DOI: 10.5061/dryad.sqv9s4nh5
title: Evolution of aquatic snails' defences resulted in clade-specific differences in egg toxicity, pigments, and warning colouration

description: Oviparous animals have evolved diverse strategies that deter egg
 predation. In terrestrial species, these often include noxious compounds
 and aposematic signalling, but little is known in freshwater environments.
 Here we unravel the evolutionary and ecological strategies of Pomacea,
 aquatic snails that lay conspicuous masses of toxic orange-pink eggs to
 reduce predation risk. We reveal the interplay among warning colouration,
 toxicity, and predator visual perception that enables the evolution of
 advanced chemical defences. We provide evidence that snails modify dietary
 carotenoids and that this controls egg colouration in a clade-specific
 manner. Snails from the canaliculata clade accumulate more and
 brighter-coloured egg carotenoid pigments than those from the bridgesii
 clade. The conspicuousness of colour signals was assessed using field
 data, spectral reflectance measurements, and visual modelling. We show
 that aposematic signal variation among species is likely noticeable to
 putative waterbird predators. Feeding egg extracts to birds adversely
 affected their gut morphology. Comparative analysis revealed a correlation
 among pigment modification, conspicuousness, and toxicity, demonstrating
 that colour acts as an honest aposematic signal in apple snail eggs. To
 our knowledge, our study provides the first example of an honest
 aposematic signal in warning colouration among freshwater invertebrates.
description: # Evolution of aquatic snails' defences resulted in clade-specific
 differences in egg toxicity, pigments, and warning colouration --- Dryad
 DOI:
 [https://doi.org/10.5061/dryad.sqv9s4nh5](https://doi.org/10.5061/dryad.sqv9s4nh5) We have submitted our raw data (**PomaceaHH_3.csv** and **Villi_DATASET.csv**). In addition, we have submitted an R script (**pomacea_tetra_3_PRLS.R** ) and a Prism code (**Morphology_analysis_villi.pzf**) ## Descriptions ### **PomaceaHH_3.csv** This matrix contains the spectral data from the egg clutches of three Pomacea snail species, plus two backgrounds on which the eggs are laid: * wavelength *nm*: Wavelength in nanometers * Pomacea samples include *P. maculata*, *P. diffusa*, *P. scalaris*, *P. canaliculata*; background samples include the leaf of *Sagitaria montevidensis* and rock substrate. *species* the first three letters of the column name code the species name epithet *sample* the first nine letters code the unique sample name *replicate* the 11th letter codes the replicate of each sample * Background spectra are coded with the first three letters of the column name as: *bkg* with "saeta_" and "tosca_" indicating *Sagitaria* or rock substrates, respectively ## Code/Software R is required to run *pomacea_tetra_3_PRLS.R*; the script was created using version 4.5.1. Annotations are provided throughout the script through 1) PREPARE ENVIRONMENT, 2) VISUALIZE SPECTRA, 3) VISUAL MODELING, and 4) MODEL CHROMATIC DIFFERENCES (CONTRASTS). ## Prism file for statistical analyses of bird villi data The file *morphology analysis villi.pzf* is a Prism file that includes the villi morphology dataset and the scripts used to perform the statistical analyses (Kruskal–Wallis test) and generate the plots shown in Figure S2, which shows the effect of ingestion of both *Pomacea* species on quail gut at different times. ## bird vililii dataset Users can access the raw data in the *Villi_DATASET.csv* file bird vililii dataset, but will need GraphPad PRISM to access statistics and graphics. The units of both vilii width and vilii length are in micrometers (μm)