The South American genus Pilsbrylia Hylton Scott, 1952 contains three species, which inhabit areas in southern Brazil and northern Argentina (Simone 2018): Pilsbrylia paradoxa Hylton Scott, 1952 (the type species), P. hyltonae Fernández & Rumi, 1980, and P. dalli Simone, 2018 (Fig. 1). Members of this genus have high-spired and narrow shells and the shell aperture bears a number of teeth and lamellae. The shells are morphologically similar to the members of family Cyclodontinidae (which was formerly part of Odontostomidae; see Salvador et al. 2023 for the revised classification within superfamily Orthalicoidea), in particular to members of genera such as Clessinia Doering, 1875 and Cyclodontina H. Beck, 1837. Thus, since its description, Pilsbrylia has been classified in that family (e.g., Hylton Scott 1952; Breure 1974; Fernández and Rumi 1980; Schileyko 1999; Cuezzo et al. 2013; Simone 2018).

The molecular phylogenetic study of Breure and Romero (2012) showed that Pilsbrylia did not belong in Cyclodontinidae and those authors proposed the genus was instead the sister taxon to all other Orthalicoidea. A more recent phylogenetic study (Salvador et al. 2023), showed that Pilsbrylia did not belong in Orthalicoidea at all, being instead related to the Clausiliidae, or door snails. That was a surprising result, considering that the door snails typically have sinistral shells (i.e., a shell that “coils” counter-clockwise or, when seen with its aperture facing the observer and the spire top pointing upwards, a shell whose aperture is on the left-hand side).

Furthermore, the two branches of Clausiliidae present in the Americas are restricted to the Caribbean (subfamily Neniinae) and northwest South America (subfamily Peruiniinae) (Uit de Weerd and Gittenberger 2013). Thus, the genus Pilsbrylia is rather geographically removed from the family’s range.

In the present study we include Pilsbrylia in a phylogenetic framework of the Clausiliidae to test if it really belongs to this family and, if so, assess how it is related to other South American door snails.

DNA sequences of Pilsbrylia paradoxa used in previous Orthalicoidea-focused studies (Breure and Romero 2012; Salvador et al. 2023) were used for the present analysis. They are available on GenBank under accession numbers JF514745 (28S) and JF514687 (H3) and stem from a voucher specimen previously housed in the collection of the Instituto Miguel Lillo, Tucumán, Argentina (registration number IFML-MOLL BD316, collected in northern Argentina, Salta province, km 1650 of Salta-Jujuy highway) but now housed in the Instituto de Biodiversidad Neotropical (IBN) of the Universidad Nacional de Tucumán, Argentina. No further specimen of Pilsbrylia spp. preserved in ethanol and suitable for DNA extraction could be found in the present study.

The sequences of P. paradoxa were included in the phylogenetic framework for the family Clausiliidae established in the study of Uit de Weerd and Gittenberger (2013). That study included a reasonable sample of South American (subfamily Neniinae) taxa across two distinct clades (tribes), which enables us to test the phylogenetic position of Pilsbrylia. Later molecular phylogenetic studies with Clausiliidae focused on Eurasian taxa (e.g., Hausdorf and Neiber 2022) and data from them was not included here.

A total of 67 species of Clausiliidae, belonging to all subfamilies and almost all tribes, was part of the analysis. Five species were selected as outgroup representing the families Cerionidae, Chondrinidae, Enidae, Rhytididae, and Urocoptidae (data from Uit de Weerd 2008; Uit de Weerd and Gittenberger 2013; Saadi and Wade 2019). A complete list of the species used in the analysis, with locality data and GenBank accession numbers is given in the Suppl. material 1.

Data from three nuclear markers were used in the present phylogenetic analysis, following Uit de Weerd and Gittenberger (2013): partial 28S rRNA gene (ca. 1700 bp), partial H3 (histone 3) gene (ca. 270 bp), and partial H4 (histone 4) gene (ca. 260 bp). Information on primers and PCR protocols can be found in Uit de Weerd and Gittenberger (2013); for Pilsbrylia, see Breure and Romero (2012).

