Selected specimens were used for DNA extraction, which was conducted using the QIAGEN DNEasy® Blood & Tissue Kit generally following the manufacturer’s standard protocol, but with a modification to the final step to increase yield and concentration, which is helpful for such small freshwater snails. The modification of the final step consisted of initially using one-quarter of the suggested amount of the buffer solution (i.e., 50 µL of buffer AE) and then repeating the elution step using the extract.

A total of six specimens were chosen for DNA extraction. Five of them were from the collection efforts in Gruna Pedro Cassiano (see above), as follows: 2 specimens of the larger-sized Idiopyrgus sp. nov. (a typical specimen and a dark morph) (from lots LES 29796 and LES 29797), 3 specimens of the smaller-sized Idiopyrgus sp. nov. (from lot LES 29820). The sixth specimen was an individual of Idiopyrgus souleyetianus Pilsbry, 1911, stemming from local survey efforts in Minas Gerais state and kindly provided to us by Paulo Ricardo Coelho from the Federal University of Minas Gerais (UFMG, Brazil), included in the present study to improve the phylogenetic analysis. The extraction process failed for the three of the smaller-sized Idiopyrgus sp. nov., which were consumed in the process. So, we refrained from spending any additional specimens of it for DNA extraction, considering it better to preserve the scarce specimens with soft parts available for the future, when better options might become available.

We selected three genetic markers to target based on those used in previous phylogenetic studies done on the group (e.g., Kameda and Kato 2011; Wilke et al. 2013; Salvador et al. 2022a; Lawrie et al. 2023): (1) the barcoding region of the mitochondrial COI gene (primers LCO and HCO of Folmer et al. 1994); (2) the mitochondrial 16S rRNA gene (primers 16SarL and 16SbrH of Simon et al. 1994); (3) a stretch of nuclear DNA containing the 3′ end of the 5.8S rRNA gene, the entire ITS2 region, and a large portion of the 5′ end of the 28S rRNA gene, sequenced in two parts (primers LSU-1 and LSU-3 for the first part and primers LSU-2 and LSU-5 for the second part; Wade and Mordan 2000; Wade et al. 2006).

The same PCR protocol was used for amplification of COI and 16S, consisting of an initial denaturation step of 3 min at 95 °C, 35 cycles of denaturation at 95 °C for 30 s, annealing at 48 °C for 1 min, and extension at 72 °C for 2 min; a final extension step of 5 min at 72 °C. The PCR protocol for the ITS2+28S section was as follows: initial denaturation step of 3 min at 95 °C; 40 cycles of denaturation at 95 °C for 30 s; annealing at either 50 °C (ITS2 portion) or 45 °C (28S portion) for 1 min; and extension at 72 °C for either 5 min (ITS2 portion) or 2 min (28S portion); final extension step of 4 min at 72 °C.

Success of PCR was assessed visually via agarose gel electrophoresis. The PCR products were then cleaned with ExoSAP-IT™ (Affymetrix Inc.) following the manufacturer’s protocol, and the samples were sent to Macrogen Europe (Amsterdam, The Netherlands) for Sanger sequencing. The resulting sequences were quality-checked and assembled using the software Geneious Prime (v.2023.2.1, Biomatters Ltd.). Consensus sequences were extracted and uploaded to GenBank; the accession numbers can be seen in Table 1.

Our initial morphological assessment of the specimens suggested a placement in the genus Spiripockia, as they seemed closely related to the species Spiripockia umbraticola Simone & Salvador, 2021, previously described from another cave in Bahia state in the same area. To test that hypothesis, we included the new species into a phylogenetic framework focusing on the families of freshwater truncatelloidean snails present in Brazil (Tateidae and Tomichiidae), as well as those families in which Brazilian species have been previously or tentatively classified (Pomatiopsidae and Cochliopidae). To that end, we relied on genetic data available from our previous studies and those of other authors. The taxa used in the phylogenetic analysis are listed in Table 1, including information on GenBank accession numbers and references.

Alignment of genetic sequences of each marker was conducted in Geneious Prime using the MAFFT plugin (Katoh et al. 2002; Katoh and Standley 2013) with default settings. The resulting alignments were visually proofed for inconsistencies and then run through Gblocks (Castresana 2000; Talavera and Castresana 2007) to eliminate poorly aligned or data-deficient positions that could affect the analysis. The resulting post-Gblocks alignments were concatenated (1779 bp in total: COI 638 bp, 16S 515 bp, ITS2+28S 626 bp) for a Bayesian inference phylogenetic analysis.

