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\begin{document}

\title{A polyvalent and universal tool for genomic studies in gastropod
molluscs (Heterobranchia: Tectipleura)}



\author[1]{Juan Moles}%
\author[1]{Gonzalo Giribet}%
\affil[1]{Harvard University Faculty of Arts and Sciences}%


\vspace{-1em}



  
  \date{May 26, 2024}


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\selectlanguage{english}
\begin{abstract}
Molluscs are the second most diverse animal phylum and heterobranch
gastropods present \textasciitilde{}44,000 species. These comprise
fascinating creatures with a huge morphological and ecological
disparity. Such great diversity comes with even larger phylogenetic
uncertainty and many taxa have been largely neglected in molecular
assessments. Genomic tools have provided resolution to deep cladogenic
events but generating large numbers of transcriptomes/genomes is
expensive and usually requires fresh material. Here we leverage a target
enrichment approach to design and synthesize a probe set based on
available genomes and transcriptomes across Heterobranchia. Our probe
set contains 57,606 70mer baits and targets a total of 2,259
ultra-conserved elements (UCEs). Post-sequencing capture efficiency was
tested against 31 marine heterobranchs from major groups, including
Acochlidia, Acteonoidea, Aplysiida, Cephalaspidea, Pleurobranchida,
Pteropoda, Runcinida, Sacoglossa, and Umbraculida. The combined Trinity
and Velvet assemblies recovered up to 2,211 UCEs in Tectipleura and up
to 1,978 in Nudipleura, the most distantly related taxon to our core
study group. Total alignment length was 525,599 bp and contained 52\%
informative sites and 21\% missing data. Maximum-likelihood and Bayesian
inference approaches recovered the monophyly of all orders tested as
well as the larger clades Nudipleura, Panpulmonata, and
Euopisthobranchia. The successful enrichment of diversely preserved
material and DNA concentrations demonstrate the polyvalent nature of
UCEs, and the universality of the probe set designed. We believe this
probe set will enable multiple, interesting lines of research, that will
benefit from an inexpensive and largely informative tool that will,
additionally, benefit from the access to museum collections to gather
genomic data.%
\end{abstract}%



\sloppy


\textbf{Introduction}

Molecular data have played an important role in elucidating molluscan
relationships in general and gastropod systematics in particular, to the
point the classification of many higher clades is now dominated by
molecular-based estimations of phylogeny. Early work on molecular
systematics of molluscs started, as in the case of many other animal
groups, using a series of markers amplified by PCR, which in another
context have been dubbed the ``workhorses'' of molecular systematics
(Sharma \& Giribet, 2009) or the ``usual suspects'' (Dimitrov et al.,
2017). From the early days of molecular systematics of molluscs (e.g.,
Giribet et al., 2006; Passamaneck, Schander, \& Halanych, 2004;
Winnepenninckx, Backeljau, \& De Wachter, 1996), including the early
days of gastropod molecular trees (e.g., Colgan, Ponder, Beacham, \&
Macaranas, 2007, 2003; Colgan, Ponder, \& Eggler, 2000; Harasewych et
al., 1997; Harasewych, Adamkewicz, Plassmeyer, \& Gillevet, 1998;
Harasewych \& McArthur, 2000; McArthur \& Harasewych, 2003), these
markers (mainly nuclear ribosomal RNAs, nuclear protein-encoding histone
H3, and mitochondrial 16S rRNA and cytochrome \emph{c} oxidase subunit
I) have helped to shape the gastropod tree. A first paradigm shift
occurred with the generalized used of ESTs (e.g., Dunn et al., 2008) and
later, large numbers of transcriptomes soon started accumulating for
molluscs (Cunha \& Giribet, 2019; Gonz\selectlanguage{ngerman}ález et al., 2015; Kocot et al.,
2011; Kocot, Halanych, \& Krug, 2013; Kocot, Poustka, Stöger, Halanych,
\& Schrödl, 2020; Kocot, Todt, Mikkelsen, \& Halanych, 2019; Lemer,
Bieler, \& Giribet, 2019; Lemer, González, Bieler, \& Giribet, 2016;
Lindgren \& Anderson, 2018; Pabst \& Kocot, 2018; Smith et al., 2011;
Tanner et al., 2017; Zapata et al., 2014). These datasets provided
resolution to deep nodes, previously poorly supported using the standard
Sanger markers. But transcriptomes are difficult to obtain for large
numbers of taxa, as they require fresh tissue and special preservation
to avoid RNA degradation, and are expensive to generate (Zaharias,
Pante, Gey, Fedosov, \& Puillandre, 2020). A third strategy, able to
make use of DNA available from many museum specimens, but avoiding
PCR-amplification, are methods based on large numbers of hybridizing
probes sequenced with high-throughput techniques, i.e., Illumina
sequencing (Crawford et al., 2012; Faircloth et al., 2012; Lemmon, Emme,
\& Lemmon, 2012; Lemmon \& Lemmon, 2012; McCormack et al., 2012). These
bait-capture techniques have been recently applied to study gastropod
phylogenetics (Abdelkrim et al., 2018; Choo et al., 2020; Zaharias et
al., 2020), but available baits have been designed specifically for a
genus of Pteropoda and a subset of Neogastropoda. These latter studies
were able to include ``museum samples'' (specimens not collected and
preserved for molecular work) (Abdelkrim et al., 2018), and more
recently these techniques have been applied to ethanol-preserved
specimens older than 100 years (Derkarabetian, Benavides, \& Giribet,
2019). Therefore, to capitalize on available museum samples and to be
able to sequence thousands of loci across heterobranch gastropods---the
most diverse subclass of gastropods, with ca. 44,000 living species
(Barker, 2001; Lydeard \& Cummings, 2019; WoRMS Editorial Board,
2020)---we have designed a new set of probes for ultra-conserved
elements (UCEs) with the aim to apply it to future studies across
heterobranch taxa.

Heterobranchs embody a diverse and charismatic group of marine, limnic,
and terrestrial snails and slugs with a plethora of ecological and
morphological adaptations to all environments, e.g., pelagic
(Klussmann-Kolb \& Dinapoli, 2006), abyssal (Chaban et al., 2019),
meiofaunal (Jörger et al., 2010), parasitic (Dinapoli, Zinssmeister, \&
Klussmann-Kolb, 2011). They represent the cornerstone of interesting
lines of research including chemical ecology and pharmaceutical
applications (reviewed in Avila, Núñez-Pons, \& Moles, 2018),
solar-powered slugs are among the only Metazoa able to incorporate
chloroplasts from dietary algae which remain photosynthetically active
in their tissues (i.e., kleptoplasty; Wägele et al., 2011), the giant
neurones of e.g. \emph{Aplysia} became a key model in neurobiology
studies (e.g., Kandel, 1979), some terrestrial snails and slugs are
detrimental pests or vectors of snail-borne human parasitic diseases
such as \selectlanguage{english}angiostrongyliasis, bilharzia or liver rot (Lu et al., 2018),
also, many species are indicators of ecosystem wellbeing and climate
change (Keul et al., 2017). Still, understanding the evolutionary
history of Heterobranchia has been difficult (reviewed in W\selectlanguage{ngerman}ägele,
Klussmann-Kolb, Verbeek, \& Schrödl, 2014), even when the monophyly of
the group has been well established.

