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\begin{document}

\title{Novel molecular resources for single-larva barcoding of enigmatic
crustacean y-larvae}



\author[1]{Niklas Dreyer}%
\author[2]{Ferran Palero}%
\author[3]{Mark  Grygier}%
\author[4]{Alexandra Savchenko}%
\author[5]{Gregory Kolbasov}%
\author[6]{Ryuji Machida}%
\author[6]{Benny Chan}%
\author[7]{Jørgen  Olesen}%
\affil[1]{Affiliation not available}%
\affil[2]{5Institut Cavanilles de Biodiversitat i Biologia, Evolutiva (ICBIBE)}%
\affil[3]{National Taiwan Ocean University Center of Excellence for the Oceans}%
\affil[4]{Lomonosov Moscow State University}%
\affil[5]{Moskovskij gosudarstvennyj universitet imeni M V Lomonosova Biologiceskij fakul'tet}%
\affil[6]{Academia Sinica}%
\affil[7]{Natural History Museum of Denmark}%


\vspace{-1em}



  
  \date{May 24, 2024}


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\selectlanguage{english}
\begin{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.%
\end{abstract}%



\sloppy


\textbf{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-Fern\selectlanguage{ngerman}ández 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 \emph{in
vivo}exposure 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., \emph{Hansenocari
furcifera} Itô, 1989, \emph{H. itoi} Kolbasov and Høeg, 2003,
and\emph{H. 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 \emph{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 \selectlanguage{greek}µ\selectlanguage{english}m 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.

\textbf{2. MATERIALS \& METHODS}

2.1 Collections, rearing, imaging \& digital sorting

About 11,000 y-larvae were collected with 75-\selectlanguage{greek}µ\selectlanguage{english}m-mesh plankton nets from
surface waters (0-5m) at three sites: the University of the Ryukyus
Sesoko Laboratory on Sesoko Island, Okinawa, Japan (JA) and Gongguan
Harbor on Green Island and WangHaiXiang Fishing Harbor in Keelung, both
in Taiwan (TA) (Olesen et al., 2022; Fig. 1; Table S1); 35 of these were
sequenced and included in this work. Supplementary material came from
Tioman Island (Malaysia; MAL), Piscinas do Pesquerio in Ponta Delgada,
Azores, Portugal (AZ, n=10), and waters off the White Sea Marine
Biological Station, Russia (RU; n=14) between 2017 and 2021. Larvae were
sorted from plankton samples with glass Pasteur pipettes under an
Olympus stereomicroscope and either (1) photographed and video-recorded
live with a Nikon ECLIPSE 80i compound microscope (JA) or either an
Olympus IX70 inverted compound microscope or a Zeiss AX10 light
microscope (TA), both of the latter being equipped with Nomarsky (DIC)
optics and a Canon EOS 5D Mark IV digital camera, or (2) bulk-fixed for
subsequent molecular work (TA, RU, AZ). All images were sorted digitally
to identify groups of morphologically similar specimens (i.e.,
morphotypes; Olesen et al., 2022), so that at least 2-3 specimens per
group were used for Sanger sequencing.

2.2 Primer design, DNA extraction, PCR, and sequencing

Reference nucleotide sequences (Ascothoracida and Cirripedia) of
selected loci were downloaded from NCBI GenBank and aligned with
in-house (see below) or NCBI Facetotecta sequences with MAFFT version
7.450 (--auto --maxiterate 1000; Katoh and Standley 2013). The
alignments were imported, visualized, and used to design primers in
Geneious Prime version 2022.0.2. To enhance specificity and
amplification success, we strived to design longer (\textgreater{}20bp)
primers with GC-contents between 30-60\%, a GC-clamp of 2 (3'
terminating in at least one G/C to promote optimal binding), a melting
temperature (T\textsubscript{m}) between 55\selectlanguage{ngerman}°C-68°C, and no runs of
\textgreater{}4 bases (homopolymeric regions, e.g., TGGGGG). We tested a
total of 28 primer pairs (23 new) and performed 2056 PCR reactions using
these. Tables 1, 2, and S2 provide an overview of the primers, their
sequences, and their amplification success with single specimens.

Our goal was to develop and test the efficacy of fast ``filter-free''
DNA-extraction methods that amplify single-specimen y-larvae while
retaining their ``exuviae'' (here understood as any cuticular remains
originating from an acutal molt of after tissue digestion during DNA
extraction) as vouchers. We therefore tested and compared DNA-extraction
using the GeneReleaser® kit (BioVentures, TN, USA), which has previously
proven to be an efficient protocol for retrieving DNA from zooplankton
(Schizas et al., 1997; Böttger-Schnack and Machida, 2011; Watanabe et
al., 2016; Olesen et al., 2022) and a simplified DNeasy method using a
subset of the reagents in the Blood and Tissue kit (QIAGEN, CA, USA). To
this end, and as a part of a larger phylogenetic and barcoding campaign,
we extracted DNA of a total 421 single y-larva specimens, of which nine
were from the Azores, 326 from Sesoko Island (Japan), 4 from Malaysia,
105 from various locations in Taiwan, and 17 from the White Sea
(Russia).

