The study area is situated in the Alto Ribeira karst area, São Paulo State, southeastern Brazil. It is geologically inserted in the Açungui Group, composed of Upper Precambrian metasedimentary rocks (Karmann and Sànchez 1979). Four NE-SW limestone outcrops extend over 20 km, with widths varying from 1 to 8 km, alternate with insoluble detrital rocks, such as phyllites (Trajano et al. 2016) (Figure 1).

Figure 1. 

Map showing the surveyed localities (basins, microbasins and caves) from Alto Ribeira karst area, Southeastern Brazil. Some localities are approximated (*) (Author: DM von Schimonsky). Caves: A1 – Aranhas, A2 – Chapéu Mirim I, A3 – Chapéu, A4 – Chapéu Mirim II, A5 – Temimina II, A6 – Gurutuva, A7 – Córrego Seco, A8 – Fendão, A9 – Paiva, A10 – Jane Mansfield, A11 – Minotauro; B1 – Areias de Cima, B2 – Areias de Baixo, B3 – Ressurgência das Areias de Água Quente; C – Ouro Grosso; D – Alambari de Baixo; E – Água Suja; F1 – Pérolas, F2 – Santana; G1 – Casa de Pedra, G2 – Água Sumida; H – Tapagem; I1 – Morro Preto, I2 – Couto; J – Pescaria; K* – Betari de Baixo; L – Jeremias; M – Colorida; N – Calcário Branco; O – Alambari de Cima. Epigean streams: a – Ouro Grosso; b – Alambari; c – Água Suja; d – Roncador; e – Maximiano; f – Ostras; g – Calcário Branco; h – Iporanga; i1 – Betari, i2 – Água Quente, i3 – Morro Preto; j1 – Bocaina, j2 – Espírito Santo, j3 – Temimina, j4 – Pescaria, j5 – Lageado, j6 – Pilões, j7 – Ribeira, j8 – Cutia de Cima.

The Alto Ribeira river valley is located in the transition between the Tropical Atlantic and Araucaria Forest domains (Ab’Sáber 1977) (Figure 2). The climate in the study area is type “Cfa”, which is defined as tropical wet to temperate humid without a dry season. Total precipitation is around 1,500 mm and precipitation in the driest and coolest month (July) is more than 30 mm. Temperatures are above 22 °C in the warmest month (January) and below 4 °C in the coolest month; mean annual temperature (as generally observed deep inside caves) is around 19–20 °C in the Parque Estadual Turístico do Alto Ribeira (PETAR), and 17–18 °C in the upper Parque Estadual Intervales (PEI), where collections were carried out (Köeppen 1948). This area is mostly covered by perennial subtropical humid forest (Hueck 1972), with typical well-oxygenated headwater streams, with rocky beds and rapids alternating with some soft-bottomed pools, which cross the rock outcrops (Figure 2).

Figure 2. 

Landscape (Atlantic Rainforest) (A), Potamolithus sp. 5 in natural habitat, Santana cave (B) and subterranean streams (C, D) from Alto Ribeira karst area, Southeastern Brazil. Photographs: PP Rizzato (A, C, D), A Gambarini (B).

Faunistic surveys were conducted in epigean and cave stream reaches in two continuous regions in the Alto Ribeira karst area, mostly protected by state parks: PETAR and PEI. Altitudes vary from 300 to 1,000 m in PETAR, which is crossed by four limestone outcrops. One of these outcrops bifurcates and extends toward the northeast, crossing PEI, where altitudes reach up to 1,200 m.

We visited 33 caves and 10 epigean streams in PETAR and four caves and two epigean streams in PEI (Figure 1). We also collected in the Tapagem Cave (a partially touristic cave, also known as Caverna do Diabo - Devil´s Cave) and the epigean stream that sinks into it, a relatively isolated karst area in the middle of the Rio Ribeira valley, protected by the Parque Estadual Caverna do Diabo (PECD). The survey areas were located in four basins, or micro-basins (Figure 1).

The caves (from northeast to southwest) are shown in Figure 1: PEI - Fendão, Paiva, Jane Mansfield, Minotauro, and Colorida; PETAR - Temimina II, Pescaria, Aranhas, Chapéu, Chapéu Mirim I, Chapéu Mirim II, Água Sumida, Casa de Pedra, Calcário Branco, Água Suja, Santana, Pérolas, Morro Preto, Couto, Córrego Seco, Alambari de Cima, Alambari de Baixo, Gurutuva, Ouro Grosso, Areias de Cima, Areias de Baixo, Ressurgência das Areias de Água Quente, Betari de Baixo, and Jeremias; PECD - Tapagem.

