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.