Evolutionary transitions between different modes of development in marine invertebrates are thought to be biased toward the loss of feeding larvae. Because the morphology of feeding larvae is complex and nonfeeding larvae or encapsulated embryos with benthic development often have simplified morphologies, it is presumed to be easier to lose a larval stage than to reacquire it. Some authors have gone so far as to suggest that feeding larvae, morphologically similar to the ancestral feeding larvae, cannot be reacquired. However, the larval structures of some groups, most notably gastropods, are often retained in the encapsulated embryos of species that hatch as benthic juveniles. Therefore the re-evolution of feeding larvae using the same structures may be possible in these groups. Here we present the first well-substantiated case for the recent re-evolution of feeding larvae within a clade of direct-developers. DNA sequence data show that Crepipatella fecunda, a species of calyptraeid gastropod with planktotrophic development, is nested within a clade of species with direct development, and that Crepipatella dilatata, a species with direct development, appears to be paraphyletic with respect to C. fecunda. Observation of the embryos of C. dilatata shows that the features necessary for larval feeding and swimming are retained in the encapsulated veligers, suggesting that heterochronic shifts in hatching time and changes in nurse-egg allotment could have resulted in the re-evolution of feeding larvae in this species.

To understand the role of gene expression in adaptive variation, it is necessary to examine expression variation in an ecological context. Quantitative real-time PCR (qPCR) is considered the most accurate and reliable technique to measure gene expression and to validate the data obtained by RNA-seq; however, accurate normalization is crucial. In Chile, the freshwater silverside fish Basilichthys microlepidotus inhabits both polluted and nonpolluted areas, showing differential gene expression related to pollution. In this study, we infer the stability of six potential reference genes (tubulin alpha, hypoxanthine-guanine phosphoribosyltransferase, glyceraldehyde-3-phosphate dehydrogenase, beta-actin, 60S ribosomal protein L13, and 60S ribosomal protein L8) in the gills and liver of silverside individuals inhabiting polluted and nonpolluted areas. To validate the reference genes selected, the most and least stable reference genes were used to normalize two target transcripts, one for each organ. The RefFinder tool was used to analyze and identify the most stably expressed genes. The 60S ribosomal protein L8 gene was ranked as the most stable gene for both organs. Our results show that reference gene selection influences the detection of differences in the expression levels of target genes in different organs and, also highlighting candidate reference genes that could be used in field studies.