The genetic sequences were aligned through the MUSCLE plugin (Edgar 2004) in Geneious Prime (v.2023.0.4, Biomatters Ltd.), using default settings (i.e., optimised for accuracy). The resulting alignments were visually proofed for inconsistencies. The alignment of the 28S marker was run through Gblocks (Talavera and Castresana 2007), using the least restrictive settings, in order to eliminate poorly-aligned or data-deficient positions that could introduce noise into the analysis. The alignments were then concatenated for a single phylogenetic analysis, with each marker being treated as an individual partition.

A Bayesian inference phylogenetic analysis was performed through MrBayes (v.3.2.7; Ronquist et al. 2012) via the CIPRES Science Gateway (v.3.3; Miller et al. 2015). Two concurrent analyses, each with 4 Markov chains of 80 million generations (the first 20% discarded as ‘burn-in’), were run with the default priors, nst = 6, rates = invgamma, temperature parameter = 0.1, sampling every 1,000 generations. Substitution model parameters were unlinked across the markers (28S, H3, and H4). MCMC convergence was assessed using the standard deviation of split frequencies (<0.01) and the potential scale reduction factor PSRF (~1.0), as well as by examining the trace plots (Ronquist et al. 2009).

The concatenated sequences of the three markers (after trimming the 28S marker using Gblocks) contained 2172 bp. The total-evidence tree resulting from the Bayesian analysis contained 73 species (including Pilsbrylia paradoxa and the outgroup) and is shown here in simplified format, with the branches of non-immediate interest collapsed (Fig. 2). The full tree can be seen in the Suppl. material 2.

All subfamilies of Clausiliidae are strongly supported (posterior probability PP=1), except for Serrulininae, which is paraphyletic (Fig. 2). As expected in a clade inflated by family-level names, many of the supposed tribes were recovered as para- or polyphyletic (e.g., Cochlodinini, Delimini; Suppl. material 2), as already noted by Uit de Weerd and Gittenberger (2013). When also considering the existence of several monotypic tribes (e.g., Garnieriini, Strumosini), the Clausiliidae would benefit from a clean-up of names on the tribe level, as many could easily be synonymized if supported by further research.

Considering that our ingroup was identical to that of Uit de Weerd and Gittenberger (2013) – barred the inclusion of Pilsbrylia paradoxa – our tree is largely similar to the one presented by those authors. However, a major difference can be observed: while Uit de Weerd and Gittenberger (2013) had the Neniinae as the sister group to all other clausiliids (though unsupported), our tree places the Laminiferinae in that position (Fig. 2). The Laminiferinae are sister to the remaining clausiliids (PP=0.97) and, inside the latter clade, the Neniinae are sister to all others, which are joined in an unsupported polytomy (PP=0.54). This polytomy includes three strongly supported branches (PP=1: Peruiniinae, Garnieriinae, and a clade formed by Phaedusinae and a paraphyletic Serrulininae) and an unsupported branch (PP=0.75) containing the Alopiinae and the Clausiliinae (each PP=1) as sister clades (Fig. 2).

Our taxon of interest, Pilsbrylia paradoxa, is included in the Peruiniinae, in a derived position (Fig. 2). It was recovered as the sister taxon to the Peruvian Incaglaia adusta (O. Boettger, 1880), although with low support (PP=0.88). Both species are the sister clade to Incania papillosa Neubert & Nordsieck, 2005 (PP=1), also from Peru. The relationships within Peruiniinae recovered by our analysis are largely the same as those in Uit de Weerd and Gittenberger (2013), with a single exception: those authors recovered Zilchiella scala Neubert & Nordsieck, 2005 and Peruinia peruana (Troschel, 1847) as sister taxa (although unsupported), while in the present tree they form a trichotomy with the remaining Peruiniinae (Fig. 2).

The results of the present phylogenetic analysis allow the conclusion that Pilsbrylia in fact belongs in the family Clausiliidae, more specifically to the South American subfamily Peruiniinae, as expected from a biogeographical perspective.