Rivers, separated by micro-basins, are also shown in Figure 1: Pilões micro-basin - Pilões, Lageado, Pescaria, Temimina, and Bocaina; Iporanga micro-basin - Iporanga, Maximiano, and Espírito Santo; Betari micro-basin – Betari, Calcário Branco, Água Suja, Roncador, Morro Preto, Ouro Grosso, Alambari, and Água Quente; Ostras and Cutia de Cima micro-basins.

Systematic field trips to the Alto Ribeira karst area were carried out during 11 months between 1996 and 1997 (Bichuette and Trajano 2003); additional collections were done in 2008, 2009, 2010 and 2013 (four samplings, one per year). Potamolithus specimens were collected by hand after visual inspection of the substrate, mostly under boulders, pebbles, and other submerged objects, and by straining of thin substrates, such as sand and clay. Specimens were fixed in 70% ethanol and brought to the laboratory for morphological and taxonomic study. Identification at the genus level was confirmed with the collaboration of LRL Simone (Seção de Moluscos, Museu de Zoologia da Universidade de Sao Paulo, MZUSP).

For a preliminary separation of species/morphs, we considered the following characters in samples containing between 15 and 82 specimens: shape of intestine, observed by transparency in an abapertural view (slightly curved to left; slightly curved to right; markedly curved to left; two marked curves, a broad fold to left; a constricted fold to left) (Figure 3); pattern of body (mantle) pigmentation; presence versus absence of eyes; shell shape (fusiform; globose; fusiform-globose); shell dimensions (height – from the apex to the base, following the columellar axis, and width – the maximum measurement at a right angle to the columellar axis); and periostracum coloration observed in live individuals (dark brown, brown, pale yellow, white, translucent). Shell measurements were taken using digital calipers with 0.01 mm precision; we analyzed only adult individuals (smaller individuals that showed mature gonads – see Bichuette and Trajano 2003). We followed the anatomical nomenclature from Simone (2006).

Figure 3. 

Schematic intestine forms observed in Potamolithus species from Alto Ribeira karst area, Southeastern Brazil. Abapertural view.

To illustrate the shell opening, shape, and ornamentation, one or two individuals per population were studied under a scanning electron microscope (SEM) (Zeiss electron microscope) and through images acquired via LAS software (Leica Application Suite v3.7). Criteria for distinction between troglobites and troglophiles, according to the Schiner-Racovitza system, were used as in Trajano and Carvalho (2017). The absence of records in the epigean environment allied to troglomorphic characters (such as translucent periostracum and absence of eyes) were considered for troglobite status; for troglophile status, we considered the occurrence of well-established (source) populations in both epigean and hypogean environments, indicated by the presence of many individuals of all size/age classes deep inside caves throughout the annual cycle.

Examined material was deposited at the Museu de Zoologia, Universidade de São Paulo (MZUSP) and the Laboratório de Estudos Subterrâneos, Universidade Federal de São Carlos (LES).

To detect statistical differences among individuals from each OTU (Operational Taxonomic Unit - Sokal and Sneath 1963), the shell sizes (adults) were visually compared using boxplot graphs. A variance analysis (One Way ANOVA), followed by Dunn’s post-hoc test, was performed using the software PAST version 3.09 (Hammer et al. 2001). Before the analysis, data were tested in relation to normality and homogeneity of variances.

The concept of a species is one of the most complicated and debatable problems in biology. For several decades now, hundreds of publications have focused on this subject but no consensus has been achieved, and probably never will be (see Wheeler and Meier 2000, for a debate on the theme). Herein we adopted the Operational Taxonomic Unit (OTU; Sokal and Sneath 1963), which most taxonomists use in their daily task of recognizing, characterizing, and describing “species” and most inclusive clades. This is the first, and necessary, step for phylogenetic studies, as well as ecological studies for conservation, among others. Describing biodiversity is urgent for several reasons, and the OTU approach is not only a practical way to begin, but has a sound theoretical basis.

In our approach, OTUs are a spin-off from the evolutionary species (“An evolutionary species is an entity composed of organisms that maintains its identity from other such entities through time and over space and that has its own independent evolutionary fate and historical tendencies”), or species-as-lineage concepts (Wiley and Mayden 2000). According to these authors, if lineages are independent, at the empirical level we can expect to eventually discover differences among them. Such differences are expressed as character states indicating the lineages’ individuality (not to be confused with statements of similarity).

Therefore, the species as recognized and characterized below are lineages distinguished by sets of diagnostic traits (that may be apomorphic or plesiomorphic, which is not relevant in our approach).