DNA extracts were kept in a freeze Eppendorf© vial rack during
preparation of the PCR assay. We pipetted 12.2 \selectlanguage{greek}µ\selectlanguage{english}L filtered
ddH\textsubscript{2}O, 5 \selectlanguage{greek}µ\selectlanguage{english}L ``hot start'' HOT FirePol\selectlanguage{ngerman}® polymerase master
mix (Solis BioDyne, Tartu, Estonia), 0.4 \selectlanguage{greek}µ\selectlanguage{english}L of each oligonucleotide
primer (usually ordered as ``desalted''), and 3 \selectlanguage{greek}µ\selectlanguage{english}L gDNA template to each
vial. For multiplex PCR reactions, which use two or more primer pairs in
combination, we subtracted the equivalent amount of added primer from
the amount of ddH\textsubscript{2}O. To increase primer annealing
sensitivity, specificity to complex templates (e.g., \textgreater{}60\%
G-C content), and yield we generally used a Touchdown PCR profile under
the following conditions: initial denaturation for 15min at 95\selectlanguage{ngerman}°C, then
10 cycles of denaturation at 95°C for 1min, annealing at
T\textsubscript{m}+10°C decreasing by 1°C/cycle for 30 seconds,
extention at 72°C for 1kb/60secs, then 30 cycles of denaturation at 95°C
for 1min, annealing at T\textsubscript{m}-2°C for 30 seconds, extension
at 72°C for 1kb/60secs, final extension at 72°C for 10min, and finally
20min at 4°C to halt the reaction. Primer details including sequences
and annealing temperatures is listed in Supplementary Table 1.

All PCR reactions were conducted in a DNA Engine Thermal Cycler
(Bio-Rad, Richmond, CA, United States) and PCR products were visualized
in agarose gel with varying concentrations depending on fragment size.
DNA sequencing was performed by Genomics BioSci \& Tech Ltd. (New Taipei
City, Taiwan) and Macrogen Europe BV (Amsterdam, The Netherlands).

We sequenced genomic DNA templates of three ribosomal loci
(mitochondrial 16S and nuclear 18S, 28S) and two protein-coding genes
(mitochondrial COX1 and nuclear Histone-3) of 74 specimens and added new
sequences to NCBI GenBank (Tables 1, 2, S2). When we sequenced PCR
amplicons, we defined ``successful amplification'' as those amplicons
that yielded clear gel electrophoresis bands and clean chromatograms
that aligned with our in-house database. Less than 10\% of amplicons
were not sequenced but nonetheless yielded clear bands, and these were
computed as successful amplicons.

2.3. Protocol 1. Complete information of live-imaged specimens with
preserved voucher exuviae

\emph{2.3.1 Culturing and morphological data.} Plankton-collected
y-larvae were sorted into petri dishes and reared further in
mini-cultures of up to five morphologically distinct types or forms as
outlined in Olesen et al., (2022). These were then followed alive daily
in culture by video under a light microscope (LM). Live specimens were
transferred between petri dishes and the light microscope in a glass
Pasteur pipette. The final naupliar exuviae of the developing specimens
were mounted in glycerin jelly on glass slides (GL; Grygier et al.,
2019), or (although only attempted for a few specimens) on SEM-stubs for
high-resolution morphological investigations (Grygier et al., 2019).

\emph{2.3.2 DNA extraction.} Specimens were extracted using both the
GeneReleaser and DNeasy kits. For GeneReleaser, we follow a slightly
modified protocol from that outlined in Schizas et al., (1997). 1 \selectlanguage{greek}µ\selectlanguage{english}L of
10xPCR buffer and 9 \selectlanguage{greek}µ\selectlanguage{english}L ddH\textsubscript{2}O were mixed in a 250 \selectlanguage{greek}µ\selectlanguage{english}L
Eppenorph tube, to which a single y-larva was added. Protein
denaturization was initiated by incubating the tubes at 94-95\selectlanguage{ngerman}°C for 2
mins, and the specimens were subsequently transferred to ice. We then
added 1 \selectlanguage{greek}µ\selectlanguage{english}L protein kinase K (QIAGEN, Chatsworth, CA, USA) and vortexed
the samples thoroughly before incubating them at 55\selectlanguage{ngerman}°C for 15 min and
70°C for 10 min. Subsequently, we added 10 \selectlanguage{greek}µ\selectlanguage{english}L of GeneReleaser
(BioVentures, Inc, Murfreesboro TN, USA) and finally incubated the
samples under the following thermal program: 65\selectlanguage{ngerman}°C for 15 sec, 8°C for 15
sec, 65°C for 45 sec, 97°C for 90 sec, 8°C for 30 sec, 65°C for 90 sec,
97°C for 30 sec, 65°C for 30 sec, 80°C for 3 min, and lastly 4°C for 10
min to halt the reaction. The tubes were then centrifuged for 1 min,
after which 15 \selectlanguage{greek}µ\selectlanguage{english}L supernatant and 10 \selectlanguage{greek}µ\selectlanguage{english}L AE-buffer (QIAGEN, Chatsworth,
CA, USA) were transferred to fresh Eppendorph tubes. To locate and
retrieve voucher ``exuviae'', the samples were spun down and inspected
under a dissection microscope. Any exuviae were recovered and mounted as
described below.