Although traditionally divided into two large gastropod subclasses,
Heterobranchia now includes the polyphyletic Opisthobranchia and the
paraphyletic Pulmonata plus some other shelled `prosobranch' lineages
(Schrödl, Jörger, Klussmann-Kolb, \& Wilson, 2011). Among the
morphological traits that define the taxon, hermaphroditism, a gill of
heterogeneous nature, a heterostrophic protoconch, spiral-shaped sperm,
and a pallial kidney are shared characteristics (Brenzinger, Haszprunar,
\& Schrödl, 2013; Haszprunar, 1985; Wägele et al., 2014). Although the
monophyly of most major taxa is well supported, some of their
interrelationships among and within subgroups remain controversial. For
instance, the uncertain systematic placement of some obscure lineages of
`lower heterobranchs', such as Acteonoidea, Rissoelloidea or
Rhodopemorpha, that lack an euthyneurous (i.e. detorted) nervous system
(Brenzinger et al., 2013; Wägele et al., 2014), awaits resolution. Among
Euthyneura, two major clades are accepted: Tectipleura (Panpulmonata +
Euopisthobranchia) and Ringipleura (Ringiculoidea + Nudipleura) (Kano,
Brenzinger, Nützel, Wilson, \& Schrödl, 2016). Panpulmonata includes
land snails and slugs (Stylommatophora), many limnic (e.g. Hygrophila),
marine intertidal (Siphonarioidea), marine interstitial (Acochlidia),
and marine ectoparasitic lineages (Pyramidelloidea), as well as the
marine solar-powered slugs (Sacoglossa) (Jörger et al., 2010; Kano et
al., 2016; Kocot et al., 2013; Zapata et al., 2014). Euopisthobranchia
comprises sea hares (Aplysiida), pelagic sea angels (Pteropoda), bubble
snails \emph{sensu lato} (Cephalaspidea), false limpets (Umbraculida),
and Runcinida (Jörger et al., 2010; Kano et al., 2016; Zapata et al.,
2014). Nudipleura includes the side-gilled slugs (Pleurobranchida) and
the colourful sea slugs (Nudibranchia) (Kano et al., 2016; Pabst \&
Kocot, 2018; Wägele \& Willan, 2000; Zapata et al., 2014). The inclusion
of novel genomic approaches to better reconstruct the evolutionary
history of Heterobranchia from high-ranking to the species level remains
crucial (Cunha \& Giribet, 2019; Goodheart, Bazinet, Collins, \&
Cummings, 2015; Kocot et al., 2013; Pabst \& Kocot, 2018; Peijnenburg et
al., 2019; Zapata et al., 2014). Moreover, the possibility to provide
molecular evidence from museum-preserved specimens thanks to UCEs will
render elusive taxa and/or type material available for study. Hence, a
new systematic framework may provide input on the mode and tempo to
interesting ecological questions such as the reduction or loss of the
shell across Heterobranchia (Medina et al., 2011; Wägele \&
Klussmann-Kolb, 2005), the acquisition of defensive mechanisms
alternative to the shell (Avila et al., 2018; Vonnemann, Schrödl,
Klussmann-Kolb, \& Wägele, 2005), the adaptation to freshwater and
terrestrial habitats (Klussmann-Kolb, Dinapoli, Kuhn, Streit, \&
Albrecht, 2008; Neusser, Jörger, Lodde-Bensch, Strong, \& Schrödl,
2016), morpho-anatomical transitions and adaptations (Brenzinger et al.,
2013; Kano et al., 2016) or evolutionary dietary patterns (Goodheart,
Bazinet, Valdés, Collins, \& Cummings, 2017; Malaquias, Berecibar, \&
Reid, 2009), among many other hot topics on this hyperdiverse group of
molluscs.

\textbf{Material and Methods}

\emph{Taxon sampling}

Genomes of selected gastropods, including the aplysiid \emph{Aplysia
californica} (base genome), the sacoglossan \emph{Elysia chlorotica} ,
the hygrophilid \emph{Radix auricularia} , and the
caenogastropod\emph{Pomacea canaliculata} (used as outgroup), and
transcriptomes of the cephalaspidean \emph{Haminoea antillarum} and the
umbraculid\emph{Tylodina fungina} were downloaded from NCBI. Additional
transcriptomes across Heterobranchia, including Acochlidia, Acteonida,
Architectonicoidea, Ellobiida, Nudibranchia, Pleurobranchida, Pteropoda,
Pylopulmonata, and Rissoelloidea, were downloaded for the \emph{in
silico} test of the bait set. Finally, two Caenogastropoda were
downloaded for matrix construction and for rooting the phylogenetic
trees (Table 1, S1).

Specimens for the \emph{in vitro} test of the bait set were obtained
from the Museum of Comparative Zoology (MCZ) and the \selectlanguage{english}University Museum
of Bergen (Department of Natural History; ZMBN, Norway) (Table 2). Most
of the material was freshly collected from the Maldives in 2019,
preserved at 95\% EtOH and kept at --20 \selectlanguage{ngerman}°C. The remaining samples were
gathered from museum collections, mostly preserved in 70\% EtOH and kept
at room temperature, sometimes for several years, and are thus
considered `degraded' samples.

\emph{Bait set design and synthesis}

All downloaded transcriptomes were assembled \emph{de novo} using the
pipeline from Cunha \& Giribet (2019). Briefly, quality threshold
filtering was conducted with Rcorrector v. 3.0 (Song \& Florea, 2015)
and Trim Galore! V. 3
(\emph{http://www.bioinformatics.babraham.ac.uk/projects/trim\_galore/}).
rRNA and mitochondrial unwanted sequences for molluscs were filtered out
using Bowtie2 v. 2.3.2 (Langmead \& Salzberg, 2012). Paired-end reads
were \emph{de novo} assembled into transcripts with Trinity v. 2.4
(Grabherr et al., 2010; Haas et al., 2013). A second Bowtie2 round and
CD-HIT-EST v. 4.6.4 were used to reduce sequence redundancy (Fu, Niu,
Zhu, Wu, \& Li, 2012). The software PHYLUCE (Faircloth, 2016) was then
used to identify UCE loci and design baits to target them using the
online tutorial
(\emph{https://phyluce.readthedocs.io/en/latest/tutorial-four.html}).
Downloaded FASTA files for the selected genomes/transcriptomes were
reformatted into 2bit using faToTwoBit and headers were modified using
Bio.SeqIO (Grüning et al., 2018) for compatibility with PHYLUCE.ART
(Huang, Li, Myers, \& Marth, 2012), which was used to simulate reads of
100 bp in length, covering the genome randomly to roughly 2X, and having
an insert size of 200 bp (150 SD), for each species. These were
individually mapped into putatively orthologous loci with a sequence
divergence of \textless{} 5\% from our base genome (\emph{A.
californica} ) using stampy v. 1 (Lunter \& Goodson, 2011) and unmapped
reads were removed with SAMtools v. 1.5 (Li et al., 2009). BEDTools
(Quinlan \& Hall, 2010) was used to convert BAM files, sort the contigs
by scaffold and position, and merge them in putative conserved regions.
Intervals where the base genome was shorter than 80 bp and where
\textgreater{} 25 \% of the base genome was masked (i.e., repetitive
regions) were deleted in PHYLUCE.

An SQLite table was created to query for conserved loci across taxa with
an optimal number of four out of five taxa, resulting in a total of
7,222 shared loci. Temporary baits were designed to capture loci shared
among the base genome and the exemplar taxa, buffering to 160 bp to
ensure designing two 120mers per locus with 3x tiling density, removing
potentially problematic baits with \textgreater{}25\% masking and GC
content outside of a 30--70\% range. Finally, potential duplicates of
\textgreater{}50\% identity and coverage were parsed and removed. In
order to include baits designed from the base genome and the exemplar
taxa, the temporary baits were also aligned against all five exemplar
taxa and conserved loci were extracted as FASTA files. An additional
SQLite table was created to check for the loci found consistently across
taxa.

We finally decided to target loci that were shared among five out of the
six taxa, totalling 2,320. Final bait design was performed using the
abovementioned steps but using both the base genome and the rest of the
exemplar taxa. A subset of locus bait set targeting only the specific
heterobranch species (excluding the caenogastropod \emph{P.
canaliculata} ) was designed using
phyluce\_probe\_get\_subsets\_of\_tiled\_probes. The final set contained
19,333 baits and targeted 2,259 loci across Tectipleura. \emph{In
silico} tests of the UCE bait set against \emph{de novo} assembled
transcriptomes were performed against a wide range of taxa belonging to
Heterobranchia and two Caenogastropoda outgroups
using\emph{phyluce\_assembly\_match\_contigs\_to\_probes} (see Table 1).

