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 in silico against 30 Trinity 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 (<0.01 μg/mL) to very high (>600
μg/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
>1,000 UCEs (auth. unpubl. data). The total number of
Illumina raw reads obtained ranged from 4 to 32 million but in the
species Creseis acicula and 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 ofAplysia californica , Elysia chlorotica , Radix
auricularia , Haminoea antillarum , and Tylodina funginaused for the bait set design were included in the trees. Additionally,Chraronia tritonis and 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 ± 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).