For the simplified DNeasy method, we transferred individual y-larval
specimens from their vials into small, individual petri dishes. After
gently vortexing the dish by hand, the specimens were often easy
locatable at the center of the dish, after which they were transferred
to a 250 \selectlanguage{greek}µ\selectlanguage{english}L Eppendorf tube in a 0.5-1.0 \selectlanguage{greek}µ\selectlanguage{english}L droplet of ethanol by using a
2.5 \selectlanguage{greek}µ\selectlanguage{english}L pipette fitted with a sterile filter tip. The vial(s), each
containing a single y-larva specimen, were then incubated for 2 min at
36\selectlanguage{ngerman}°C to evaporate the remaining ethanol. 40 \selectlanguage{greek}µ\selectlanguage{english}L AE buffer and 4uL
Protease K (Qiagen, DNeasy kit) were subsequently added to each vial,
which were then incubated for 1h at 56\selectlanguage{ngerman}°C followed by enzyme inactivation
at 72°C for 12 min. The vials were then spun down and placed in a rack.
The ``exuviae'', now located at the bottom of their vial, were removed
in a 1\selectlanguage{greek}µ\selectlanguage{english}L droplet by using a 2.5\selectlanguage{greek}µ\selectlanguage{english}L pipette fitted with a filter tip and
either placed directly in pre-heated glycerin jelly on a glass slide or
in a droplet in a petri dish with a small note written on the lid above
the droplet indicating the specimen's ID code for later mounting. We
usually performed PCR of ``legacy'' markers (12S, 16S, 18S, and 28S
rDNA, COX1, and H3) before mounting the exuviae to avoid potential
contamination prior to PCR. Using the pipette tip or an eyelash taped to
a stick, y-naupliar exuviae (those of the last y-nauplius or an earlier
stage) were rotated so the dorsal side faced upward, and y-cyprids were
placed on their lateral side. We found intact exuviae in vials that had
been stored at -18\selectlanguage{ngerman}°C for up to 3 years, suggesting that continued
freezing does not impact the quality of prospective vouchers.

We also tested the non-destructive protocol provided by Cornils (2015),
which uses Protease K and ATL-buffer instead of AE buffer for DNA
extraction. Their protocol consistently worked for a range of copepod
species, but for y-larvae yielded faint bands, fragmented exuviae, and
noisy chromatograms (i.e., low quality DNA template amplicons).

\emph{2.3.3 PCR.} PCR was done as described in the Methods and Materials
section. The vials were sequenced with their PCR-identifier, complete
specimen-designation, and gene listed sequentially (e.g.,
1-JA-2019-001-Hansenocaris-demodex\_COX1,
2-JA-2019-100-Hansenocaris-demodex\_COX1, etc).

2.4 Protocol 2: Assorted information of specimens with or without
voucher exuviae

\emph{Culturing and morphological data.} This method is clearly
distinguished from Protocol 1 by retaining notably fewer data layers
than Protocol 1. For example, while the WS-specimens are represented by
all data layers, except for live images of the sequenced specimens, the
AZ-material is exclusively represented by exuvial vouchers photographed
in LM. Although admittedly artificial, we decided to lump all cases with
varying degrees of morphological information, but no live imaging, into
one category. Protocol 2 was applied to the three live-imaged specimens
of \emph{H. demodex} from Green Island (TA) in Olesen et al., (2022),
the specimens of \emph{H. itoi} from the White Sea (WS; for which all
data layers except live-images of single specimens exist), and the
Azores specimens (AZ; for which only y-naupliar exuviae are available).

\emph{2.4.1 DNA extraction.} We tested three extraction procedures: that
used exclusively for Protocol 1, the DNeasy\selectlanguage{english} kit (as described in detail
above and in the Supplementary Material) following the manufacturer's
specifications, and the GeneReleaser\selectlanguage{english} kit (as described in detail
above).

2.5 Protocol 3: Anecdotal information on non-imaged, non-vouchered
specimens.

\emph{2.5.1 Culturing and morphological data.} This protocol is clearly
distinguished from any other by the lack of any image data for the
specimens. Information about single/bulk-sequenced specimens is,
therefore, anecdotal at best. We did not apply this method, but it was
used previously by others to generate the published y-larva sequences in
NCBI GenBank of ``Facetotecta spp.'' and \emph{H. itoi} (Perez-Losada et
al., 2002, 2009; Gallego et al., 2015). These authors used different DNA
extraction kits but successfully amplified long and short ribosomal and
protein-coding mitochondrial (16S and COX1, respectively) and nuclear
(18S, 28S, and H3, respectively) markers.

2.6 Phylogeny estimation and barcoding of individual y-larvae

Chromatograms of new forward and reverse sequences were trimmed and
assembled in Geneious Prime. Protein-coding genes were translated
(invertebrate mitochondrial code for COX1 and universal code for H3) to
proteins, aligned, and manually checked for stop codons. Using MAFFT
(--auto --maxiterate 1000), nucleotides of each locus and specimen were
then aligned with outgroup sequences from Ascothoracida and previously
sequenced material of Facetotecta (Perez-Losada et al., 2009).
Individual locus alignments were first inspected by eye, then
subsequently curated by assessing ambiguities with the less stringent
options in GBLOCKS version 0.91b (-t=p -b1=24 -b2=39 -b3=4 -b4=10 -b5=n
-b6=y; Castresana 2000).