In order to synthesize the designed bait set, each bait candidate was
BLASTed against the base genome in order to filter non-specific or
over-represented regions and a hybridization melting temperature
(defined as the temperature at which 50\% of molecules are hybridized)
was estimated for each hit assuming standard myBaits® (Arbor
Biosciences, MI, USA) buffers and conditions. There were 129 baits that
matched a portion of the genome that was \textgreater{}25\% soft masked
for repeats and 5 baits failed out Moderate BLAST analysis (candidates
pass if they have at most 10 hits at 62.5--65 °C and 2 hits above 65 °C,
and fewer than 2 passing baits on each flank), indicating they had
multiple hits to the genome at the hybridization temperature and, thus,
were removed. Due to technical difficulties in synthesizing the 120mer
set, three overlapping 70mers for each 120mer were designed (1 bait
every 25 nt), both providing the same coverage of the original design
targets with the same capture efficiency as the 120mer design.
Interestingly, shorter fragments have the ability to be used at a range
of hybridization temperatures, thus, being more effective on degraded
museum samples. The total design had 57,606 baits (out of the original
19,202 120mer set).

\emph{Molecular data collection}

Genomic DNA from freshly preserved material was extracted from the
parapodia and/or foot using the DNeasy Blood and Tissue kit (Qiagen
Inc., Valencia, CA, USA) following the manufacturer's protocol. In order
to obtain a higher DNA yield for millimetric specimens and a
non-destructive DNA extraction of mostly `degraded' whole museum
individuals we used the protocol described in Tin, Economo \& Mikheyev
(2014) using silica-based beads, with some in-lab modifications
(Derkarabetian et al., 2019). \selectlanguage{english}Extractions were quantified using the
Qubit 2.0 fluorometer (Life Technologies, Inc.) dsDNA High Sensitivity
kit and visualized on a 2200 Tape Station (Agilent) to assess DNA
degradation. Up to 500 ng of DNA in 130 \selectlanguage{greek}µ\selectlanguage{english}L for all fresh specimens was
sonicated for 80 s with a Covaris S220 Focused-ultrasonicator for a
target peak of 500--600 bp, \selectlanguage{english}with a Peak Incidence Power of 50, Duty
Factor of 10\%, and 200 cycles per burst. Sonication time was adjusted
depending on the Tape Station results for each sample since museum
samples were naturally degraded to the appropriate size for sequence
capture library preparation (see Table 2).

Library preparation followed the general protocol on the UCE website
(\selectlanguage{english}https://www.ultraconserved.org/) and some in-lab modifications
(Derkarabetian et al., 2018). Libraries were prepared using the KAPA
Hyper Prep kit following the manufacturer's protocol and using up to 250
ng of DNA as starting material. For samples with lower DNA yield, we
used it all (down to 5 ng). We used fresh Serapure Speed-beads for all
clean-up steps (Rohland \& Reich, 2012) washing with freshly prepared
80\% EtOH. After the first clean-up, 25 \selectlanguage{greek}µ\selectlanguage{english}L of fragmented,
double-stranded DNA was assembled for end repair and A-tailing, 20 \selectlanguage{ngerman}°C
for 30 min and 65 °C for 30 min, respectively. Immediately afterward we
proceeded to adapter ligation, samples with \textgreater{}200 ng of DNA
were run at 20 °C for 30 min using the universal iTru Stubs at 10 \selectlanguage{greek}µ\selectlanguage{english}M,
while samples \textless{}200 ng were run up to 1h using 5 \selectlanguage{greek}µ\selectlanguage{english}M iTru Stubs.
An immediate post-ligation clean-up step was carried at 0.8X for
high-yield, fresh samples, and at up to 3X for degraded, low-yield
samples. Library amplification was conducted using 25 \selectlanguage{greek}µ\selectlanguage{english}L of post-ligated
libraries, using individual iTrue i5/i7 dual index adaptors (8 bp long ;
Glenn et al., 2016), with an initial denaturation step at 98 \selectlanguage{ngerman}°C for 45
s, then 6--14 PCR cycles (adjusted to post-ligated Qubit concentrations)
of 98 °C for 15 s, 60 °C for 30 s and 72 °C for 1 min, and a final
extension step of 72 °C for 5 min. Libraries were then washed,
quantified, and 125 ng of each were pooled in batches of eight samples
for a total of 1000 ng. Pools were then speed-vacuumed if necessary, to
a final volume of 14 \selectlanguage{greek}µ\selectlanguage{english}L.

Hybridization capture for targeted UCEs was carried following the
myBaits\selectlanguage{ngerman}® v.4 user's manual and the target enrichment for Illumina
standard workflow protocols
(\emph{https://www.ultraconserved.org/\#protocols}). Pooled libraries
were hybridized with the synthesized bait set at 60 °C for 24 h. Hybrid
pools were then bound to streptavidin beads\selectlanguage{english} (Dynabeads MyOne C1,
Invitrogen), washed, and eluted in 31 \selectlanguage{greek}µ\selectlanguage{english}L of NF Water. Post-hybridization
amplification was carried in a 50 \selectlanguage{greek}µ\selectlanguage{english}L reaction using 15 \selectlanguage{greek}µ\selectlanguage{english}L of hybridized
pools, with same post-ligation PCR conditions but for a total of 16
cycles. Immediately followed a bead clean up with 70\% EtOH, Qubit 2.0
quantification, and visualization and molarity calculation using a 2200
Tape Station. \selectlanguage{english}Post-hybridized pools were pooled in equimolar amounts
and sequenced in the Illumina NovaSeq 6000 SP platform (paired-end, 150
bp) at the Bauer Core Facility, Harvard University. New sequences were
deposited in the NCBI Sequence Read Archive (BioProject PRJNA612319);
voucher information and assembly statistics are available in Table 2 and
S2.

\emph{Species and matrix assembly}

Raw reads were demultiplexed per individual and the software PHYLUCE v
1.6.8 (Faircloth, 2016) was used for each species assembly. Raw reads
were trimmed for adapter contamination and low-quality bases with
trimmomatic v 0.39 (Bolger, Lohse, \& Usadel, 2014) implemented in
illumiprocessor v 2.0.9 (Faircloth, 2013). We then run cd-hit-dup v
4.6.4 for removing duplicates from sequencing reads (Fu et al., 2012).
We used the three assemblers: ABySS v 2.0 (Jackman et al., 2017),
Trinity v. 2.1.1 (Haas et al., 2013), and Velvet v 1.2.10 (Zerbino \&
Birney, 2008) for comparative purposes (see Table S2). In order to
recover a higher number of contigs, we combined both Velvet and Trinity
assemblies and matched the contigs to the probe set with the modified
version of the original script by B.
Faircloth\emph{phyluce\_assembly\_match\_contigs\_to\_probes\_duplicates}
with\emph{--min-coverage} and \emph{--min-identity} of 80\emph{.}

Assembly QC statistics are shown in Table 3. Contigs that represent the
targeted UCE loci were captured and duplicated loci --either different
probes hitting the same locus or a probe hitting multiple loci-- were
removed. A list of the enriched UCE loci in each taxon, including
incomplete loci (not found in all the taxon set), was generated and
individual FASTA files for these were extracted (Table 2; see Table S2
for summary statistics for each species using each of the three
assemblers). In this step, we also included the UCE loci captured for
all genomes and transcriptome assemblies used for the probe set design
and two transcriptomes from caenogastropods. Selected UCE loci were
aligned in MAFFT v 7.455 (Katoh \& Standley, 2013). Edge and internal
trimming of the resulting alignments was conducted using Gblocks v 0.91
(Castresana, 2000; Talavera \& Castresana, 2007), \selectlanguage{english}specifying mid-level
arguments, ideal for higher-level taxonomic ranking phylogenies,
i.e.\emph{--b1} 0.5, \emph{--b2} 0.5, \emph{--b3} 6, \emph{--b4} 4. A
matrix was then built using a percentage of completeness of 50\% (see
Table 3 for the number of genes per species in the final datasets).

