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).