The nucleotide alignments were then concatenated in Geneious Prime
(6584bp and 3415bp before/after GBLOCKS), partitioned (each codon
position in protein-coding genes), and used to estimate the best-fit
substitution model with ModelFinder (Kalyaanamoorthy et al., 2017) by
allowing partitions to be merged if this increased model fit (-m
MFP+MERGE). Maximum Likelihood (ML) phylogenetic trees were built with
IQ-TREE multicore version 2.0.3 (Minh et al., 2020) and node support was
assessed with 10,000 ultrafast bootstrap (UFBoot) replicates and 10,000
replicates of the SH-like approximate likelihood ratio test (SH-aLRT;
--alrt 10,000). To alleviate issues inherent to the UFBoot approximation
method we specified the ``-bnni'' option. We allowed each partition to
have its own substitution rate (-spp) and performed a more thorough
nearest-neighbour interchange search (-allnni). We also estimated a
phylogeny with Bayesian inference in Mr. Bayes 3.2 (Ronquist et al.,
2012) using the concatenated dataset. We estimated posterior
probabilities through six independent runs with four Markov chains for
10,000,000 generations and with sampling every 1000 generations. We used
the `invgamma' model as the lset rate and computed the potential scale
reduction factor with the `sump' command. We estimated a consensus
phylogenetic tree with the first 25\% of the iterations discarded as
burn-in.

The unrooted tree estimates were visualized in Geneious Prime, rooted in
Ascothoracida, and further annotated in Corel Draw(c) with morphological
information from live and exuvial specimens. These illustrations were
adjusted individually for brightness, contrast, and background color.

\textbf{3 RESULTS}

3.1 Loci selection, DNA extraction, and primer efficacy

We tested 28 primer pair combinations by performing 2056 individual PCR
reactions and provided 74 new sequences of Facetotecta that have been
deposited in NCBI GenBank with the accession numbers XXXX-YYYY (Table
S1).

We also tested the efficacy and amplification success of the two
DNA-extraction protocols (Fig. 2A-C; Tables 1, S2). We performed 815 PCR
reactions with the GeneReleaser kit and 1241 reactions with the
simplified DNeasy kit (Table S2). The minimum and maximum amplification
rates varied between 0\% (DNeasy method, 16S 1471F/1427R,
COI\_Palerointernal, both n=6; Table S2) and \textasciitilde{}98\%
(Dneasy method, 18Sface2F/2R, n=109; Table S2). Both protocols produced
nearly identical overall amplification rates (\textasciitilde{}68\%; Fig
2A, C; Table S2). The GeneReleaser protocol yielded at least
\textasciitilde{}42\% and at most \textasciitilde{}90\% amplification
success across loci (Fig 2; Table S2). Extraction and PCR amplification
with the simplified DNeasy method yielded a slightly higher minimum
amplification success (\textasciitilde{}47\%) and a slightly lower
maximum (\textasciitilde{}88\%) (Fig 2A; Table S2); because different
primer combinations were tested with the two protocols, these results
may be biased. Combining the data from the two extraction methods, we
computed an overall amplification success rate of 68\% across all primer
combinations (2096 successfully amplified fragments; Tables 1, S2; Fig
2A). Average (mean) successful amplification rates were
\textasciitilde{}60\% for COX1 (296 out of 495 reactions; Fig. 2; Tables
1, S2), \textasciitilde{}87\% for 18S (515 of 589 reactions; Fig. 2C;
Tables 1, S2), \textasciitilde{}85\% for 28S (361 of 426 reactions; Fig.
2C; Tables 1, S2), \textasciitilde{}52\% for 12S (73 of 139 reactions;
Fig. 2C; Tables 1, S2), and 56\% for 16S (168 of 300 reactions; Fig. 2C;
Tables 1, S2). The amplification rate difference between the two
extraction methods was notably higher for 16S and H3 than any other
locus (Fig 2B, C; Table 1, S2), with 12S, COX1 and 18S yielding more
similar amplification success rates between the two methods (Fig 2B, C;
Tables 1, S2). For all primer combinations, 18S and 28S expectedly had
the highest amplification rates, but we did not observe this pattern for
H3 (\textasciitilde{}40\% with GeneReleaser and \textasciitilde{}75\%
with the simplified DNeasy method; Tables 1, S2).

