Flexible oviposition behavior enabled the evolution of terrestrial reproduction

Abstract

Among vertebrates, nearly all oviparous animals are considered to have either obligate aquatic or terrestrial oviposition, with eggs that are specialized for developing in those environments. The terrestrial environment has considerably more oxygen but is dry and thus presents both opportunities and challenges for developing embryos, particularly those adapted for aquatic development. Here, we present evidence from field experiments examining egg-laying behavior, egg size, and egg jelly function of 13 species of Central and South American treefrogs in the genus Dendropsophus, which demonstrates that flexible oviposition (individuals laying eggs both in and out of water) and eggs capable of both aquatic and terrestrial development are the likely factors which enable the transition from aquatic to terrestrial reproduction. Nearly half of the species we studied had previously undescribed degrees of flexible oviposition. Species with obligate terrestrial reproduction have larger eggs than species with aquatic reproduction, and species with flexible reproduction have eggs of intermediate sizes. Obligate terrestrial breeding frogs also have egg masses that absorb water more quickly than those with flexible oviposition. We also examined eight populations of a single species, Dendropsophus ebraccatus, and document substantial intraspecific variation in terrestrial oviposition; populations in rainy, stable climates lay fewer eggs in water than those in drier areas. However, no differences in egg size were found, supporting the idea that the behavioral component of oviposition evolves before other adaptations associated with obligate terrestrial reproduction. Collectively, these data demonstrate the key role that behavior can have in facilitating major evolutionary transitions.

Keywords: evolutionary transition; flexible behavior; life on land; phenotypic plasticity; terrestrial reproduction.

Fig. 1.

Results of behavioral assays for reproduction and phylogenies of 13 species of Dendropsophus treefrogs and one outgroup species showing ancestral state estimation of reproductive mode. (A) The results of behavioral assays for reproduction in shaded (dark) or unshaded (light) breeding enclosures. Boxplots show the proportion of eggs laid out of water in assays of each species and points represent individual females. The box-and-whisker plot shows the median (thick horizontal line), interquartile range (top and bottom of the box), and the most extreme values (whiskers). (B) Bayesian phylogeny. Numbers at nodes represent posterior probabilities in the consensus tree. (C) A maximum likelihood phylogeny. Numbers at nodes indicate bootstrap support generated from 1,000 replicates. In both trees, the color of the branches in the phylogeny indicates estimated ancestral reproductive mode, from obligate aquatic reproduction shown in blue to obligate terrestrial reproduction shown in green, and degrees of flexible reproduction in shades of orange. Mode of reproduction, shown by branch colors, was estimated by treating reproduction as a continuous variable ranging from fully aquatic (0) to fully terrestrial (1). Pie diagrams at nodes indicate the estimated probability of the ancestral state being either aquatic (blue), flexible (orange), or terrestrial (green) reproduction when considering reproduction of extant taxa as a categorical variable.

Fig. 2.

Results of behavioral assays for reproduction and population structure and variation in reproduction of Dendropsophus ebraccatus in Central America and Ecuador. (A) The results of behavioral assays for reproduction in shaded (dark) or unshaded (light) breeding enclosures. Box-and-whisker plots show the proportion of eggs laid out of water in assays of each population and points represent individual females. The box-and-whisker plot shows the median (thick horizontal line), interquartile range (top and bottom of the box), and the most extreme values (whiskers). (B) Median joining haplotype network of eight populations of D. ebraccatus based on 1,442 base pairs of mitochondrial DNA (ND1 and 16S). Each colored circle shows a unique haplotype whereas small black circles represent hypothetical, unsampled haplotypes. Different colors indicate localities, and the diameter of the circle represents the number of individuals with that haplotype. Tick marks or numbers represent the number of substitutions between haplotypes. (C) Estimated population genetic clusters as generated in STRUCTURE from an average of 59,198 variable SNP loci per site obtained through RAD-seq. Each bar represents a different individual and the colors of each bar represent the estimated probability of belonging to one of four genetic clusters. Localities included in each cluster are listed at the top.

Fig. 3.

Variation in egg diameter across frogs in general, species of Dendropsophus and populations of D. ebraccatus. (A) A box-and-whisker plot showing egg diameter of frog species with obligate aquatic or terrestrial oviposition. The box-and-whisker plot shows the median (thick horizontal line), interquartile range (top and bottom of the colored box), and either the most extreme values (whiskers without points) or 1.5 times the interquartile range and outliers (ends of the whiskers followed by points). (B) The relationship between egg diameter and the proportion of eggs laid terrestrially within the genus Dendropsophus. Large colored circles represent the average egg diameter and degree of terrestrial reproduction for each species, whereas small colored circles represent oviposition for individual clutches laid in shaded and unshaded behavioral assays. (C) Egg size variation across eight populations of D. ebraccatus in relation to mode of reproduction. Large colored circles represent the average egg diameter and degree of terrestriality for each population of D. ebraccatus, whereas small colored circles represent oviposition for individual clutches laid in shaded and unshaded behavioral assays. Different colors in (B) and (C) represent different species or populations, respectively. The values for egg diameter of individual clutches in (B) and (C) represent the average of five eggs removed from each clutch laid at that site or by that species. Curves were calculated from generalized linear mixed effects models using a binomial error distribution. The gray shaded region is a 95% confidence interval.

Fig. 4.

Maximum rates of water absorption by frog egg masses after submergence in water. (A) Absorption rates for four species of Dendropsophus. (B) Absorption rates for eight populations of D. ebraccatus. Mass gain via absorption of water was measured by submerging terrestrially laid egg masses in water and recording the increase in mass over time. Curves were calculated from linear mixed effects models; the shaded region is the 95% confidence interval.

Fig. 5.

Degree of terrestrial reproduction in Dendropsophus ebraccatus in relation to latitude and climate. Data for eight populations are plotted against (A) latitude, (B) precipitation during the wettest quarter of the year, and (C) isothermality (the ratio of the mean daily temperature fluctuation to the annual temperature range). Curves were calculated from generalized linear mixed effects models using a binomial error distribution; the gray shaded region is the 95% confidence interval.