging to high-acidity species may also be more successful at prey capture, assuming no bias in fungus gnat occurrence throughout the glasshouse; and/or (3) acidic pitchers may also be better at digestion, thus leaving less physical evidence of their prey capture success. In addition to these sources of intra-specific variability, pH differences are largely explained by species differences, as even with prey induction, it has been demonstrated that not all species are capable of achieving the same levels of acidity75. Also, the samples with the greatest number of visible gnats had relatively moderate to high pH (pH 4–6, Fig. 1). Future studies examining how prey abundances in pitchers correlate to 18S rRNA sequence counts could help to clarify this10. The effect of pH on eukaryotes is likely not limited to effects on prey DNA, though, as pH still has a significant effect on eukaryotic community composition after removing fungus gnats from the OTU table. Microbial eukaryotes living symbiotically in the fluid such as fungi, algae, and amoebae likely experience physiological challenges in acidic conditions similar to the bacteria, and/or appear to be similarly affected due to their interactions with the bacteria themselves. Future experimental work can distinguish direct effects of pH on microbial taxa, effects due to interactions between taxa, and effects due to prey.

It was surprising that viscosity, a biotic factor and definitively plant-regulated trait, had only a weak relationship to community structure, with no significant difference in community composition for either bacteria or eukaryotes between species with different fluid viscosities. The only effect we noted was that viscous pitchers had lower alpha diversity for both bacteria and eukaryotes. This might suggest that viscous fluid presents a harsher environment for inquilines, similar to how low pH environments lead to reduced diversity. Notably, 18S extractions from viscous samples were more likely to fail than those from non-viscous samples (Table 2). This was similar to lower 16S PCR amplification success for more acidic fluids (Table 2), mirroring the trend of decreasing bacterial alpha diversity with increasing acidity. However, without qPCR data directly measuring numbers of ribosomal RNA genes, it is not possible to ascertain whether PCR amplification failure can be attributed to reduced microbial abundance or to some form of bias introduced by the extraction process. In any case, viscosity might have a larger impact on individual OTUs than on community composition, as our ANCOM results revealed OTUs with significant differential abundances between viscous and non-viscous fluids. However, it should be noted that we determined viscosity visually and recorded it as a binary trait39; quantitative rheological measurements40 might have revealed continuous variation in viscoelasticity across samples, which may have had more explanatory power. As another caveat, levels of viscosity in this experiment possibly differ from natural conditions, as viscosity can be plastic76,77.

For eukaryotes, but not for bacteria, colorful pitcher fluids had significantly lower alpha diversity than clear fluids, suggesting fluid color to be a more important factor for eukaryotic communities. However, the effects of fluid color on eukaryotic community composition and alpha diversity are not robust, and disappear when fungus gnats are removed from the dataset. Like viscosity, fluid color appears to be more important at the individual OTU level than it is at the community level. Fluid color could be an indication of the production of droserone and 5-O methyl droserone. Past studies have shown that the presence of these compounds results in reddish37,78 or yellowish78 fluid coloration. Droserone and 5-O methyl droserone are anti-fungal agents induced by prey capture, specifically in response to chitin37,78,79. This could explain the higher abundance of fungus gnat DNA in colored samples relative to clear fluid samples, accompanied by a decrease in the relative abundance of certain fungal OTUs (Fig. 4). Without confirmation by chemical analyses like Gas Chromatography-Mass Spectrometry (GCMS) this explanation remains somewhat speculative, but the pattern is suggestive. Pitcher fluid coloration is not a well-documented trait in the literature; to our knowledge, plant-produced colored fluid has only been reported for N. khasiana37,78,79. Our observation of colored fluid in several species is novel, and future work should investigate this this trait, both in the field and in cultivation, as it might impact fungal colonization and survival.

Interspecific differences in pH regulation may be linked to functional/ecological differences between species. Future work should determine what ecological functions link the species that regulate their fluid pH levels in similar ways. In addition to interspecific trait variation, intraspecific variation is characteristic of Nepenthes. Most species produce two distinct pitcher morphs, “lower pitchers” from the terrestrial rosette phase and “upper pitchers” from the aerial climbing phase, occasionally with intermediate morphs produced during the transition between growth phases22. In some species the morphological and ecological differences between lower and upper morphs can be quite extreme, e.g. the insectivorous lower and coprophagous upper pitchers of N. lowii23. Thus, it may be valuable to examine between-morph fluid trait differences within species in future studies. Unfortunately, we were unable to adequately sample both morphs within species in order to meaningfully compare fluid traits between morphs; our species are generally represented by single morphs (Table 1).

In this study, it was not possible to assess the functional significance of differentially abundant microbial OTUs, but these could be probed by future transcriptomic or proteomic work. The observation of certain OTUs frequently occurring in Nepenthes pitchers in both natural and artificial situations could indicate that these particular associations are ecologically significant, so the common Nepenthes symbionts like Acidocella found here merit further research from a functional perspective. Pitchers may also modify other abiotic features of the fluid such as dissolved oxygen levels or temperature, so additional fluid properties should be examined in future work as well.

Our research supports the hypothesis that Nepenthes pitcher plants regulate abiotic factors, potentially as a means of maintaining species-specific microbial associations. This is important in considering the possibility of community codiversification80,81. From the perspective of the host, as long as an abiotic factor is under host control, it functionally becomes an extended phenotype with the same potential for evolution in response to interspecies interactions82 as any other biotic phenotype. However, from the perspective of the microbial symbionts, evolution in response to host conditions becomes much less tight. Microbes that respond to a purely biotic factor, such as secondary metabolites, can be considered to be necessarily linked to the evolution of the host, as the exact biochemical compounds involved are unlikely to be found in other environmental contexts. On the other hand, when microbes respond to an abiotic factor, such as fluid pH, those microbes may have been pre-adapted to live in a wide range of environments that incidentally fit that factor, such as other small aquatic environments. So even if the abiotic factor is a product of host evolution in one context, the symbionts may not have evolved in response to the host. Thus, the evolutionary implications of biotic and abiotic filters can be quite different from the perspective of the symbiont, despite having similar implications from the perspective of the host.