Results

We identified 134 CHC compounds in total from our representative termite and cockroach species (Tab. S1). The six major CHC compound classes detected were n -alkanes, n -alkenes, alkadienes as well as mono-, di-, and tri-methyl-branched alkanes (Fig. 2). The different compound classes greatly vary in relative amounts across all species and not all classes were observed in each species. On average,n -alkanes show higher relative abundancies in termites (29.1 %) than in cockroaches (15.43 %), whereas di-methyl-branched alkanes show the reversed pattern with much higher average abundancies in cockroaches (25.96 %) than in termites (0.027 %). Tri-methyl alkanes, on the other hand, only occur in detectable quantities in B. germanica . Alkadienes were only found in Rf, M. darwiniensis and N. castaneus , with minimal trace occurrences also in B. orientalis . Generally, mono-methyl-branched alkanes were the most abundantly detected compound class across the tested cockroach and termite species, however, they occurred in comparably low quantities in M. darwiniensis and N. castaneus . Unsaturated compounds, generally considered to be among the structurally more complex CHCs indicating higher chemical complexity together with methyl-branched alkanes (Martin and Drijfhout 2009; Kather and Martin 2015), occur inconsistently in two high (ST) and one low (OPT) social complexity termite species, but only in traces in the cockroaches. However, the most structurally complex methyl-branched alkanes with three methyl branches occur exclusively in just the cockroach species B. germanica . Moreover, the structurally most simple CHC profile, consisting almost exclusively ofn -alkanes and mono-methyl-branched alkanes, was found in the high social complexity termite C. formosanus .
The molecular phylogeny of our tested termite and cockroach species mostly mirrors their respective levels of social complexity except forM. darwiniensis and C. formosanus , which represents the most basal termite group despite displaying a high level of social complexity (Fig. 3). This is not at all reflected in their chemical phylogeny based on the average CHC divergence between the species. Namely, the most highly supported cluster (99 Bootstrap) encompasses a solitary (B. orientalis ), highly social (C. formosanus ) and lowly social (K. flavicollis ) species. Moreover, all levels of social complexity that cluster together in the molecular phylogeny are basically broken off in the chemical phylogeny, with no recognizable pattern. Unsurprisingly, a Mantel test found no significant correlation between the molecular and chemical phylogeny (r = 0.3971, P = 0.1291).
Comparing overall CHC biosynthesis gene transcript counts with the total number of CHC compounds detected in each species, we found no significant correlation between the quantities (χ2 – test, r=0.12, p=0.79; Fig. 4). R. flavipes has the highest transcript counts (191) but the third lowest CHC compound count (37), conversely,M. darwiniensis has the lowest transcript count (111) but the second highest CHC compound count (59). Again, no trend towards the different levels of social complexity could be detected, which is best exemplified in the three high social complexity termite species, where both the highest (R. flavipes ) and the lowest (M. darwiniensis ) number of transcripts as well as the second highest (M. darwiniensis ) and the lowest number (C. formosanus ) of CHC compounds was detected. Across all investigated CHC biosynthesis gene transcripts (Tab. 1), counts did not vary systematically by social complexity level (χ2= 1.11, df = 1, p = 0.29), which is also apparent in a heat map representing the individual transcript counts per species normalized by their average relative abundancies (Fig. 5).