\emph{Phylogenetic analyses}

Phylogenetic trees were reconstructed using maximum likelihood (ML)
implemented in IQ-TREE v. 1.5.5 (Nguyen, Schmidt, Von Haeseler, \& Minh,
2015) under the general time-reversible (Tavar\selectlanguage{ngerman}é, 1986), four class
mixture model (GTR+FO*H4) on the unpartitioned matrix. This is a General
Heterogeneous evolution On a Single Topology model (GHOST) that infers
separate base frequencies per class and accounts for heterotachy or rate
variation across sites and lineages (Crotty et al., 2020). Bootstrap
support values (bs) were estimated via the ultrafast bootstrap algorithm
with 1500 replicates (Minh, Nguyen, \& Haeseler, 2013). \selectlanguage{english}Bayesian
inference (BI) was assessed in ExaBayes v 1.5 (Aberer, Kobert, \&
Stamatakis, 2014) with the implemented GTRGAMMA model with four coupled
\selectlanguage{english}Markov chain Monte Carlo (MCMC) runs, each with 10 million generations,
and sampling every 500 generations. Convergence was checked based on the
average standard deviation of split frequencies (ASDSF
\textless{}0.2\%). \selectlanguage{english}The first 25\% of the trees were discarded as
burn-in for each MCMC run prior to convergence. \selectlanguage{english}Topological robustness
was assessed using posterior probabilities (pp). Trees were visualized
in FigTree v. 1.4.4 (Rambaut, 2014) and edited in Adobe Illustrator CC
2018 (Fig. 1).

\textbf{Results \& Discussion}

Here we provide a relatively inexpensive and universal probe set for
sequencing ca. two thousand loci across Heterobranchia, using a
particular subset of genomes and transcriptomes of relevant clades
within Tectipleura (Euopisthobranchia and Panpulmonata). The final bait
set contains 57,606 70mer baits, overlapping threefold with the
originally designed 19,333 120mer baits, with the advantage of better
capturing degraded DNA from museum material. This was designed to target
a total of 2,259 loci across some of the major clades of Tectipleura,
i.e. a sea hare (Aplysiida), a bubble snail (Cephalaspidea), a false
limpet (Umbraculida), a hygrophil limnic snail (Hygrophila), and a
solar-powered slug (Sacoglossa). Prior to synthesis, the bait set was
tested \emph{in silico} against 30 Trinity \emph{de novo} transcriptome
assemblies of multiple lineages across Heterobranchia, with a number of
captured UCEs of 136 to 966 (6--43\%). Congruently, the number of loci
captured increased when approaching our core taxon Tectipleura (Table
1).

For testing the efficiency of the designed probe set, 31 taxa across the
major groups of marine Heterobranchia were sequenced. Samples were
collected during 2001--2019 and were either preserved in 70\% or 95\%
EtOH. Regular Qiagen or the `degraded' museum specimens' extractions for
millimetric specimens were carried out. DNA concentrations ranged from
undetectable (\textless{}0.01 \selectlanguage{greek}μ\selectlanguage{english}g/mL) to very high (\textgreater{}600
\selectlanguage{greek}μ\selectlanguage{english}g/mL) and the Covaris sonication shearing time was adjusted on samples
with degraded DNA (Table 2). Also, additional museum samples older than
40 years, preserved in 70\% EtOH, and kept at room temperature, yielded
\textgreater{}1,000 UCEs (auth. unpubl. data). The total number of
Illumina raw reads obtained ranged from 4 to 32 million but in the
species \emph{Creseis} \emph{acicula} and \emph{Pontohedyle
milaschewitchii} the number of reads decreased substantially after
trimming. These were excluded from the final matrix construction. In
order to increase the yield of captured UCEs, both Trinity and Velvet
assemblies were combined since they recovered larger contig lengths than
the ABySS assembly (Table S2). The genomes and transcriptomes
of\emph{Aplysia californica} , \emph{Elysia chlorotica} , \emph{Radix
auricularia} , \emph{Haminoea antillarum} , and \emph{Tylodina
fungina}used for the bait set design were included in the trees.
Additionally,\emph{Chraronia tritonis} and \emph{Crepidula navicella}
were used as outgroups. The final matrix contained 36 taxa and captured
all our 2,259 originally targeted UCEs, with species of Aplysiida
displaying a 92--98\% efficiency in captured UCEs, 67--95\% in
Cephalaspidea, 93--95\% in Umbraculida, 83--92\% in Sacoglossa, and
35--72\% in Runcinida (Table 3). Samples from the most distantly related
groups to our core taxon captured 73--86\% UCEs in Nudibranchia, up to
81\% in Pleurobranchida, and 76--87\% in Acteonoidea (Table 3). The
final alignment of the concatenated data set contained 525,599 bp with a
mean contig size per UCE of 233 \selectlanguage{ngerman}± 5 bp with 273,694 informative sites
(52\%) across all loci and less than 21\% missing data (mostly found in
the downloaded transcriptomes; Table 4). The final matrix contained
12,557,760 nucleotides out of 14,352,347 possible characters, not
accounting for the sequences not represented in each alignment (less
than 13\% of missing data). The 50\% occupancy matrix contained 2,156
loci. Overall, our probe set opens up to the possibility to access old
museum collections of usually not-well preserved specimens for molecular
analysis to be used in future phylogenetic assessments across
Heterobranchia (see Derkarabetian et al., 2019). Type taxa and obscure
lineages, sometimes seldom recorded again since their original
description, could potentially be available for genomic studies
henceforth.

Both BI and ML analyses recovered the monophyly (bs = 100, pp = 1.00) of
the orders and superorders Pleurobranchida, Nudibranchia, Sacoglossa (bs
= 100, pp = 0.94), Acteonoidea, Umbraculida, Aplysiida, Runcinida, and
Cephalaspidea with maximum support (Fig. 1). The monophyly of
Euopisthobranchia (Aplysiida + Runcinida + Umbraculida; bb = 100, pp =
1.00), Panpulmonata (Sacoglossa + Hygrophila; bb = 100, pp = 1.00), and
Nudipleura (Nudibranchia + Pleurobranchida; bb = 100, pp = 1.00), as
well as for our core group of study Tectipleura only in the BI
(Euopisthobranchia + Panpulmonata; pp = 1.00). The phylogenetic position
of Acteonoidea was ambiguous, placed as the sister group to Tectipleura
in the ML analysis (bs = 98) or as the sister group to Euthyneura in the
BI (pp = 1.00). Our results are congruent with previous studies using
multilocus Sanger sequencing and transcriptomic assessments (Jörger et
al., 2010; Kano et al., 2016; Schrödl et al., 2011; Zapata et al.,
2014). We have proven the versatility of UCEs combined with
transcriptomic and genomic available data and it is a matter of time
that a comprehensive and thorough genomic dataset is amassed to better
establish the evolutionary history of Heterobranchia and its
interrelationships. Big questions remain to be answered, for instance,
the position of the non-Euthyneura groups (Brenzinger et al., 2013),
such as the controversies on the Acteonoidea. Sanger sequencing
assessments have placed them as the sister group to Nudipleura (i.e.,
Acteopleura; Medina et al., 2011), or in a stemward position the sister
group to Euthyneura (Kano et al., 2016), the latter result also
supported by anatomical evidence and our BI analysis. Transcriptomic
data and our ML results recover Acteonoidea as the sister group to
Tectipleura (Pabst \& Kocot, 2018; Zapata et al., 2014), although
sometimes with little or no support. The latter scenario could imply
acteonoids having retained plesiomorphic anatomical characters such as
the torted nervous system (i.e. streptoneury). Evidently, a
comprehensive taxon sampling, including Rissoellida (+ Acteonidae
=Acteonimorpha) among other taxa, will help clarify the disagreeing
topologies recovered. The position of Runcinida as the sister group to
Cephalaspidea has also been recently supported (Malaquias,
Mackenzie-Dodds, Bouchet, Gosliner, \& Reid, 2009; Oskars, Bouchet, \&
Malaquias, 2015) and here we recovered that same topology. Additional
taxa remain to be included in order to resolve deeper relationships,
such as Pteropoda and major Panpulmonata groups, i.e. Acochlidia,
Amphiboloidea, Eupulmonata (Stylommatophora, Systellommatophora,
Ellobioidea), Glacidorboidea, Hygrophila, Pyramidelloidea, and
Siphonarioidea.