The analysis was conducted with MrBayes (v.3.2.7, Ronquist et al. 2012) through the CIPRES Science Gateway (Miller et al. 2015) with two concurrent runs, each with four Markov chains of 80 million generations (the first 20% discarded as ‘burn-in’), the default priors, nst = 6, rates = invgamma, temperature parameter = 0.1, sampling every 1,000 generations, and with substitution model parameters unlinked across the markers (COI, 16S, ITS+28S). MCMC convergence was assessed by examining the standard deviation of split frequencies (<0.001) and the potential scale reduction factor (PSRF ~1.0), as well as trace plots in Geneious (Ronquist et al. 2009).

Family Tomichiidae

Genus Idiopyrgus Pilsbry, 1911

 Idiopyrgus eowynae sp. nov.

https://zoobank.org/733E29CA-FF4B-4138-A3A9-05396C501716

Fig. 4

Type material

LES 29795 (holotype), LES 29796 (4 paratypes), LES 29797 (2 dark morphs paratype), MNRJ 37168 (3 paratypes), MZSP 168419 (3 paratypes). All specimens collected on 10.ix.2021 (M.E. Bichuette & J.E. Gallão leg.); all paratypes from type locality (except dark morphs, which are from the same cave but from the entrance zone).

Type locality

Brazil, Bahia state, Carinhanha municipality, Gruna do Pedro Cassiano (“Pedro Cassiano Cave”), 13°47'48.0"S, 43°54'50.0"W.

Etymology

The species is named in honour of Éowyn, from J.R.R. Tolkien’s “The Lord of the Rings.” Éowyn exemplifies courage, resilience, and resistance against darkness, both internal and external, standing against Gríma Wormtongue and the Witch-king of Angmar.

Diagnosis

Shell conical to turriform and relatively tall in comparison to congeners. Body whorl is translucent yellow; previous whorls are slightly darker and brownish. Teleoconch sculpture consisting of few (4–7) radial rows of thorn-like hairs on the apical portion of whorl and a multitude of fine irregular spiral lines below them, reaching all the way to the umbilicus. Aperture large, rounded.

Description

Shell conical to turriform, 5–5¼ whorls, 4.5–4.6 mm high, ~2.7 mm wide. Colour pale beige or yellowish on body whorl, but slightly darker and brownish on earlier whorls; body whorl translucent; early whorls typically with flaked off periostracum. Protoconch of 1¼ whorl, rounded, smooth (Fig. 4G, I). Suture deep. Whorls increasing uniformly in width but more rapidly in height. Teleoconch sculptured by minute triangular thorn-like ‘periostracal hairs’ arranged in equidistantly spaced spiral rows (4 to 7 rows) and restricted to apical portion of body whorl (Fig. 4A–D, H). Below the rows of periostracal hairs, there is a multitude of fine, irregular spiral lines, reaching all the way to the umbilicus (Fig. 4H). Periostracal hairs can be worn out in older specimens (Fig. 4C, D), particularly in the early whorls. Peristome complete, not covering penultimate whorl, expanded, wider in abapical region, narrower in columellar region, of same colour as body whorl or slightly whiter. Aperture rounded, with light apical angulation, but with smooth, rounded contour. Umbilicus rimate, nearly closed. Operculum oval, paucispiral; corneous, thin, and translucent, of same colour as body whorl. Soft body completely white in colour.

Figure 4. 

Idiopyrgus eowynae sp. nov. A, B. Holotype LES 29795;. C. Paratype (typical morph) #1 LES 29796; D. Paratype (typical morph) #2 LES 29796; E, F. Paratype (dark morph) LES 29797; G. Paratype (typical morph) #3 LES 29796, juvenile, showing the protoconch in greater detail as well as the sculpture of the first teleoconch whorls; H. Detail of the body whorl of holotype in dorsal view, showing the teleoconch sculpture, in particular the multitude of spiral lines; I. Paratype #3 in apical view, showing the protoconch.

Dark morph (Fig. 4E, F) differs from the above-described typical morph by having a black shell with thicker walls, in which the teleoconch sculpture is nearly invisible: a few remnants of the spiral rows of hairs are visible in the adapical portion of the whorls; traces of the sculpture can also be seen through the shell wall of the aperture (Fig. 4E).

Distribution

Known only from type locality (Fig. 1), the twilight and dark (aphotic) zones of the cave (typical morph) and at the cave entrance (dark morph).