Based on the highest observed primer efficacy (i.e., amplification
success), as well as the overlap with publicly available primers, the
fragment lengths obtained, and the quality of the sequences (e.g., the
presence of clear and distinct chromatogram peaks), we recommend
sequencing the following five ``legacy'' loci in future investigations
of facetotecta systematics. COX1: F1new and R1 with the internal
``Leray''-segment mlCOIInF/jgHCO2198 (\textasciitilde{}93\%
amplification success; \textasciitilde{}750bp; Leray et al., 2013; Fig.
3; Tables 2, S2). 18S: initially with Face1F/2R (\textasciitilde{}85\%
success; \textasciitilde{}1800bp; Fig. 3; Tables 2, S2); subsequently,
depending on the amplification rate of the latter, Face1F/1R
(\textasciitilde{}83\% success; \textasciitilde{}300 bp; Fig. 2; Tables
2, S2) and Face 2F/2R (\textasciitilde{}98\% success;
\textasciitilde{}300bp; Fig. 3; Tables 2, S2). 28S: 84F
(\textasciitilde{}74\% success; \textasciitilde{}880bp; Fig. 3; Tables
2, S2) with the internal primer Face3F/3R (\textasciitilde{}94\%
success; \textasciitilde{}350bp; Fig. 3; Tables 2, S2). 12S: 3F
(\textasciitilde{}83\% success; \textasciitilde{}300bp; Fig. 3; Tables
2, S2). 16S: SF/SR (\textasciitilde{}60\% success; 700bp; Tsang et al.,
2009; Fig. 3; Tables 2, S2) with 137F/548R (\textasciitilde{}50\%
success; \textasciitilde{}400bp; Fig. 3; Tables 2, S2).

\textbf{3.2 \emph{Morphotype assignment and phylogeny estimation}}

Based on LM images we recovered two named species and six undescribed
morphotypes (\emph{Hansenocaris demodex} from Green I., Taiwan and
Sesoko I., Okinawa; \emph{H. itoi} from the White Sea, Russia; Type A*
from Green I., Sesoko I., and the Azores; Type C from Sesoko I., Type D*
from WangHaiXiang harbor, Taiwan and Sesoko I.; Type I* from the Azores;
and Type AE* from Taiwan), with morphotype designations following Dreyer
et al., submitted). To this were added four specimens that could not be
morphotyped prior to sequencing due to lack either of images or any
voucher material (Facetotecta sp. 1 from Antarctica and Facetotecta sp.
5 from Sesoko Island; Fig. 1).

Our alignment contained 81 specimens (of which seven are non-imaged and
voucherless ``Facetotecta sp.'' sequences), 6730 bps, 1680 distinct
patterns, 1443 parsimony-informative sites, 384 singleton sites and 4903
constant sites (Table 3). Following the best substitution-model fit
inferred by ModelFinder, we merged the partitions as follows: TNe+R2
(18S rDNA; BIC-score 14930.890, LnL: -24693.263), TIM2+F+R2 (H3, all
codons; BIC-score 6798.637, LnL: -24693.133), TN+F+I+G4 (28SrDNA;
BIC-score 12621.319; LnL N/A), TPM2+F+I (12SrDNA; 3435.374, LnL N/A) and
GTR+F+G4 (16S rDNA, COX1, all codons; BIC-score11902.261, LnL:
-24705.039) (Table 3). We recovered two clades (I and II), although the
monophyly of the second one was not well-supported (Clade I: 98.6\%
Ultrafast Bootstrap (UFBoot), 93\% SH-aLRT support; Clade II: 66.6\%
UFBoot, 81\% SH-aLRT; Fig. 1).

In Clade I the planktotrophic morphotype A* is paraphyletic (97.5\%
UFBoot/96\% SH-aLRT). The placement of three Type A* specimens from the
Pacific (TA-2018-096, JA-2019-126) and the Azores (AZ-2019-5) within a
highly supported, monophyletic subclade comprising pigmented and yolky
lecithotrophs (95.8\% UFBoot/71\% SH-aLRT support). The relationship
between Types C, D*, and \emph{H. demodex} remains unresolved in our
data set (Fig. 1; 69.2\% UFBoot/32\% SH-aLRT). In our dataset, all the
Clade II specimens and morphotypes are planktotrophic, although the
feeding type of Facetotecta sp. 5 and Antarctic species 1 and 2 is
unknown. All multi-specimen morphotypes in the clade are monophyletic
with high support, including an unnamed morphotype from the Azores
(``Unnamed y-nauplius''; AZ-2019-1, 2, 4, 6, 7, 9; 90.3\% UFBoot/99\%
SH-aLRT), Type AE* (100 UFBoot/100\% SH-aLRT) and \emph{H. itoi} (99.9\%
UFBoot/100\% SH-aLRT).

\textbf{4 DISCUSSION}

The enigmatic facetotectans (commonly referred to as ``y-larvae'') are a
severely understudied crustacean subclass, and the notable dearth of
molecular resources and published morphological voucher data concerning
them has impeded reconstruction of the group's evolutionary history of
(Perez-Losada et al., 2002, 2009; Kolbasov et al., 2022). To overcome
the challenges inherent in inferring systematic relationships based on
unvouchered, unphotographed specimens, as exemplified by Perez-Losada et
al., (2002, 2009) and Gallego et al., (2015), we developed a cheap,
fast, and voucher-preserving protocol capitalizing on the widely-used
DNeasy\selectlanguage{english} extraction kit. Our aim was to develop a rearing and extraction
protocol that maximizes morphological and molecular information for
single y-larval specimens spanning what is emerging as a complex and
wide phylogenetic range (Olesen et al., 2022; Kolbasov et al., 2022;
Dreyer et al., submitted; data herein). By relying on a suite of light
microscopic images of live larvae (Olesen et al., 2022) and by
developing primers targeting both nuclear and mitochondrial genomic
markers, we demonstrate a way to infer phylogenetic relationships based
on larval instars of a crustacean taxon with a complicated and partly
unknown lifecycle. In the following we discuss three central topics
related to the development of these new molecular resources: (1) The
efficacy and usefulness of the new primers and the two new DNA
extraction methods, (2) how three overall protocols and their varying
yields of morphological resolution may affect systematic accounts and
future taxonomic classification, and finally (3) interesting features of
the preliminary phylogeny, for example the molecular and biogeographical
extent of morphotypes, and how to use such information in building a
taxonomy that, for now, must rely entirely on larval stages. We believe
that our protocols should also work on other molting invertebrate
larvae.

