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.