Remarks

Idiopyrgus eowynae sp. nov. can be easily distinguished from most of its congeners (including the type species I. souleyetianus) by its much wider shell and the presence of teleoconch sculpture. It closely resembles I. meriadoci sp. nov. (see below) and I. umbraticola. It differs from I. meriadoci sp. nov. by a much larger size and larger number of whorls, as well as by having a different teleoconch sculpture, which includes periostracal hairs. Conchologically, I. eowynae sp. nov. is most similar to I. umbraticola comb. nov., from which it can be distinguished by its smaller size (~4.5 mm vs. ~5.0 mm in I. umbraticola), higher whorls with a more strongly convex outline, a larger and rounder aperture, and by having a different teleoconch sculpture. In I. eowynae sp. nov., there are few spiral rows of the triangular thorn-like hairs, restricted to the apical portion of the whorl, and which are then replaced by irregular spiral lines that continue towards the umbilicus (also visible in juveniles). In I. umbraticola, there is a larger number of rows of hairs, stretching the median portion of the whorl; the spiral lines are absent. Notably, a dark morph has also been observed in I. adamanteus Salvador, Silva & Bichuette, 2022, and I. cf. walkeri Pilsbry, 1924 (Salvador et al. 2022a).

Except for the existence of two morphs (typical and black), there is little variation in shell shape, form, and proportions among the presently available specimens of I. eowynae sp. nov. (Fig. 4A–F). Notably, the sequences of the COI barcoding gene fragment of the two morphs were 100% identical, while the sequences of both morphs had a pairwise identity of around 90% in relation to I. adamanteus and I. minor. According to the results of the phylogenetic analysis (Fig. 3), I. eowynae sp. nov. is sister to I. adamanteus; both species together are sister to I. minor.

 Idiopyrgus meriadoci sp. nov.

https://zoobank.org/B70A02D0-9ABD-42F4-89FD-DE52950C7E44

Fig. 5

Type material

LES 29798 (holotype), LES 29820 (4 paratypes), MZSP 168420 (2 paratypes). All specimens collected on 10.ix.2021 (M.E. Bichuette & J.E. Gallão leg.); all paratypes from type locality.

Type locality

Brazil, Bahia state, Carinhanha municipality, Gruna do Pedro Cassiano (“Pedro Cassiano Cave”), 13°47'48.0"S, 43°54'50.0"W.

Etymology

The species is named in honour of Meriadoc “Merry” Brandybuck, from J.R.R. Tolkien’s “The Lord of the Rings.” Besides standing with Éowyn against the Witch-king in the Battle of the Pelennor Fields, Merry is also an example of the fight for nature conservation in Middle-earth, pushing the Ents into action and ultimately ending Saruman’s threat to Fangorn Forest.

Diagnosis

Shell minute, whitish, entirely translucent. Teleoconch sculptured by numerous spiral rows of minute vertical pustulae, arranged roughly equidistantly from one another, which do not form thorn-like periostracal hairs. Aperture oval, adapically and abapically angulated.

Description

Shell conical, 4½–4¾ whorls, 2.8–2.9 mm high, 1.6–1.8 mm wide. Colour pale yellow to white; shell entirely translucent. Protoconch of 1¼–1½ whorl (transition to teleoconch not easily observable), rounded, smooth (Fig. 5E,G). Suture deep. Whorls increasing uniformly in height and width. Teleoconch sculptured by numerous spiral rows of minute vertical pustules, arranged roughly equidistantly from one another (Fig. 5E,F); they do not develop thorn-like periostracal hairs as seen in some of its congeners (e.g., I. eowynae sp. nov.), though a few pustules on the body display what seem to be minute truncated hairs in two of the more apically-positioned rows (Fig. 5F). Peristome complete, not covering penultimate whorl, expanded, wider in abapical region, narrower in columellar region, of same colour as body whorl. Aperture oval, adapically and abapically angulated, but with smooth, rounded contour. Umbilicus rimate, nearly closed. Operculum oval, paucispiral; corneous, thin, and translucent, of same colour as body whorl. Soft body completely white in colour.

Figure 5. 

Idiopyrgus meriadoci sp. nov. A, B. Holotype LES 29798; C. Paratype #1 LES 29820; D. Paratype #2 LES 29820; E. Detail of the spire of the holotype in apertural view, showing the teleoconch sculpture in the early whorls; F. Detail of the body whorl of holotype in apertural view, showing the teleoconch sculpture; F. Detail of the protoconch of the holotype.

Distribution

Known only from type locality (Fig. 1).