\emph{4.1. Efficacy of DNA extraction protocols and primers}

We first tested two different DNA extraction methods using a series of
novel and published nuclear and mitochondrial primer pairs. Although
primer amplification success varied between 0\% and
\textasciitilde{}98\% (Table S2), the average amplification success of
our ``legacy'' primers (Fig 3; Tables 3, S1) was \textasciitilde{}80\%.
The lowest amplification rates overall were seen for legacy primers 16S
(average \textasciitilde{}62\%) and H3 (average \textasciitilde{}60\%),
which is likely caused by divergent priming sites or too low copy
numbers for individual y-larvae, respectively. Importantly, maximum
amplification rates were not restricted to nuclear loci as amplification
with the COX1 ``Folmer'' and ``Leray'' primers yielded
\textasciitilde{}93\% success. In combination with nuclear ribosomal
markers that demonstrated high amplification rates, this will likely
positively influence phylogeny estimation and robust species
delimitation in future systematic efforts.

DNA extraction relied on either the GeneReleaser kit, which had
previously been used to retrieve high-quality DNA templates from minute
zooplankton specimen (Schizas et al., 1997; Bottger-Schnack and Machida,
2011; Watanabe et al., 2016), including y-larvae (Olesen et al., 2022),
or a simplified yet highly effective modification of the QIAGEN DNeasy
Blood and Tissue kit. There are important advantages and disadvantages
related to both protocols. On the one hand, GeneReleaser yields higher
amplification rates for certain loci. On the other hand, the
GeneReleaser kit only yields \textasciitilde{}15-20 \selectlanguage{greek}µ\selectlanguage{english}L DNA extract and
its thermal incubation program passes through high temperatures
(\textgreater{}90\selectlanguage{ngerman}°C). This may have a severe impact on successful
retrieval voucher ``exuviae'' because (1) in initial trials with various
crustacean larvae (data not shown), incubation at such temperatures
almost always resulted in the disintegration of the ``exuviae'', and (2)
the ``exuviae'' may easily become trapped in the GeneReleaser powder
grains when the tubes are spun down after incubation. Less than 5\% of
the vials contained ``exuviae'' after transferring the GeneReleaser
supernatant to new vials. Moreover, the GeneRelaser-kit is more
expensive (US\$125 for a kit on 20 October 2022), considering that it
does not come with buffers and protease K included, which thus have to
be acquired elsewhere. By contrast, the simplified DNeasy kit contains
all relevant products for fast and efficient DNA extraction and produces
bright gel-bands in the expected size ranges, clean chromatograms, and
high rates of both overall and locus-specific amplification (Figs. 2, 3;
Table S2). For these reasons, we recommend using our extraction method
with the simplified DNeasy kit and with the legacy primers. We are
currently designing a series of primers for nuclear protein-coding genes
in order to expand this approach to higher-level phylogenetics.

\emph{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
(\textgreater{}1500bp) and short (\textless{}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 \emph{Hansenocaris
demodex}was 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 \emph{Baccalaureus} sp. nests as a sister species to \emph{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).

\emph{4.3. Implications for classification, taxonomy and phylogeography}

Our sequence data matrix recovered the same two clades found earlier
(Pérez-Losada et al., 2009; Olesen et al., 2022), but we provide
enhanced phylogenetic resolution by sequencing more markers for multiple
new MOTUs, some of which may be putative species awaiting taxonomic
description (Fig. 1). Importantly, most morphotypes are supported by
molecular and morphological data in concert, testifying to the
usefulness of combining these two data layers for phylogeny estimation
and evaluation. Given the molecular diversity in the phylogeny and the
morphological disparity of the larval forms, these two clades may
represent high-level taxonomic units (e.g., families), but more study is
required.

