Characterization of gut length and content
In January 2015, 250 guppies preserved in ethanol at -20°C were dissected with sterile instruments in the laboratory. The tubular digestive tract was removed, washed in 70% ethanol, measured for gut length with a ruler in a sterile petri dish, and cut to delineate anterior and posterior sections. Anterior and posterior guts were preserved in ethanol and DNA was extracted within 2 weeks. A separate set of 105 intact guppies were shipped to Universidade do Estado do Rio de Janeiro for analysis of gut length and gut content. Upon dissection, guts were removed and preserved in 10% formalin, then weighed, photographed and measured using the software ImageJ (Schneider et al. 2012).
Methodology used for gut content analysis was the same used in Zandonàet al. (2011). Briefly, we analyzed the first third of each gut, which included the stomach and a small part of the foregut, because here items are not fully digested and thus identifiable. The gut contents were then distributed evenly onto a 64-square gridded slide squares and examined under a dissecting scope to identify invertebrates to the lowest possible taxonomic degree, generally the family (Mugnai et al. 2010). The slide was then analyzed under a compound microscope, where 10 squares were randomly chosen among the 64 to estimate the area covered by each food item: invertebrates, detritus, diatoms and filamentous algae. Detritus and filamentous algae areas were estimated for each square, while diatoms were counted. We assigned an average size for diatoms to calculate the total area occupied in each square. Due to the patchier distribution of invertebrates on the gridded slide and their lower occurrence but bigger sizes, we estimated the area covered by them in all 64 squares. Subsequently, we estimated the total area and relative percentages covered by each food item in the whole slide. Invertebrates are the most nutritious item and, together with detritus, they made up most of the gut contents. Because invertebrate and detritus percentages were correlated, we only considered invertebrates in the statistical analysis.