1. INTRODUCTION
Increased availability of diverse phylogenomic resources has transformed pancrustacean (Hexapoda and ‘Crustacea’) systematics, yet numerous clades remain recalcitrant to analysis due to a critical lack of molecular resources (Regier et al., 2010; von Reumont et al., 2012; Oakley et al., 2013; Schwentner et al., 2017, 2018; Lozano-Fernández et al., 2019; Bernot et al., 2022). This particularly applies to the so-called ‘dark’ taxa (Hartop et al., 2022), of which the enigmatic crustacean y-larvae (Pancrustacea: Thecostraca: Facetotecta) constitute one of the most remarkable examples.
Y-larvae are fascinating invertebrates that are almost exclusively known from planktonic larval stages (Glenner et al., 2008; Dreyer et al., 2022). They develop through a series of dispersive stages (y-nauplii: Itô, 1986; Kolbasov et al., 2021; Olesen et al., 2022) and a single putative attachment stage (y-cyprid: Kolbasov et al., 2022). A subsequent stage, called the ypsigon, which lacks segmented appendages, a gut, and compound eyes, was recently induced through in vivoexposure of y-cyprids to crustacean molting hormone (Glenner et al., 2008; Pérez-Losada et al., 2009; Dreyer et al., 2022). As the ypsigon exites the y-cyprid, it is tentatively regarded as an early instar/juvenile of a hypothetical endoparasitic adult stage (Pérez-Losada et al., 2009; Dreyer et al., 2022). The y-larva life cycle thus envisaged is reminiscent of that of parasitic barnacles (Cirripedia: Rhizocephala; Dreyer et al., in press).
Few studies have cast light on the systematics and evolution of facetotectans, despite their discovery more than a century ago and their global distribution at depths of 0-6000m (Hansen, 1899; Grygier, 1987; Dreyer et al., 2022; Kolbasov et al., 2022). Morphological studies based on wild-caught specimens have proven unsuccessful in delimiting species within geographically widespread “types” such as type IV (Hansen, 1899; Schram, 1972), “Pacific Type I” (Itô, 1986), and type VIII with its three subtypes VIII-a, -b, and -c (Itô, 1987), which are each practically identical wherever found. Formal cladistic analyses using morphological characters have not resolved facetotectan phylogeny (Pérez-Losada et al., 2009; Kolbasov et al., 2022), and the need for DNA sequences of live-imaged and properly vouchered material is inescapable (Olesen et al., 2022). Phylogenetic analyses show that y-larvae form a distinct and monophyletic group (Grygier, 1987; Chan et al., 2021; Dreyer et al., 2022), but conflicting datasets currently place Facetotecta as sister either to Ascothoracida (parasitic “gall barnacles”: Petrunina et al., 2014; Dreyer et al., 2022) or a monophyletic group composed of Ascothoracida and Cirripedia (the stalked, acorn, burrowing, and parasitic barnacles; see Pérez-Losada et al., 2009). These studies are all problematic in that they have mostly used unvouchered “ghost” sequences of y-larvae or only a few quite conservative molecular markers (Pérez-Losada et al., 2009; Petrunina et al., 2014; Dreyer et al., 2022). No facetotectan species description has been supplemented by both nuclear and mitochondrial sequence data, and a complete or near-complete series of naupliar and cyprid instars has been described for only three species to date (viz., Hansenocari furcifera Itô, 1989, H. itoi Kolbasov and Høeg, 2003, andH. demodex Olesen, Dreyer, Palero and Grygier in Olesen et al., 2022). The incompletely known life cycle of y-larvae, their small size, and a dearth of molecular data have hampered an accurate assessment of their evolution and systematics.
The current systematic resolution of y-larvae is unsatisfactory and problematic for several reasons. Considering that their local and global diversity may be significantly larger than what is described (Hansen 1899; Glenner et al., 2008; Dreyer et al., in press), authentic sequences of vouchered and named species are essential for biodiversity inventories. Currently only 17 species are formally described, in the single genus Hansenocaris Itô, 1985 (Olesen et al., 2022; Olesen and Grygier, 2022). Solving phylogenetic relationships is, moreover, crucial for robust macroevolutionary modeling of life histories and ecomorphological traits, especially when compelling life-history evidence suggests that y-larvae attain maturity as endoparasites in unknown hosts (Glenner et al., 2008; Pérez-Losada et al., 2009; Dreyer et al., 2022).
Destructive DNA-extraction methods often result in complete digestion of the specimen, which limits adequate morphological species delimitation and museum storage of vouchers. Here, we present novel molecular resources and optimized protocols for successful DNA extraction, voucher exuvium retainment, and PCR-amplification of single-specimen y-larvae below 500 µm in size. This is intended to supplement the single-specimen rearing and live-imaging protocol previously developed by us (Olesen et al., 2022). Through extensive laboratory experimentation, we developed an optimal DNA-extraction and PCR-amplification protocol for Facetotecta and here compare it to two other methods that can also yield single-specimen nucleotide sequences. To this end, and as part of a larger campaign investigating the phylogeny and evolution of Facetotecta (Olesen et al., 2022; Dreyer et al., submitted), we designed novel oligonucleotide primers to amplify nuclear and mitochondrial genes and tested 28 primer pairs in more than 2000 PCR reactions. We thereby show that up to 6700 aligned nucleotide resolution can be achieved for a single specimen. We demonstrate the utility of our protocol by estimating a preliminary phylogeny of Facetotecta that expands upon previous phylogenetic analyses. This estimate includes more markers (n=6) and specimens (n=74) than previous efforts, using material obtained from Pacific, Northeast Atlantic, Antarctic (NCBI data), and Arctic waters. Finally, we discuss the importance of our protocols for further advancing the knowledge of y-larva systematics and evolution.