Results
Environmental differences among HP and LP environments varied mostly as
expected based on what is already known about Trinidadian streams.
Namely, lower elevation, high-predation (HP) sites had higher stream
water temperature (HP: 24.87°C, ±0.08 vs. LP: 22.82°C, ±0.03) and pH
(HP: 7.5 ±0.03, LP: 6.7 ±0.03) compared to low-predation sites
(p<0.01 for both comparisons), which include LP native, old,
and new introductions (see Fig. S1 for site-level stream data). Fish
morphology also fit expectations: fish in LP environments had longer gut
lengths relative to body size (Fig. 2A) and were larger overall (Fig.
2B). Site-specific variation overwhelmed significant effects of site
category on gut content (as measured by percent invertebrates, Fig. 2C),
and fish condition (weight/length, Fig. 2D). If anything, at the time of
sampling, LP fish were eating more invertebrates (Fig. S1D), contrary to
expectations. HP fish had diets richer in detritus, as well as higher
relative abundance of cyanobacteria (according to 16S) in their guts
(p<0.001, Fig. S2, Table S4).
After pre-filtering steps (Table S1), we identified 12,068 bacterial
OTUs in 485 guppy gut microbiome samples. In general, the posterior
portion of the gut harbored bacterial communities that were more diverse
(Fig. S3) and compositionally distinct from the anterior portion
(PerMANOVA p<0.001). Across site categories, however, gut
position explained less variation than other factors (Table 1), and
differences in diversity and composition across site categories were
similar in anterior and posterior communities (e.g. Fig. S2, S4, S5).
Thus, we focused remaining analyses on the posterior gut microbiome,
which may be less transient than the anterior gut community.
Site category had a significant effect on gut microbiome community
composition (Fig. 3A; PerMANOVA p=0.001). The presence or absence of
bacterial taxa in the gut was largely driven by stream water temperature
(which correlated closely to stream pH) and gut length (Fig. 3 inset,
Table S2). When HP source populations were removed and only LP
environments were considered (LP native, old, and recent introduction
sites), diet was a larger contributor to microbiome composition (Fig.
3B), but not in the dimension that corresponded with site category
(arrows in Fig. 3B), for which water temperature was more important.
Diet also had elevated importance when changes in relative abundances of
taxa were accounted for (Fig. S6), compared to presence-absence alone,
as also found by Sullam et al. (2015).
There were larger differences between gut microbiomes of HP source
populations and those in LP environments (i.e. LP native and
introduction sites) than among LP sites. When we compared microbiomes in
LP environments to microbiomes of their respective HP source
populations, microbiomes of introductions (e.g. Intro 2008, 2009, 1957,
etc.) more closely resembled each other, and LP native populations, than
their respective HP source site (Fig. 4, distance from 0 in y-axis). Gut
microbiomes in introduced populations sometimes, but not always became
more similar microbiomes of reference LP native populations (Fig. 4 does
not convey this, but see Fig. S7). In two of four drainages, gut
microbiome composition of introduced populations remained significantly
different from those of LP native sites (Fig. S7, Table S3), with no
effect of time since introduction.
Variation in microbiome composition across individuals was highest in HP
sites (Fig. S8, betadispersion test p<0.01), and lower in the
introductions than native sites, even though we might expect it to be
higher in more genetically varied populations. Higher betadispersion was
not driven by a greater among-individual diet diversity
(p>0.05 for correlation, data not shown). While
betadispersion differences can influence betadiversity tests (i.e.
PerMANOVA), these differences were small so not likely to be the sole
reason we observed significant differences in composition (Anderson &
Walsh 2013).
Mean number of species within individual guts also differed across site
categories. Gut bacterial richness was significantly lower in native
populations (HP source and LP native) and higher in introductions (Fig.
5A), in both anterior and posterior gut positions (Fig. S4), while
community evenness and alpha diversity were less responsive (Fig. S4).
Richness was positively correlated to relative gut length
(r2=0.43, p<0.001), which may explain
previously-observed positive correlations between fish length and
microbiome diversity (Bolnick et al. 2014a; Forberg et al.2016), and was not explained by higher diet diversity (Bolnick et
al. 2014a).
Gut communities were dominated by Proteobacteria, Actinomycetes, and
Firmicutes (Fig. S2), similar to other fish microbiomes (Sullam et
al. 2012), and previous dominant phyla in guppies at this site (Sullamet al. 2015). Changes in the relative abundances of the most
dominant taxa (Table S4) largely drove trends in weighted Unifrac
betadiversity. Five of the 10 most abundant taxa across the entire
dataset were from the order Rhizobiales, a genus able to fix nitrogen to
create nutrients. While we did not measure fitness, we did observe
patterns that suggest this additional source of nutrients could have
increased fitness. For instance, the significantly more abundant
Rhizobiales species in El Cedro (Fig. S9) could explain why these fish
were in better condition than their LP native counterparts, despite a
lower quality diet (Fig. S1D,E). The total Rhizobiales abundance (Fig.
5B, S9), as well as the abundances of these highly abundant taxa (Fig.
5C), tended to be highest in the old introduction streams, and lowest in
HP source streams.