Due to the polyvalent nature of UCEs and the universality of the probe
set designed we believe this study will lead to multiple, interesting
lines of research in gastropod molluscs, not only resolving the
phylogenetic conundrum between many large clades but also aiming at
establishing the systematics within major subgroups, which still present
many unresolved relationships at species, genus or family level
(Carmona, Pola, Gosliner, \& Cervera, 2013; Epstein, Hallas, Johnson,
Lopez, \& Gosliner, 2018; Goodheart, 2017; Korshunova et al., 2020;
Krug, Vendetti, \& Valdés, 2016; Moles, Avila, \& Malaquias, 2019;
Oskars et al., 2019; Padula et al., 2016).

\textbf{Acknowledgements}

We are indebted to the members of the Giribet lab, especially to Shahan
Derkarabetian for his advice during lab work and UCEs assembly. We are
also grateful to the Bauer Core and the RC Computing team. We are
especially thankful to Tauana J. Cunha and the MaRHE Center staff for
their help while collecting in the Maldives and for providing
photographs of live specimens. We appreciate the samples donated from
the GROC Association (\emph{https://opistobranquis.org/en/home}) and M.
A. E. Malaquias (University Museum of Bergen). Specimen collection was
funded by a Putnam Expedition Grant from the Museum of Comparative
Zoology to JM. Lab and analytical work were supported by internal funds
from the Faculty of Arts and Sciences, Harvard University, to GG. JM
postdoctoral fellowships were supported by the Ramón Areces (Spain) and
the Alexander von Humboldt (Germany) Foundations.

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\selectlanguage{english}\textbf{Data accessibility statement}

\textbf{} \selectlanguage{english}Raw reads for all newly sequenced samples have been deposited
\selectlanguage{english}in the NCBI Sequence Read Archive (BioProject PRJNA612319). The final
concatenated matrices, all individual locus alignments, and resulting
phylogenies are available from the Harvard Dataverse \selectlanguage{english}https://
doi.org/XXX.

\textbf{Author contributions}\selectlanguage{english}

\textbf{} \selectlanguage{english}J.M. designed and performed research, analysed data, and
wrote the paper. G.G. contributed with laboratory reagents, and
financial support. Both authors edited and approved the final
manuscript.

\textbf{Table 1.} Number of unique recovered loci from the \emph{in
silico} test of the baits after removing duplicates from genomes and
transcriptomes, including number of contigs and N50. Species in bold
were used for the bait set design.