Remarks

Idiopyrgus meriadoci sp. nov. is easily distinguished from all its congeners by its unique teleoconch sculpture and much smaller shell; it is smaller than even Idiopyrgus minor Salvador, Silva & Bichuette, 2022, which was previously the species with the smallest body size in the genus. The whitish and entirely translucent shell, along with the smaller number of whorls, is likewise useful to easily distinguish it from Idiopyrgus eowynae sp. nov. (see above) and I. umbraticola. The latter two species also display a teleoconch sculpture that seems to be homologous to that of I. meriadoci sp. nov.; the latter, however, lacks the periostracal hairs observed in the former two species.

As for I. eowynae sp. nov. above, there is little variation in shell shape and proportions among the presently available specimens of I. meriadoci sp. nov. (Fig. 5A–D).

In view of the presently available information, the new species described herein are endemic to a single cave (they have not been found in collections in other caves). Idiopyrgus meriadoci sp. nov. can be found in the twilight zone and dark (aphotic) zone of the cave; the typical morph of I. eowynae sp. nov. is found in the twilight and dark zones, while the dark morph is found closer to the cave entrance.

Considering the classification of subterranean organisms (Romero 2009; Christiansen 2012; Trajano and Carvalho 2017), we identify both I. eowynae sp. nov. and I. meriadoci sp. nov. as troglobitic species. Besides their endemicity, there are other morphoanatomical features to support this, some of which are of particular importance for freshwater gastropods (Grego et al. 2020; Gladstone et al. 2018, 2021; Salvador et al. 2022b, 2024) as follows. Idiopyrgus meriadoci sp. nov. has a white translucent and fragile shell and unpigmented body; it also has a minute size, smaller than all other congeners. These are features typical of specialised cave fauna (Gladstone et al. 2018, 2021; Grego et al. 2020). Likewise, I. eowynae sp. nov. has an unpigmented body, and its typical morph has a translucent and fragile shell.

The dark morph of I. eowynae sp. nov. displays a sturdier black shell, which has also been observed in I. adamanteus and I. cf. walkeri (Salvador et al. 2022a). While the significance of this is yet unknown, similar colour polymorphism is also known in other troglobitic animals, particularly in gastropods of the genus Potamolithus and in fish (Bichuette and Trajano 2004, 2005, 2018). Considering that there is negligible genetic distance between the two morphs in their mitochondrial markers (Fig. 3), we can only hypothesise that would be true for the rest of the genome and that the morphological differences observed in the shell are likely due to phenotypic plasticity and/or have direct environmental causes.

Both I. eowynae sp. nov. and I. meriadoci sp. nov. have a unique teleoconch sculpture (i.e., rows of periostracal hairs), so far unknown in surface-dwelling freshwater snails in Brazil but known from other cave-dwelling snails in the country: I. umbraticola and Spiripockia punctata (Simone 2012; Simone and Salvador 2021; Salvador et al. 2022b). However, we are presently unable to say whether this is somehow related to life in a cave environment or not, but the fact that only cave snails display it might be an indication of that. Other members of Idiopyrgus, including cave- and surface-dwelling species, lack the sculpture (Salvador et al. 2022a), so future studies could, for instance, show that the sculpture is a feature related to a clade within the genus (e.g., I. eowynae sp. nov. + I. meriadoci sp. nov. + I. umbraticola). Even so, according to our present phylogenetic analysis (Fig. 3), the cave-dwelling species I. eowynae sp. nov., I. adamanteus, and I. minor form a single clade. While this apparently could indicate that cave species are closely related and descend from a common ancestor, it should be noted that our coverage of the genus is still quite reduced, and future studies with a better sampling across more species and populations could paint a different picture.

The facts that Idiopyrgus is a relict genus and that it has radiated into caves, alongside the intraspecific (and potentially non-genetic) differences in shell morphology, make species in this genus good models for studies that go beyond phylogenetics and biogeography and into evolutionary ecology. Thus, we hope that our account will bring more attention to these intriguing animals.

The genus Idiopyrgus has a reasonable geographic range throughout Brazil, occurring mostly in the eastern part of the country (except for I. brasiliensis (Rey, 1959) in the west) and across five different hydrographic regions: São Francisco (where most diversity and records are), East Northeast Atlantic, Southeast Atlantic, Tocantins-Araguaia, and Paraguay (Salvador et al. 2022a: fig. 4). There is little data on the distribution of most species, but they seem to have restricted ranges, often known only from their type localities (Salvador et al. 2022a). The exception is I. souleyetianus, which is a widespread species, distributed across three states (Bahia, Minas Gerais, and Espírito Santo) and two hydrographic regions (São Francisco and Southeast Atlantic) (Salvador et al. 2022a).

