Compositional changes
We next explored if the increased gas production could be explained by compositional differences resulting from the addition of the pre-adapted inocula. At the end of the experiment, community composition had changed in parallel across both the control and adaptation treatments (and the pre-adaptation source communities for the adaptation treatment), indicative of strong selection associated with the transition to a novel environment. There was a 60% drop in the mean number of taxa detected per community, compared to the ancestral community, but we did not find a significant difference in diversity loss across treatments (one-way ANOVA on ASV reduction from the ancestral sample; F(2,32)=2.93, P value = 0.07; Figure 3A). In particular, there were reductions in the abundance of taxa belonging to the Firmicutes, Proteobacteria, Spirochaetes and Tenericutes.
Despite these parallel changes, treatments differed in terms of their community composition (Figure 3B, PERMANOVA on Bray-Curtis dissimilarities: F(2,32) = 15.8, R2 = 0.49, Unweighted UniFrac: F(2,32) = 5.6, R2 = 0.26, Weighted UniFrac: F(2,32) = 26.9, R2 = 0.63, p-value = 0.001 and, 999 iterations per test in all cases), with the frequency of methanogens, the organisms directly responsible for methane production, remaining constant across treatments. Most notably, control samples had a higher fraction of Bacteriodia and Gamma-proteobacteria compared to both the Pre-Adaptation and Adaptation samples (Figure 4). We note however that the magnitude of compositional differences between control and adapted communities are similar to that observed between pre-adapted versus control communities, which did not differ in gas production.
The compositional changes and associated increases in gas production resulting from the addition of the pre-adapted inocula could have arisen through selection acting on novel ecological composition (ecology), genetic variants (evolution) or both. Given that directly attributing causal changes in composition and function to evolution in such complex communities is not possible, we instead sought to determine if ecological processes were sufficient to explain these changes. First, our experimental design - adding only 1% of the pre-adapted communities - should have had only a minimal direct impact on the ecology of the recipient communities. To confirm this, we simulated the addition of 1% of the pre-adapted communities to control communities and unsurprisingly found that this caused non-significant changes in community composition (adonis2, F1,23 = 0.017, R2<0.001 P  = 1). Furthermore, adapted communities were no more similar to directly linked pre-adapted communities, compared to unlinked (randomly selected pre-adapted community) or control communities (Fig. 2C).
Previous work has shown that rare taxa can play an important role in methane production in methanogenic and other communities . As such, while adding only 1% of the pre-adapted community had no detectable direct impact on community composition, it could have had a direct effect on gas production if functionally important rare taxa increased in frequency by orders of magnitude during pre-adaptation. To investigate this possibility, we first identified taxa that had significantly greater abundance in adapted versus control communities, as their presence could explain between-treatment differences in gas production. Next, we determined whether any of those taxa increased between the start (i.e., the ancestral community) and end of the pre-adaptation treatment to an extent where they could have enriched the adaptation treatment. Using this two-pronged approach, we found one candidate taxon, belonging to the Ruminococcus genus (Table 1) – this taxon had a higher frequency in the adaptation versus control treatment and was enriched over the course of the pre-adaptation treatment. It is however highly unlikely that this organism could explain the difference in gas production between control and adaptation treatments: it was only detected in a subset of replicates in the adaptation treatment (10 out of 12), so its presence cannot contribute to all observed increases in gas production. The taxon is also present in the pre-adaptation treatment (in 6 replicates) and we did not find a significant difference in its abundance when comparing pre-adapted versus adapted communities (t(21) = -0.21; P = 0.84, means of 92.8 for pre-adaptation and 104.5 for adaptation treatment, 95% CI -138.1 to 114.1). Despite that, there is a significant difference in gas production between those two treatments.