With more than 60 troglobitic species, the Alto Ribeira is a high-diversity area for troglobites (sensuTrajano et al. 2016, Gallão and Bichuette 2018). This is among the highest genetic diversities recorded for Brazil, and is likely related to three factors: the high degree of habitat fragmentation, limited mobility leading to low capacity for dispersal, and habitat preferences.

Potamolithus gastropods prefer relatively shallow (0.6 m maximum depth) lentic locations, with clear waters, neutral to basic pH (values between 7 and 8), high conductivity (0.211 ms/cm), and relatively high temperatures (20 °C average) for Ribeira standards (Bichuette and Trajano 2003). These snails appear to be bioindicators of good quality water, since no specimen was observed in locations close to sewage discharges. Indeed, the spatial distribution of Potamolithus in the Ribeira Valley seems to be constrained by environmental conditions, since we did not record specimens in sites with strong currents, domestic pollution, at depths greater than 1 m, or devoid of substrates for attachment (such as pebbles and boulders). The pH and conductivity values were high in the different caves/epigean rivers with Potamolithus records.

Solem and van Bruggen (1984) observed significant positive correlations between the species richness of freshwater gastropods and high values of pH, conductivity, hardness, and alkalinity on the one hand and, on the other, negative correlations with high levels of suspended particles. According to Macan (1949) and Dillon (2000), good conditions for the occurrence of operculate snails in freshwater are a large water volume, moderately warm and alkaline habitats, with little or no organic solids, and no pollution. Among truncatelloids from temperate regions, Bithynia tentaculata (Linnaeus, 1758) are extremely sensitive to low oxygen concentrations and avoid water with strong currents, showing a preference for backwaters (branches of rivers) with depths between 70 and 180 cm. Polluted waters, with low dissolved oxygen, are avoided by Peringia ulvae (Pennant, 1777) (Fretter and Graham 1962), another truncatelloid species. The troglobitic Pseudotricula eberhardi Ponder, 2002, from Tasmania, set up on smooth rocks that occur in small hypogean streams, preferring shallow sites (Ponder 1992).

The main tributaries of the Alto Ribeira basin in São Paulo State (left bank) cross limestone outcrops, forming several, semi-isolated micro-basins. Most of the rivers flowing across the limestone of the Alto Ribeira valley form cave systems, and almost all of them have their own troglophilic, or troglobitic, populations (Trajano et al. 2016). Moreover, insoluble rocks, such as phyllites, are intercalated with limestone outcrops (Karmann and Sànchez 1979), which probably limit subterranean dispersal (Fernandes et al. 2013). Insoluble rocks represent barriers, which may be facilitating in the isolation and speciation process. Furthermore, the fact that there are no observed cases of syntopy, points to allopatric speciation events.

In addition to the hydrogeology, another factor that may contribute to the restricted distribution observed in Potamolithus in Alto Ribeira is the limited capacity for dispersal due to the small size and slow locomotion of these animals, favoring isolation in confined areas. Ponder (1994) observed a high degree of endemism in mollusks in Southeast Australia and argued that limited mobility and restriction to some habitats (in that case, springs and ponds) would allow for relatively rapid speciation. The same was observed in Tasmania when epigean and cave fauna were compared (Ponder et al. 2005).

The paucity of records at higher altitudes in the Ribeira Valley was probably due to the lower temperatures, since the other variables (pH, conductivity, dissolved oxygen) were within the ranges observed elsewhere and the waters were even more pristine (Bichuette and Trajano 2003). In the Intervales karst area (PEI), despite the extensive collecting effort expended by E. Trajano and colleagues since the 1980s, Potamolithus specimens have been found in just one cave, and, in Espirito Santo-Caboclos area, located between PEI and Betari valley, these gastropods have been found in only two caves. The winter temperatures may drop to negative values in PEI, much lower than those observed in the Betari and Iporanga valleys located in PETAR (ca. 4 °C), which could be limiting the colonization of PEI by Potamolithus gastropods. It is possible that the PEI region represents the northern boundary of the genus distribution, at least in the Upper Ribeira Valley.

The mosaic distribution of character states observed encompasses not only the classic troglomorphic traits (presence vs. absence of eyes; body and periostracum pigmentation), but also apparently neutral characters under the hypogean selective regime (shell shape) and those with a less clear relationship with the subterranean way of life (body size and intestine shape - see below). This observation, allied to the high degree of habitat fragmentation, provides evidence for an independent origin of these species from one or more unknown epigean ancestors living in some of the main tributaries of the Upper Ribeira River. These ancestral populations would have colonized the micro-basins upstream and possibly became isolated in epigean headwaters during the dry phases of paleoclimatic cycles, originating in species such as P. karsticus and Potamolithus spp. 1 to 6). Further steps would be the colonization of subterranean habitats, with the establishment of troglophilic populations (all of the above except for Potamolithus sp. 1), and isolation in the subterranean realm followed by troglobitic speciation. In the case of P. troglobius and Potamolithus spp. 8 to 12, these originated from unknown epigean ancestors; we could not relate any of these morphs to the epigean and troglophilic ones based on distribution and morphology.