\textbf{Clade}

\textbf{Species}

\textbf{Number of}

contigs

\textbf{N50}

\textbf{Number of unique contigs}

\textbf{\% UCEs}

Acochlidia

\emph{Microhedyle glandulifera}

131,208

621

520

23

Acochlidia

\emph{Turbonilla} sp\emph{.}

260,564

827

406

18

Acteonoidea

\emph{Hydatina physis}

111,189

1,132

196

8.7

\textbf{Aplysiida}

Aplysia californica

\textbf{164,545}

\textbf{9,586}

\textbf{2,107}

\textbf{93.3}

\textbf{Caenogastropoda}

Pomacea canaliculata

\textbf{14,655}

\textbf{81,153}

-

-

Architectonicoidea

\emph{Architectonica perspectiva}

31,468

803

259

11.5

Caenogastropoda

\emph{Charonia tritonis}

142,385

761

554

24.5

Caenogastropoda

\emph{Crepidula navicella}

160,229

871

348

15.4

\textbf{Cephalaspidea}

Haminoea antillarum

\textbf{133,818}

\textbf{933}

\textbf{714}

\textbf{31.6}

Ellobiida

\emph{Ophicardelus sulcatus}

127,388

856

618

27.4

Nudibranchia

\emph{Antiopella barbarensis}

55,493

635

151

6.7

Nudibranchia

\emph{Armina californica}

112,779

1,572

694

30.7

Nudibranchia

\emph{Dendronotus venustus}

126,496

923

459

20.3

Nudibranchia

\emph{Dirona picta}

75,472

944

194

8.6

Nudibranchia

\emph{Dondice occidentalis}

109,230

704

170

7.5

Nudibranchia

\emph{Doris kerguelenensis}

68,349

1,010

561

24.8

Nudibranchia

\emph{Flabellina iodinea}

115,498

978

200

8.9

Nudibranchia

\emph{Phylliroe bucephala}

69,583

954

393

17.4

Nudibranchia

\emph{Prodoris clavigera}

99,994

892

482

21.3

Nudibranchia

\emph{Tritonia tetraquetra}

63,261

465

136

6.0

Pleurobranchida

\emph{Bathyberthella antarctica}

56,335

880

231

10.2

Pleurobranchida

\emph{Pleurobranchaea californica}

246,413

1,222

409

18.1

Pteropoda

\emph{Clio pyramidata}

34,131

744

331

14.7

Pteropoda

\emph{Limacina antarctica}

86,747

1209

759

33.6

Pteropoda

\emph{Peracle reticulata}

133,448

882

853

37.8

Pteropoda

\emph{Spongiobranchaea australis}

100,691

1419

766

33.9

\textbf{Hygrophila}

Radix auricularia

\textbf{69,921}

\textbf{24,354}

\textbf{2,208}

\textbf{97.7}

Pylopulmonata

\emph{Phallomedusa solida}

141,712

993

628

27.8

Rissoelloidea

\emph{Rissoella caribaea}

142,330

1,709

421

18.6

\textbf{Sacoglossa}

Elysia chlorotica

\textbf{41,686}

\textbf{30,474}

\textbf{1,994}

\textbf{88.3}

Sacoglossa

\emph{Elysia timida}

101,458

1,286

728

32.2

Sacoglossa

\emph{Oxynoe viridis}

61,143

617

204

9

Systellommatophora

\emph{Onchidella floridana}

145,935

874

573

25.4

\textbf{Umbraculida}

Tylodina fungina

\textbf{79,344}

\textbf{751}

\textbf{966}

\textbf{42.8}

\textbf{Table 2.} List of species used in the \emph{in vitro} test of
designed baits with collection and DNA extraction details and UCE
assembly summary statistics.\selectlanguage{english}
\begin{longtable}[]{@{}llllllllllllllllllllllllll@{}}
\toprule
\textbf{Taxonomy} & \textbf{Taxonomy} & & \textbf{Collection} &
\textbf{Collection} & \textbf{Collection} & \textbf{Collection} &
\textbf{Collection} & \textbf{Extraction} & \textbf{Extraction} &
\textbf{Extraction} & & & & & \textbf{Trinity assembly} &
\textbf{Trinity assembly} & \textbf{Trinity assembly} & \textbf{Trinity
assembly} & & & \textbf{Velvet assembly} & \textbf{Velvet assembly} &
\textbf{Velvet assembly} & \textbf{Velvet assembly} &\tabularnewline
\midrule
\endhead
\textbf{Order} & \textbf{Species} & & \textbf{Voucher} &
\textbf{Locality} & \textbf{Locality} & \textbf{Year} & \textbf{Year} &
\textbf{Type} & \selectlanguage{greek}\textbf{Concentration (μg/mL)}\selectlanguage{english} & \textbf{Shearing
Covaris (s)} & \textbf{Shearing Covaris (s)} & \textbf{\# raw reads} &
\textbf{\# raw reads} & \textbf{\# trimmed reads} & \textbf{\# trimmed
reads} & \textbf{\# contigs} & \textbf{Mean contig length} & \textbf{\#
UCEs} & \textbf{\# UCEs} & & & \textbf{\# contigs} & \textbf{Mean contig
length} & \textbf{\# UCEs} & \textbf{\# UCEs}\tabularnewline
Aplysiida & \emph{Akera bullata} & & ZMBN 83032 & Italy & Italy & 2009 &
2009 & Qiagen & 29.7 & 60 & 60 & 6,845,394 & 6,845,394 & 6,140,197 &
6,140,197 & 218,855 & 314.6 & 1,307 & 1,307 & & & 1,364,834 & 174.4 &
1,457 & 1,457\tabularnewline
Aplysiida & \emph{Aplysia dactylomela} & & ZMBN 84917 & Venezuela &
Venezuela & 2010 & 2010 & Qiagen & 24.5 & 80 & 80 & 16,278,867 &
16,278,867 & 14,152,131 & 14,152,131 & 579,031 & 341.4 & 1,560 & 1,560 &
& & 1,426,074 & 232.0 & 1,711 & 1,711\tabularnewline
Pleurobranchida & \emph{Berthella martensi} & & MCZ 393762 & The
Maldives & The Maldives & 2019 & 2019 & Qiagen & \textgreater{}600 & 80
& 80 & 7,264,881 & 7,264,881 & 6,867,701 & 6,867,701 & 307,983 & 358.2 &
1,179 & 1,179 & & & 750,030 & 224.9 & 1,511 & 1,511\tabularnewline
Pleurobranchida & \emph{Berthellina} \emph{citrina} & & MCZ 393775 & The
Maldives & The Maldives & 2019 & 2019 & Qiagen & \textgreater{}600 & 80
& 80 & 293,381 & 293,381 & 80,793 & 80,793 & 1,546 & 257.4 & 38 & 38 & &
& 28,350 & 163.7 & 294 & 294\tabularnewline
Cephalaspidea & \emph{Bulla vernicosa} & & MCZ 393827 & The Maldives &
The Maldives & 2019 & 2019 & Qiagen & 9.57 & 80 & 80 & 7,058,179 &
7,058,179 & 5,861,958 & 5,861,958 & 315,980 & 337.7 & 1,247 & 1,247 & &
& 877,161 & 211.9 & 1,709 & 1,709\tabularnewline
Cephalaspidea & \emph{Colpodaspis thompsoni} & & MCZ 393765 & The
Maldives & The Maldives & 2019 & 2019 & Museum & 0.68 & 80 & 80 &
4,411,010 & 4,411,010 & 2,130,402 & 2,130,402 & 58,341 & 283.9 & 510 &
510 & & & 536,106 & 153.4 & 1,077 & 1,077\tabularnewline
Pteropoda & \emph{Creseis} \emph{acicula} & & MCZ 393923 & Spain & Spain
& 2018 & 2018 & Museum & 0.29 & 80 & 80 & 3,708,796 & 3,708,796 &
678,909 & 678,909 & 2,878 & 258.6 & 11 & 11 & & & 53,377 & 173.5 & 150 &
150\tabularnewline
Sacoglossa & \emph{Cyerce elegans} & & MCZ 393754 & The Maldives & The
Maldives & 2019 & 2019 & Qiagen & 11.1 & 80 & 80 & 10,361,171 &
10,361,171 & 9,247,804 & 9,247,804 & 489,150 & 372.0 & 1,317 & 1,317 & &
& 1,094,420 & 233.6 & 1,672 & 1,672\tabularnewline
Aplysiida & \emph{Dolabella auricularia} & & MCZ 393823 & The Maldives &
The Maldives & 2019 & 2019 & Qiagen & 100 & 80 & 80 & 32,564,674 &
32,564,674 & 28,918,755 & 28,918,755 & 1,300,173 & 405.0 & 1,512 & 1,512
& & & 2,011,703 & 327.4 & 1,548 & 1,548\tabularnewline
Aplysiida & \emph{Dolabrifera dolabrifera} & & MCZ 393712 & The Maldives
& The Maldives & 2019 & 2019 & Qiagen & 21.2 & 80 & 80 & 21,612,295 &
21,612,295 & 19,184,922 & 19,184,922 & 1,035,945 & 352.8 & 1,407 & 1,407
& & & 2,198,601 & 240.7 & 1,688 & 1,688\tabularnewline
Nudibranchia & \emph{Doto cervicenigra} & & MCZ 393347 & Spain & Spain &
2018 & 2018 & Qiagen & 1.74 & 80 & 80 & 10,193,363 & 10,193,363 &
9,258,214 & 9,258,214 & 302,361 & 355.6 & 762 & 762 & & & 757,873 &
