Genetic and morphological identification of formalin fixed, preserved larval fishes; can we have the best of both worlds?

Abstract

Surveys of larval fishes require accurate identifications of larvae, which are essential to understand early life history of fish, fish ecology and fisheries. However, the identification of larval fishes requires microscopic examination that is substantially more difficult than that of juvenile and adult fishes, as many larval stages remain undescribed. Furthermore, the traditional, formalin fixation of larval fishes were previously thought to prevent genetic sequencing compared to ethanol preserved larvae. In this study, we used an integrative taxonomic approach based on morphology, imaging and DNA barcoding of the mitochondrial (mtDNA) cytochrome c oxidase subunit (COI) gene. We used this approach in both cultured yellowtail kingfish, Seriola lalandi and wild sourced fish larvae that had been fixed in 5% formalin. Based on controlled and in-field formalin treatments, DNA barcoding and genetic species identification was 100% successful in cultured yellowtail kingfish fixed in formalin for up to 6 months, while barcoding of wild caught fish larvae enabled species identification of 93% of up to 8-weeks formalin fixed specimens. Furthermore, we demonstrated the viability of using either whole larval individuals or a single eyeball (<1 mm diameter, thus retaining the specimen intact) from formalin fixed larval fish for genetic species identification. While COI genetic identifications from the in-field experiments were patchier than the controlled experiments, our study highlights the possibility of recovering suitable DNA from larvae that have been fixed in formalin for up to six months. This was achieved by applying DNA extraction methods that use de-cross-linking steps and species identification based on both full-length reference and mini-barcodes. Our study provides the larval fish research community with a practical framework for undertaking both morphological and genetic identifications of larval fish assemblages, particularly when geographic relevant reference sequence databases (based on vouchered adult fishes) are available for interrogation. It also simplifies field-based collection of samples allowing their preservation in formalin without compromising the genetic identification of species.