Three of the previously known species (I. adamanteus, I. minor, and I. umbraticola) are so far known only from caves, while I. souleyetianus and potentially I. walkeri have wider distributions and have also been recorded from caves (Salvador et al. 2022a). Notably, I. umbraticola and I. minor occur in the Serra do Ramalho region near the municipality of Carinhanha, in which both new species described herein (as well as Spiripockia punctata) can be found (Fig. 2). The region of Central, to the north of Carinhanha and where I. adamanteus can be found, is dominated by the Caatinga biome with a dry vegetation type, and the caves have been hypothesised as refuges for these animals (Salvador et al. 2022a).

Considering the difficulty of accessing the caves and conducting detailed observations of live animals, so far only snapshots of their biology are known. We collated here all the information currently available from the literature and from field observations by MEB.

Idiopyrgus umbraticola occurs only in the aphotic zone of Gruna do Domingão cave, attached to hard surfaces such as rocks and logs in the water (Simone and Salvador 2021). This species co-occurs with (and uses the same microhabitat as) Xangoniscus aganju Campos-Filho, Araujo & Taiti, 2014 (Campos-Filho et al. 2014), an amphibious troglobitic isopod.

Both I. eowynae sp. nov. and I. meriadoci sp. nov. were found side by side in a small stream inside the Gruna do Pedro Cassiano cave, attached to the small roots of plants (e.g., “gameleiras”) that penetrate the caves from the surface in search of water; the deep of the small lentic drainage is formed by silt and has a depth of 10 to 15 cm. Values of pH and temperature measured on site varied, respectively, from 7.5 to 8.2 and from 21.3 to 22.4 °C. Both snail species occur in the cave’s twilight zone and dark (aphotic) zones; however, the dark morph of I. eowynae sp. nov. lives close to the cave entrance. It is interesting that both species also co-occur with a species of the isopod genus Xangoniscus, probably X. aganju (Fig. 1G), and the cave catfish species Trichomycterus, probably T. rubbioli (Bichuette & Rizzato, 2012) (Fig. 1H).

Idiopyrgus minor lives in small pools resulting from infiltration water in the aphotic zone of two close-by caves (Gruna da Pingueira cave and Gruna do Engrunado cave) in Feira da Mata municipality and is apparently restricted to this type of habitat (Salvador et al. 2022a). Idiopyrgus adamanteus occurs in four close-by caves in Central municipality (Toca Bonita cave, Toca da Mulher cave, Toca de Candeias cave, and Toca da Machadinha cave) (Salvador et al. 2022a); individuals of this species were always found in phreatic waters, in twilight and aphotic zones of the caves, attached to rocks and submerged logs.

Spiripockia punctata lives attached to rocks inside a small stream in the aphotic zone in the Lapa dos Peixes cave (Simone 2012), also co-occurring with the isopod X. aganju and a species of cave catfish, Trichomycterus rubbioli. Both the isopod and catfish are troglobites (Bichuette and Rizzato 2012; Campos-Filho et al. 2014).

The repeated co-occurrence of Idiopyrgus and the isopod Xangoniscus aganju is interesting. The genus Xangoniscus is composed only by troglobitic species with high endemicity (Campos-Filho et al. 2014; Cardoso et al. 2020), and the somewhat more widespread X. aganju is suspected of being a cryptic species complex (MEB, pers. obs.). This partially parallels the story of Idiopyrgus in the region (Salvador et al. 2022a). Also, the observation that Idiopyrgus spp. and Xangoniscus spp. share the same microhabitats begs the investigation of other potential relationships between these species, such as modes of dispersal and cave colonisation.

Considering the co-occurrence of two species in the same cave and sharing the same microhabitat, the threats observed regionally and locally such as water use, deforestation in the cave surroundings, and the progressively decreasing recharge of water to the cave likely due to climate changes (Fig. 1A; Gallão and Bichuette 2018), we can consider both species to be in a vulnerable situation. Some impacts are specific, such as the extraction of water (Fig. 1D) for use in villages and plantations, but they are nonetheless worrying (Gallão and Bichuette 2018). The fact that both species are endemic to the same locality already makes them fragile to any environmental disturbance. In addition, the cave is about 2 km long, considering the mapped passageways (Fig. 1C, cave gallery of Gruna do Pedro Cassiano; Grupo Bambuí de Pesquisas Espeleológicas, pers. comm.), reaching a potential extension area, for both species, of 2,000 m². However, from a small-scale perspective, despite the high abundance observed, with population densities reaching an average of 30 individuals/m² (estimated in loco during fieldwork), both species use specific microhabitats with a high concentration of roots (Fig. 1E, F), which implies a small area of occupation not exceeding 20 m² and which further increases their risk of extinction.