Pilsbrylia, however, has a much different shell morphology from other members of the Peruiniinae. First of all, the shell is dextral. Dextral shells are rare in Clausiliidae, but are more frequent in subfamily Alopiinae (Nordsieck 2007; Páll-Gergely et al. 2019). Within the genera Alopia Adams & Adams, 1855 and Albinaria Vest, 1867, eniantomorph taxon pairs are known, and dextral lineages have evolved from sinistral ancestors multiple times independently (Fehér et al. 2013; Kornilios et al. 2015). While those shells are usually near mirror images of their congeners or conspecifics, Pilsbrylia, on the other hand, presents a shell morphology that is more similar to Cyclodontinidae than to most Clausiliidae, notably by not having a complete peristome (Fig. 1), which justified its previous classification.

Hylton Scott (1952) described the radular and genital anatomy of P. paradoxa, the only species in the genus for which anatomical data is available. Notably, that author did not mention a clausilium, which would be a reasonably obvious structure if present (unless it was present as a reduced structure). The single columellar lamella of Pilsbrylia spp. only extends about half whorl inside the shell along the columella (Hylton Scott 1952; Simone 2018), which could indicate a reduced or absent clausilium. Clausiliidae have two columellar lamellae that stretch throughout the entire body whorl (Nordsieck 2007, 2015).

Species of the sister genus to Pilsbrylia in the present phylogeny, Incaglaia Pilsbry, 1949 (Fig. 2), have a typical clausiliid shell morphology. However, members of the next closest genus, Incania Poliński, 1922, can exhibit a simplified peristome, reduced lamellae and reduced clausilial apparatus (Neubert and Nordsieck 2005). Notably, some fossil species of European Ryllia Munier-Chalmas, 1883 and Rillyarex Nordsieck, 1985 have reduced or absent lamellae and a peristome with similar configuration to Pilsbrylia spp. (Nordsieck 2015).

Thus, the epithet ‘paradoxa’ given to the first species described in this genus reaffirms itself as a very appropriate name: genetically, it is a Clausiliidae, but it does not possess the two typical traits of the family, i.e., a sinistral shell and a clausilial apparatus (or might present it in a reduced state). Nevertheless, the genetic similarity of Pilsbrylia with clausiliids is observed across all markers analysed and thus, a fortuitous similarity of a single marker biasing the phylogenetic analysis is unlikely. In view of the present evidence, we conclude that Pilsbrylia is a member of Peruiniinae defined by its dextral shell and hypothesize that the simplification of the dextral shell (i.e., aperture and lamellae) is related to the reduction or loss of the clausilium.

The Clausilioidea are considered to have arisen in Europe, as both the extinct Filholiidae and Palaeostoidae, as well as one of the first branches of Clausiliidae (i.e., Laminiferinae; Fig. 2) are from that continent (Nordsieck 2007, 2015; Uit de Weerd and Gittenberger 2013). The Palaeostoidae date back to the Late Cretaceous of western Europe and so does Clausiliidae thanks to the extinct tribe Rillyini Nordsieck, 1985 (Nordsieck 2015).

Still, Clausiliidae has a clear Laurasian component, with Neniinae in the Caribbean and Peruiniinae in South America (Fig. 2) and an origin in the latter cannot be entirely excluded (Uit de Weerd and Gittenberger 2013). It has since become known that the presence of the family Clausiliidae (and purportedly of subfamily Peruiniinae) in South America is equally old. Namely, there is a fragmentary fossil from the Late Cretaceous of Uruguay classified as Clausiliidae indet. (Salvador et al. 2018; Cabrera et al. 2020). The next oldest fossil is Temesa ? magalhaesi (Trindade, 1953) from the Early Eocene of Rio de Janeiro, Brazil (Salvador and Simone 2013; Salvador et al. 2018). The latter species was provisionally assigned to a recent genus, but it likely belongs to a different (and still undescribed) genus, potentially close to the basal node of Peruiniinae.

Considering the present findings, we propose a revised classification of the genus Pilsbrylia Hylton Scott, 1952, placing it inside subfamily Peruiniinae of the Clausiliidae. Thus, this extends the known range of this subfamily in South America to the south (Argentina and Uruguay) and to the east (Brazil, Minas Gerais state).