204.7 & 1,259 & 1,259\tabularnewline
Sacoglossa & \emph{Elysia pusilla} & & MCZ 393737 & The Maldives & The
Maldives & 2019 & 2019 & Qiagen & 2.53 & 80 & 80 & 3,943,273 & 3,943,273
& 2,853,145 & 2,853,145 & 102,222 & 309.3 & 1,233 & 1,233 & & & 342,899
& 186.5 & 1,695 & 1,695\tabularnewline
Nudibranchia & \emph{Glossodoris acosti} & \emph{Glossodoris acosti} &
MCZ 393870 & MCZ 393870 & The Maldives & The Maldives & 2019 & Qiagen &
5.42 & 80 & 80 & 10,388,101 & 10,388,101 & 9,684,855 & 9,684,855 &
394,185 & 323.4 & 1,192 & 1,192 & 1,192 & & 1,242,851 & 204.0 & 1,403 &
1,403\tabularnewline
Nudibranchia & \emph{Godiva quadricolor} & & MCZ 393921 & France &
France & 2018 & 2018 & Qiagen & \textgreater{}600 & 80 & 80 & 11,485,405
& 11,485,405 & 10,018,439 & 10,018,439 & 392,392 & 327.6 & 739 & 739 & &
& 1,178,406 & 192.7 & 1,329 & 1,329\tabularnewline
Cephalaspidea & \emph{Lamprohaminoea cymbalum} & \emph{Lamprohaminoea
cymbalum} & MCZ 393734 & MCZ 393734 & The Maldives & The Maldives & 2019
& Qiagen & 10.1 & 80 & 80 & 5,266,226 & 5,266,226 & 4,820,721 &
4,820,721 & 223,514 & 351.1 & 1,229 & 1,229 & 1,229 & & 682,073 & 210.5
& 1,766 & 1,766\tabularnewline
Acteonida & \emph{Hydatina} sp. & & MCZ 382639 & Australia & Australia &
2016 & 2016 & Museum & 16.3 & 50 & 50 & 22,924,435 & 22,924,435 &
20,233,947 & 20,233,947 & 633,094 & 328.4 & 581 & 581 & & & 1,000,492 &
148.2 & 578 & 578\tabularnewline
Acteonida & \emph{Micromelo undatus} & & MCZ 378584 & Philippines &
Philippines & 2001 & 2001 & Museum & 0.2 & 80 & 80 & 25,320,664 &
25,320,664 & 21,944,350 & 21,944,350 & 688,782 & 303.7 & 449 & 449 & & &
2,211,068 & 239.0 & 1,166 & 1,166\tabularnewline
Sacoglossa & \emph{Oxynoe antillarum} & & MCZ 393908 & Caribbean &
Caribbean & 2018 & 2018 & Qiagen & 92.3 & 80 & 80 & 19,491,422 &
19,491,422 & 18,392,155 & 18,392,155 & 1,048,490 & 359.8 & 1,153 & 1,153
& & & 2,019,745 & 263.8 & 1,766 & 1,766\tabularnewline
Cephalaspidea & \emph{Phanerophthalmus batangas} &
\emph{Phanerophthalmus batangas} & MCZ 393707 & MCZ 393707 & The
Maldives & The Maldives & 2019 & Qiagen & \textgreater{}600 & 80 & 80 &
7,856,796 & 7,856,796 & 7,249,054 & 7,249,054 & 361,163 & 342.1 & 616 &
616 & 616 & & 992,214 & 199.7 & 1,303 & 1,303\tabularnewline
Nudibranchia & \emph{Phestilla lugubris} & & MCZ 393735 & The Maldives &
The Maldives & 2019 & 2019 & Qiagen & 2.98 & 80 & 80 & 11,205,917 &
11,205,917 & 10,383,830 & 10,383,830 & 394,272 & 346.2 & 868 & 868 & & &
1,058,739 & 211.4 & 1,234 & 1,234\tabularnewline
Cephalaspidea & \emph{Philine orca} & & MCZ 393842 & The Maldives & The
Maldives & 2019 & 2019 & Museum & 1.73 & 80 & 80 & 6,918,732 & 6,918,732
& 6,178,528 & 6,178,528 & 200,330 & 317.8 & 433 & 433 & & & 1,247,660 &
161.8 & 959 & 959\tabularnewline
Nudibranchia & \emph{Phyllidia elegans} & & MCZ 393746 & The Maldives &
The Maldives & 2019 & 2019 & Qiagen & \textgreater{}600 & 80 & 80 &
5,474,431 & 5,474,431 & 5,121,941 & 5,121,941 & 200,094 & 321.7 & 1,140
& 1,140 & & & 1,415,157 & 172.3 & 1,319 & 1,319\tabularnewline
Sacoglossa & \emph{Polybranchia jensenae} & \emph{Polybranchia jensenae}
& MCZ 393773 & MCZ 393773 & The Maldives & The Maldives & 2019 & Qiagen
& 97.5 & 80 & 80 & 36,770,678 & 36,770,678 & 33,536,568 & 33,536,568 &
1,425,061 & 404.1 & 1,405 & 1,405 & 1,405 & & 2,112,862 & 311.7 & 1,519
& 1,519\tabularnewline
Nudibranchia & \emph{Polycera hedgpethi} & & MCZ 393920 & France &
France & 2018 & 2018 & Qiagen & 96.4 & 80 & 80 & 17,668,809 & 17,668,809
& 15,611,315 & 15,611,315 & 570,352 & 356.6 & 1,152 & 1,152 & & &
1,321,012 & 218.8 & 1,553 & 1,553\tabularnewline
Acochlidia & \emph{Pontohedyle milaschewitchii} & \emph{Pontohedyle
milaschewitchii} & MCZ 387577 & MCZ 387577 & Croatia & Croatia & 2011 &
Museum & \textless{}0.01 & 80 & 80 & 1,887,780 & 1,887,780 & 1,466,109 &
1,466,109 & 47,463 & 262.8 & 66 & 66 & 66 & & 360,777 & 159.9 & 182 &
182\tabularnewline
Runcinida & \emph{Pseudoilbia avellana} & \emph{Pseudoilbia avellana} &
MCZ 393926 & MCZ 393926 & Spain & Spain & 2016 & Museum &
\textless{}0.01 & 80 & 80 & 9,503,161 & 9,503,161 & 9,221,129 &
9,221,129 & 547,779 & 523.2 & 229 & 229 & 229 & & 844,389 & 170.4 & 588
& 588\tabularnewline
Runcinida & \emph{Runcina adriatica} & & MCZ 393924 & Spain & Spain &
2015 & 2015 & Museum & 0.55 & 80 & 80 & 7,386,985 & 7,386,985 &
6,838,071 & 6,838,071 & 335,442 & 323.0 & 347 & 347 & & & 777,283 &
171.1 & 1,075 & 1,075\tabularnewline
Runcinida & \emph{Runcina africana} & & MCZ 393925 & Spain & Spain &
2014 & 2014 & Museum & 1.83 & 80 & 80 & 4,199,676 & 4,199,676 &
3,921,154 & 3,921,154 & 163,650 & 329.3 & 463 & 463 & & & 687,419 &
155.1 & 1,050 & 1,050\tabularnewline
Sacoglossa & \emph{Thuridilla gracilis} & & MCZ 393755 & The Maldives &
The Maldives & 2019 & 2019 & Qiagen & 4.11 & 80 & 80 & 9,574,678 &
9,574,678 & 8,543,671 & 8,543,671 & 423,800 & 371.1 & 1,306 & 1,306 & &
& 978,738 & 228.8 & 1,602 & 1,602\tabularnewline
Umbraculida & \emph{Tylodina perversa} & & MCZ 392614 & Canary Islands &
Canary Islands & 2013 & 2013 & Qiagen & 5.66 & 80 & 80 & 12,475,220 &
12,475,220 & 11,029,076 & 11,029,076 & 490,678 & 339.5 & 1,223 & 1,223 &
& & 1,215,005 & 203.9 & 1,771 & 1,771\tabularnewline
Umbraculida & \emph{Tylodina rafinesquii} & & MCZ 392619 & Canary
Islands & Canary Islands & 2013 & 2013 & Qiagen & 25.2 & 80 & 80 &
31,872,504 & 31,872,504 & 28,721,228 & 28,721,228 & 879,709 & 352.9 &
1,481 & 1,481 & & & 2,087,549 & 210.9 & 1,451 & 1,451\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table 3.} Assembly statistics and loci present in final trimmed
dataset and 50\% occupancy matrix, with combined Trinity and Velvet
assemblies and including the genomes and transcriptomes used for the
bait set design and the transcriptomes of two caenogastropod outgroups.\selectlanguage{english}
\begin{longtable}[]{@{}llllllllll@{}}
\toprule
\textbf{Species name, voucher \#} & \textbf{Total \# contigs} &
\textbf{Total bp} & \textbf{Mean length} & \textbf{95 CI length} &
\textbf{Min length} & \textbf{Max length} & \textbf{Median length} &
\textbf{Contigs \textgreater{}1kb} & \textbf{UCEs 50\%
matrix}\tabularnewline
\midrule
\endhead
\emph{Akera bullata} ZMBN83032 & 2,190 & 410,372 & 187.4 & 2.04 & 27 &
1,674 & 173 & 1 & 2,120\tabularnewline
\emph{Aplysia californica} & 2,107 & 478,446 & 227.1 & 2.27 & 102 &
1,009 & 203 & 1 & 2,055\tabularnewline
\emph{Aplysia dactylomela} ZMBN84917 & 2,211 & 469,687 & 212.4 & 2.19 &
33 & 1,676 & 193 & 1 & 2,139\tabularnewline
\emph{Berthella martensi} MCZ393762 & 1,820 & 373,732 & 205.3 & 2.33 &
59 & 959 & 186 & 0 & 1,791\tabularnewline
\emph{Berthellina citrina} MCZ393775 & 318 & 41,377 & 130 & 2.1 & 24 &
308 & 131 & 0 & 313\tabularnewline