It is an inescapable fact that Facetotecta is in critical need of a
novel higher-level classificatory scheme, as all species are currently
placed in the single genus \emph{Hansenocaris.} These new species are
both morphologically and phylogenetically so diverse that it is highly
unsatisfactory to retain them within the same genus. We expect that
single-specimen culturing, live-imaging, amplification, and sequencing
will be the key step towards a new classification of Facetotecta (Olesen
et al., 2022). For planktotrophic specimens, which we are still unable
to culture \emph{in vitro} , this poses a significant challenge that may
postpone detailed taxonomic descriptions. It is exceedingly difficult to
morphologically separate Types A* and AE*, for example, into what are
likely multiple ``species'' (or MOTUs) that together occupy a
geographical range that extends from Japan and Taiwan to the Azores. It
is highly unlikely that each Type constitutes one, big breeding
population. Wide biogeographical ranges of morphotypes could occur in
some planktotrophic y-nauplii, however, because of their long
developmental times and putative long-distance dispersal capabilities.
For example, completion of the larval development of the Arctic
facetotectan \emph{Hansenocaris itoi} requires three months (April
through June: Kolbasov et al., 2021) and this species may have a wide
distribution. Hansen (1899) identified y-nauplii that are virtually
identical to \emph{H. itoi} in the Baltic Sea (Kiel Bay), a place
separated from the type locality in the White Sea by thousands of
kilometers of open ocean. The lack of sequence data for the Baltic
population, if it still exists, illustrates the importance of Protocol
1, in which molecular sequence data are linked to live image data.

In conclusion, the easy-to-follow and inexpensive Protocol 1 described
herein allows the extraction of maximal molecular and morphological
information from single y-larval specimens, although we expect that this
method should work for any invertebrate taxon with free-living larvae.
It should be used in future efforts to, for example, model life-history
evolution in the Facetotecta or other groups. With a growing inventory
of sequence data from cultured, imaged, and vouchered y-larva
specimens/species from geographically diverse places, the evolution of
Facetotecta, as well as a comprehensive new taxonomic scheme for them,
may finally be within reach.

ACKNOWLEDGEMENTS

The authors extend their gratitude to Dr. Danny Eibye-Jacobsen
(University of Copenhagen, Denmark) for assisting collections in Taiwan
and Japan, sometimes under trying weather conditions. Members of the
Coastal Ecology lab at Academia Sinica are also thanked for assistance
with sampling in Taiwan. This work was supported by a double degree
graduate grant from the Natural History Museum of Denmark and the Taiwan
International Graduate Program to ND and a Villum Experiment grant to
JO.

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\textbf{FIGURE LEGENDS}

\emph{Fig. 1.} \textbf{Multilocus phylogeny estimation for Facetotecta
with single-specimen resolution. A} Maximum-likelihood tree from an
alignment spanning 6730bp and six loci with Ascothoracida chosen as
outgroup, showing relationships of Facetotecta and its division into at
least two major clades. \textbf{B} Putative life cycle of Facetotecta
consisting of seven y-nauplius stages, one y-cyprid stage, one or more
ypsigons, and as yet unknown adults. \textbf{C} Examples of
morphological information extracted from Protocols 1, 2, and 3,
represented by branch colors (red, grey, and yellow, respectively).
Protocol 2 lumps different subprotocols that lack live image data, here
illustrated by three examples.

\emph{Fig. 2. \textbf{Comparison of primer amplification efficacy with
two filter-free DNA extraction protocols for single-larva systematics.}}
\textbf{A} Amplification success of all primers using GeneReleaser and
Simplified DNeasy extraction kits. \textbf{B} Amplification success of
all primers partitioned to locus using GeneReleaser and the Simplified
DNeasy extraction kits. The large difference for 16S and H3 may be
caused by the different primers used for each extraction protocol (Table
1S). \textbf{C} density plot of amplification success of all primers for
both extraction kits combined, showing uniformly higher amplification
rates for 12S, 18S, and COX1 and uniformly lower amplification rates for
16S and H3.

\emph{Fig. 3. \textbf{Amplification success and efficacy of ``legacy''
primers}} \textbf{.} \textbf{A} Combined amplification success using
both GeneReleaser and Simplified DNeasy extraction kits. \textbf{B}
Summary of \textbf{A} but with amplification success partitioned to
locus, hence the comparatively larger difference for 28S and 18S
primers.

AUHTOR CONTRIBUTIONS

Niklas Dreyer (ND) designed and conceived the study. ND participated in
sampling, co-designed and tested primers, extraction methods and
protocols. ND performed all sequence, phylogenetic and statistical
analyses, co-funded the project, designed graphical outputs, took
specimen photos and wrote the first draft. Ferran Palero supervised the
project, designed primers, assisted statistical analyses, participated
in sampling and edited manuscript drafts. Mark J. Grygier (MJG)
participated in sampling, co-developed protocols, originally developed
the exuvium-voucher strategy and edited manuscript drafts. Alexandra S.
Savchenko provided the valuable Azores-material and edited the
manuscript drafts. Gregory A. Kolbasov provided the valuable White
Sea-material and edited the manuscript drafts. Ryuji J. Machida
co-supervised the project, designed DNA extraction methods, assisted
primer testing, and edited manuscript drafts. Benny K. K. Chan
supervised the project, led sampling in Taiwan and co-funded the
project. Jorgen Olesen supervised the project, co-conceived the study,
led sampling in Japan, participated in sampling in Taiwan, co-designed
protocols, co-funded the project, assisted phylogenetic analyses,
designed graphical outputs, took specimen photos, and edited manuscript
drafts.

