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 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; González 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. 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 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 Wägele,
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 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.