4.2. Comparing protocols and paving the road for comprehensive
single-specimen systematics
Besides providing the first multi-locus, vouchered phylogeny estimate
for Facetotecta, our preliminary phylogeny of this group (Fig. 1)
demonstrates the advantages and disadvantages of the three protocols
that we outlined above. Protocol 1 outperformed all previous attempts to
study the molecular phylogenetics and evolution of Facetotecta. First of
all, it retained the information obtained from living specimens
(y-nauplii, y-cyprid) throughout their larval development and also
retained voucher exuviae of single specimens after their DNA extraction.
Secondly (Fig. 1) it allowed for consistent amplification of long
(>1500bp) and short (<400bp) DNA templates of
protein-coding and ribosomal loci of both the mitochondrial and nuclear
genomes (Table 1, 2, 3, S2). Protocol 1 thus ensured maximal gathering
of morphological and molecular information from individual specimens and
allowed the scoring both y-naupliar and y-cyprid characters. Given the
remarkably high diversity at local scales such Sesoko Island (Glenner et
al., 2008; Olesen et al., 2022; Dreyer et al., in press), this protocol
will surely become an important asset to understanding Facetotecta
diversity. It has already proven invaluable for species descriptions
when dealing with sympatric distributions of multiple species (Olesen et
al., 2022). The last-stage nauplius (LSN) is an unambiguously homologous
and easily recognizable stage, of which there is never more than one for
a given taxon (Olesen et al., 2022). The exuviae of the LSN (shed during
metamorphosis to the y-cyprid), can serve as either a primary or
complementary voucher specimen whether it is mounted on a glass slide or
fixed and preserved in a liquid storage medium.
For example, the integrative description of Hansenocaris demodexwas based on a series of nearly identical specimens that were first
reared and live-imaged and subsequently amplified and sequenced
individually with a short 18S fragment (Olesen et al., 2022). To this
was added crucial details of the y-cyprid as observed by both LM and
SEM, the latter admittedly not relying on sequenced material, but on
specimens confidently assigned to the species due to similarities of
their preceding nauplii (in particular the LSN) to those of sequenced
individuals. In future work, it is not unlikely that several data
layers, including live images, LM images of voucher exuviae (of
y-nauplii), SEM images (of y-cyprids and some LSN voucher exuviae,
either naturally molted or remaining after DNA extraction), and DNA
sequence data, will be harvested from a single specimen (Grygier et al.,
2019). We recommend mounting naupliar exuviae in glycerin jelly as it
solidifies at room temperature, thus easing maintenance in museum
collections and tropical research facilities. Some LSN exuvial specimens
may also be imaged by confocal laser-scanning microscopy (CLSM),
although we have not attempted this yet for DNA-extracted specimens (for
information on mounting exuviae for SEM, see Grygier et al., 2019;
Olesen et al., 2022; Kolbasov et al., 2022).
Protocol 2 lumps together a series of culturing and sequencing
strategies, all of which retain a less informative and less complete
assortment of visual information than Protocol 1. It is typically
applied to y-larvae with planktotrophic nauplii, such as y-naupliar
Types A* and AE* and the planktotrophic y-nauplii from the Azores and
the White Sea, as these cannot yet be reared in the lab. The
morphological information is sufficient for screening morphotype
affinities established by molecular data but, in our view, does not
provide a comprehensive basis for species descriptions. Namely,
different instars in the life cycle (e.g., y-nauplii and -cyprids)
cannot easily be linked together, especially at localities Sesoko Island
with many sympatric morphotypes. We also demonstrate that protocol 2
works on other Thecostraca larvae, e.g., an ascothoracid larva identifed
as Baccalaureus sp. nests as a sister species to B.
maldivensis (Fig. 1). Thus, we anticipate that procols 1 and 2 should
work on other marine invertebrate larvae that molts and leaves exuviae.
Protocol 3, which involves the use of non-cultured y-larvae and has been
used for all specimens with sequences currently deposited in GenBank,
except for those of Olesen et al., (2022). It does not produce any
images, and thus no vouchered data, and is therefore impractical for all
levels of y-larva phylogenetics. At best, it retains anecdotal
information about the specimens obtained prior to sequencing. Despite
these limitations, the present work has allowed us to determining the
identity of a least some of the y-larval material (“Facetotecta sp.
1-6”) used by Pérez-Losada et al., (2009), as some matches to the
vouchered material sequenced herein from the same locality (Sesoko
Island) are apparent. For example, their “Facetotecta spp. 1, 2, and
6” cluster within our Type A* and “Facetotecta sp. 4” is molecularly
identical to Type D*. On the other hand, “Facetotecta sp. 5” and the
Gallego et al’s (2012, 2015) two sequences from Antarctica are still
‘ghost’ sequences and remains unlinked to known, imaged Facetotecta.
Although these specimens are unvouchered and lack any kind of published
morphological information, they have played a critical role at higher
taxonomic levels, helping to place Facetotecta systematically within
Thecostraca and Pancrustacea (Pérez-Losada et al., 2002, 2009; Petrunina
et al., 2014).