\emph{Bulla vernicosa} MCZ393827 & 2,040 & 423,241 & 207.5 & 2.23 & 49 &
1,676 & 188 & 1 & 2,010\tabularnewline
\emph{Charonia tritonis} & 554 & 106,918 & 193.0 & 3.74 & 38 & 800 & 178
& 0 & 540\tabularnewline
\emph{Colpodaspis thompsoni} MCZ393765 & 1,519 & 231,709 & 152.5 & 1.36
& 33 & 496 & 148 & 0 & 1,493\tabularnewline
\emph{Crepidula navicella} & 348 & 64,753 & 186.1 & 3.66 & 61 & 791 &
178 & 0 & 346\tabularnewline
\emph{Cyerce elegans} MCZ393754 & 1,986 & 406,416 & 204.6 & 2.34 & 54 &
1,676 & 183 & 1 & 1,947\tabularnewline
\emph{Dolabella auricularia} MCZ393823 & 2,083 & 467,651 & 224.5 & 2.38
& 59 & 1,676 & 200 & 1 & 2,023\tabularnewline
\emph{Dolabrifera dolabrifera} MCZ393712 & 2,166 & 456,281 & 210.7 &
2.29 & 42 & 1,676 & 191 & 1 & 2,111\tabularnewline
\emph{Doto cervicenigra} MCZ393347 & 1,953 & 382,009 & 195.6 & 2.34 & 53
& 1,667 & 179 & 1 & 1,912\tabularnewline
\emph{Elysia chlorotica} & 1,994 & 452,430 & 226.9 & 2.33 & 89 & 1,009 &
203 & 1 & 1,944\tabularnewline
\emph{Elysia pusilla} MCZ393737 & 1,871 & 358,391 & 191.6 & 1.88 & 38 &
807 & 176 & 0 & 1,840\tabularnewline
\emph{Glossodoris acosti} MCZ393870 & 1,649 & 342,197 & 207.5 & 2.61 &
62 & 1,676 & 186 & 1 & 1,636\tabularnewline
\emph{Godiva quadricolor} MCZ393921 & 1,933 & 368,323 & 190.5 & 2.13 &
55 & 973 & 177 & 0 & 1,895\tabularnewline
\emph{Lamprohaminoea cymbalum} MCZ393734 & 2,102 & 444,021 & 211.2 &
2.40 & 29 & 1,670 & 189 & 1 & 2,056\tabularnewline
\emph{Haminoea antillarum} & 714 & 159,424 & 223.3 & 4.09 & 58 & 908 &
197 & 0 & 696\tabularnewline
\emph{Hydatina} sp. MCZ382639 & 1,963 & 388,553 & 197.9 & 1.74 & 51 &
934 & 186 & 0 & 1,927\tabularnewline
\emph{Micromelo undatus} MCZ378584 & 1,717 & 316,028 & 184.1 & 1.41 & 53
& 600 & 175 & 0 & 1,685\tabularnewline
\emph{Oxynoe antillarum} MCZ393908 & 2,039 & 417,540 & 204.8 & 2.38 & 39
& 1,676 & 185 & 1 & 1,996\tabularnewline
\emph{Phanerophthalmus batangas} MCZ393707 & 2,154 & 408,491 & 189.6 &
2.23 & 43 & 1,670 & 174 & 1 & 2,097\tabularnewline
\emph{Phestilla lugubris} MCZ393735 & 1,723 & 347,786 & 201.8 & 2.44 &
36 & 1,675 & 185 & 1 & 1,701\tabularnewline
\emph{Philine orca} MCZ393842 & 1,895 & 307,630 & 162.3 & 1.58 & 33 &
700 & 153 & 0 & 1,854\tabularnewline
\emph{Phyllidia elegans} MCZ393746 & 1,677 & 325,661 & 194.2 & 2.48 & 29
& 1,676 & 176 & 1 & 1,652\tabularnewline
\emph{Polybranchia jensenae} MCZ393773 & 2,075 & 460,829 & 222.1 & 2.31
& 65 & 1,676 & 198 & 1 & 2,026\tabularnewline
\emph{Polycera hedgpethi} MCZ393920 & 1,978 & 412,049 & 208.3 & 2.35 &
41 & 1,676 & 187 & 1 & 1,939\tabularnewline
\emph{Pseudoilbia avellana} MCZ393926 & 788 & 110,138 & 139.8 & 1.51 &
25 & 517 & 138 & 0 & 775\tabularnewline
\emph{Radix auricularia} & 1,965 & 450,223 & 229.1 & 2.40 & 83 & 1,009 &
204 & 1 & 1,915\tabularnewline
\emph{Runcina adriatica} MCZ393924 & 1,618 & 268,956 & 166.2 & 1.64 & 33
& 894 & 158 & 0 & 1,585\tabularnewline
\emph{Runcina africana} MCZ393925 & 1,573 & 252,858 & 160.7 & 1.49 & 32
& 921 & 155 & 0 & 1,538\tabularnewline
\emph{Thuridilla gracilis} MCZ393755 & 2,050 & 419,745 & 204.8 & 2.23 &
45 & 992 & 185 & 0 & 2,004\tabularnewline
\emph{Tylodina fungina} & 966 & 196,356 & 203.3 & 2.88 & 59 & 960 & 184
& 0 & 935\tabularnewline
\emph{Tylodina perversa} MCZ392614 & 2,141 & 439,598 & 205.3 & 2.30 & 8
& 1,676 & 186 & 1 & 2,082\tabularnewline
\emph{Tylodina rafinesquii} MCZ392619 & 2,099 & 469,179 & 223.5 & 2.38 &
73 & 1,676 & 198 & 1 & 2,060\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table 4.} Alignment summary data, including informative sites,
taxon representation, missing data, character count, and alignments
found in occupancy matrices.\selectlanguage{english}
\begin{longtable}[]{@{}lllllll@{}}
\toprule
\textbf{Alignment summary} & \textbf{Alignment summary} &
\textbf{Alignment summary} & \textbf{Alignment summary} &
\textbf{Alignment summary} & \textbf{Alignment summary} &\tabularnewline
\midrule
\endhead
Loci: & Length: & Mean: & 95\% CI: & Min: & Max: &\tabularnewline
2,259 & 525,599 & 232.67 & 4.84 & 73 & 1,621 &\tabularnewline
\textbf{Informative sites summary} & \textbf{Informative sites summary}
& \textbf{Informative sites summary} & \textbf{Informative sites
summary} & \textbf{Informative sites summary} & \textbf{Informative
sites summary} &\tabularnewline
Loci: & Total: & Mean: & 95\% CI: & Min: & Max: &\tabularnewline
2,259 & 273,694 & 121.16 & 2.41 & 0 & 659 &\tabularnewline
\textbf{Taxon summary} & \textbf{Taxon summary} & \textbf{Taxon summary}
& \textbf{Taxon summary} & & &\tabularnewline
Mean: & 95\% CI: & Min: & Max: & & &\tabularnewline
27.44 & 0.2 & 3 & 36 & & &\tabularnewline
\textbf{Missing data from trim summary} & \textbf{Missing data from trim
summary} & \textbf{Missing data from trim summary} & \textbf{Missing
data from trim summary} & & &\tabularnewline
Mean: & 95\% CI: & Min: & Max: & & &\tabularnewline
0 & 0 & 0 & 0 & & &\tabularnewline
\textbf{Character counts (times present)} & \textbf{Character counts
(times present)} & \textbf{Character counts (times present)} &
\textbf{Character counts (times present)} & \textbf{Character counts
(times present)} & \textbf{Character counts (times present)} &
\textbf{Character counts (times present)}\tabularnewline
'-' & 'A' & 'C' & 'G' & 'T' & All characters &
Nucleotides\tabularnewline
1,794,587 & 3,320,248 & 2,973,180 & 2,962,790 & 3,301,542 & 14,352,347 &
12,557,760\tabularnewline
\textbf{Data matrix completeness summary (\# alignments)} & \textbf{Data
matrix completeness summary (\# alignments)} & \textbf{Data matrix
completeness summary (\# alignments)} & \textbf{Data matrix completeness
summary (\# alignments)} & \textbf{Data matrix completeness summary (\#
alignments)} & \textbf{Data matrix completeness summary (\# alignments)}
& \textbf{Data matrix completeness summary (\#
alignments)}\tabularnewline
Matrix 50\% & Matrix 60\% & Matrix 70\% & Matrix 75\% & Matrix 80\% &
Matrix 90\% & Matrix 95\%\tabularnewline
2,156 & 1,994 & 1,760 & 1,514 & 1,166 & 210 & 92\tabularnewline
\bottomrule
\end{longtable}

\textbf{Figure 1.} Heterobranchia phylogeny inferred from the 50\%
occupancy matrix (2,156 UCEs) based on BI using the GTRGAMMA model
implemented in ExaBayes and ML using the GHOST model on unpartitioned
alignments implemented in IQ-TREE. High-level taxonomic ranking
groupings depicted in boxes in the right. The tree is rooted with the
caenogastropods \emph{Charonia tritonis} and \emph{Crepidula navicella}
. Only support values for nodes without full support are depicted. (*)
denotes a conflicting topology between BI and ML, see Results for
details.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\selectlanguage{english}
\FloatBarrier
\end{document}

