Novel molecular resources for single-larva barcoding of enigmatic
crustacean y-larvae
Abstract
The enigmatic “y-larvae” (Pancrustacea: Facetotecta) still have an
incompletely understood lifecycle, and their adult forms remain unknown
despite their discovery more than 100 years ago and their documented
global occurrence from shallow waters to the deep-sea. Only two of the
17 formally described species, all based on larval stages, have been
investigated using an integrative taxonomic approach that, besides
providing descriptions of the morphology of the naupliar and cyprid
stages, also made use of exuvial voucher material and DNA barcodes. To
improve our knowledge about the systematics and phylogenetics of
y-larvae, we developed a novel protocol that maximizes the amount of
morphological, ecological, and molecular data that can be harvested from
single individuals of these tiny larvae. This revolves around single
larva barcoding, and includes daily imaging of y-nauplii reared in
culture dishes, mounting of their last naupliar exuviae on a slide as a
reference voucher, live imaging of the y-cyprid instar that follows, and
fixation, DNA extraction, amplification, and sequencing of the y-cyprid
specimen. By developing and testing a suite of new primers for both
nuclear and mitochondrial protein-coding and ribosomal genes, we
estimated the most comprehensive phylogeny of Facetotecta to date. We
expect that our novel procedure will help to unravel the complex
systematics of y-larvae and show how these fascinating larval forms have
evolved. Moreover, we posit that our protocols should work on larval
specimens of a diverse array of molting marine invertebrate taxa.