NIKLAS DREYER\textsuperscript{1,2,3,4}, FERRAN P.
PALERO\selectlanguage{english}\textsuperscript{5+*}, MARK J. GRYGIER\textsuperscript{6,7},
ALEXANDRA S. SAVCHENKO\textsuperscript{8}, GREGORY A.
KOLBASOV\textsuperscript{9}, RYUJI J. MACHIDA\textsuperscript{1}, BENNY
K. K. CHAN\selectlanguage{english}\textsuperscript{1+*} \& JORGEN OLESEN\selectlanguage{english}\textsuperscript{1+*}

TABLES

\textbf{Table 1.} Amplification success of all primers with single
larval Facetotecta specimens using Protocols 1 and 2.\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
\textbf{Locus} & \textbf{Total tested} & \textbf{Total amplified} &
\textbf{\% Success}\tabularnewline
\midrule
\endhead
\emph{COX1} & 495 & 296 & 59.79\tabularnewline
\emph{18S} & 589 & 515 & 87.43\tabularnewline
\emph{28S} & 426 & 361 & 84.74\tabularnewline
\emph{12S} & 139 & 73 & 52.51\tabularnewline
\emph{16S} & 300 & 168 & 56\tabularnewline
\emph{H3} & 107 & 70 & 65.42\tabularnewline
Total & \textbf{2056} & \textbf{1483} & \textbf{67.65}\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table 2.} Amplification success of preferred ``legacy'' loci for
Facetotecta phylogenetics.\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
\textbf{Locus} & \textbf{Primers} & \textbf{\# Tested} & \textbf{\#
Amplified} & \textbf{\% Success}\tabularnewline
\midrule
\endhead
\emph{COX1} & F1new/R1 + mlCOIInF/jgHCO2198 & 126 & 117 &
92.85714286\tabularnewline
\emph{18S} & Face1F/Face1R & 132 & 110 & 83.33333333\tabularnewline
\emph{18S} & Face2F/Face2R & 109 & 107 & 98.16513761\tabularnewline
\emph{18S} & Face1F/Face2R & 76 & 65 & 85.52631579\tabularnewline
\emph{28S} & Face3F/Face3R & 154 & 145 & 94.15584416\tabularnewline
\emph{28S} & 28S\_83F & 111 & 82 & 73.87387387\tabularnewline
\emph{12S} & 3F & 12 & 10 & 83.33333333\tabularnewline
\emph{16S} & 16S 137F/548R & 181 & 115 & 63.5359116\tabularnewline
\emph{16S} & SF/SR & 68 & 41 & 60.29411765\tabularnewline
\emph{H3} & H3af/H3ar & 96 & 67 & 69.79166667\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table 3} . Alignment and nucleotide substitution moedls chosen
for phylogeny estimation after GBLOCKS.\selectlanguage{english}
\begin{longtable}[]{@{}llllll@{}}
\toprule
\textbf{Locus} & \textbf{\# Sequences} & \textbf{\# Sites} & \textbf{\#
Informative sites} & \textbf{\# Invariable sites} &
\textbf{Model}\tabularnewline
\midrule
\endhead
\emph{12S} & 21 & 404 & 122 & 250 & TVM+F+I+G4\tabularnewline
\emph{16S} & 52 & 321 & 160 & 138 & TVM+F+I+G4\tabularnewline
\emph{18Sface1} & 46 & 1383 & 299 & 1037 & Tne+R2\tabularnewline
\emph{18Sface2} & 70 & 240 & 36 & 196 & K2P+I+G4\tabularnewline
\emph{28S} & 72 & 648 & 76 & 552 & K2P+I+G4\tabularnewline
\emph{COX1} & 33 & 419 & 173 & 214 & TVM+F+I+G4\tabularnewline
\emph{H3} & 25 & 260 & 73 & 169 & TIM2+F+I+I+R2\tabularnewline
Total & \textbf{294} & \textbf{3415} & \textbf{866} & \textbf{2387} &
\textbf{N/A}\tabularnewline
\bottomrule
\end{longtable}

ELECTRONIC SUPPLEMENTARY TABLES (can be obtained directly through
FigShare: https://figshare.com/s/9a08fa4635dd4f5c4b14)

\emph{Table S1}\textbf{.} Overview of specimens, collection data and
GenBank accession numbers of specimens used in the present study.

Table S2 . Primer amplification success for the two DNA extraction
methods.

DATA ACCESSIBILITY STATEMENT

All new sequences have been deposited in NCBI GenBank under accession
numbers XXXX-YYYY.

All figures, tables and supplementary material can be found at:
https://figshare.com/s/9a08fa4635dd4f5c4b14

BENEFIT-SHARING STATEMENT

Benefits generated: This project is part of a larger research
collaboration seeking to understand the biodiversity and evolution of
the enigmatic crustacean group Facetotecta. The collaboration was
developed with scientists from diverse countries, all of which provided
unique samples and are co-authors of the present paper. Our research
targets providing molecular resources for understanding y-larva
evolution, but the protocols should work for any invertebrate taxon with
molting larvae. Our project will therefore benefit the wider community
in evolutionary ecology. Our research group is committed to broad,
diverse, and inclusive scientific collaborations, evidenced by our team
members and samplings originating from diverse countries.\selectlanguage{english}
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