It is noteworthy that troglobitic Potamolithus from the Alto Ribeira karst are not generally smaller than the troglophilic and epigean ones. For instance, the troglobitic Potamolithus from Pescaria cave reached larger sizes than all the troglophiles, as did the epigean Potamolithus sp. 1. On the other hand, all Potamolithus spp. recorded in the study area, including the epigean one from the Betari river basin and the troglobites from the Iporanga and Bocaina river basins, were smaller than P. ribeirensis, found exclusively in the Iporanga river. Simone and Moracchioli (1994) selected four epigean Potamolithus species occurring near the Alto Ribeira, in addition to P. ribeirensis, for comparison with P. karsticus and P. troglobius, and all of them were larger than the latter. Therefore, even without a phylogeny, we feel confident in proposing that the small size of these species is due to miniaturization.

The present data corroborate Bichuette and Trajano (2003) in that the small size of Potamolithus gastropods in the Alto Ribeira would be a preadaptation (in the sense of exaptation) favoring the colonization of subterranean habitats and establishment of troglophilic populations, and explains the wide distribution of these mollusks in the area.

Morphological data from Potamolithus spp. from the Alto Ribeira do not support the hypothesis of a strict correlation, or a single cause-and-effect relationship, between miniaturization and intestine coiling. The species reaching the largest size, the epigean P. ribeirensis, presents a U-shaped intestine, and the second largest one, the troglobitic Potamolithus sp. 9, from Pescaria cave, has a type-3 intestine, a coiling condition that is more advanced than that observed in the smaller troglobitic Potamolithus spp. 11 (type 1) and 12 (type 2). Other factors besides body size may affect intestine shape, for instance selection for an increase in intestine length (increase in absorptive surface) as an adaptation to increase the efficiency of nutrient intake. This hypothesis is corroborated by the observed tendency of troglobites to have more coiled intestines (types 3, 4, or 5, except the aforementioned cases, versus types 1 or 2 in the epigean and troglophilic populations). In fact, a coiled condition of the intestine is also observed in deep-sea mollusks, and this is related to an increase in the area for nutrient absorption (Sanders and Allen 1973).

The relatively large size of Potamolithus sp. 9 may be a plesiomorphic trait, i.e., the retention of the non-miniaturized state present in a population that was extinct in the epigean habitat, or it may be the result of a secondary increase in size in a formerly miniaturized population (character reversal). Trajano and Bichuette (2016) hypothesized that the catfish Ituglanis passensis Fernández & Bichuette, 2002, from Passa Três cave in São Domingos karst area, Central Brazil, originated from a miniaturized population that vertically colonized the subterranean habitat through the epikarst (see Bichuette et al. 2015). As food input drastically increased—due to the opening of large conduits and collapse of the ceiling which then formed a typical lotic habitat with large amounts of organic matter carried into the cave—intraspecific competition without the constraints of food limitation would have selected larger sizes, resulting in a gigantic miniaturized species. There is no evidence of vertical colonization of caves by Potamolithus gastropods in the Alto Ribeira karst area; on the contrary, observations of epigean and troglophilic populations indicate that these organisms colonize the hypogean environment mostly through cave sinkholes and resurgences. However, the Pescaria cave is ecologically similar to Passa Três cave, and the model proposed for I. passensis may also account for the large size of Potamolithus sp. 9. The type-3 intestine in the latter corroborates this hypothesis because it seems inconsistent with the present-day conditions; apparently the cave habitat is not food-limited, and the relatively large size of the shell does not require coiling. A proper test for the two hypotheses—large size as a plesiomorphic condition versus character reversal—would start with a character optimization in a phylogeny of Potamolithus gastropods including the species of interest. Unfortunately, none is available to date.

The degree of troglomorphism, usually relative to visual structures and dark pigmentation, has frequently been used to infer the phylogenetic age of troglobitic taxa (species or higher), i.e., its time of isolation in the subterranean environment (Poulson 1963, Wilkens 1982, Trajano 1995). Therefore, Potamolithus spp. 8 and 9, from Morro Preto and Couto caves in the Betari micro-basin, and from Pescaria Cave in the Pilões micro-basin respectively, were isolated in the hypogean habitat later than the other troglobitic species (they would be “younger troglobites”). However, as pointed by Trajano (2007), this reasoning assumes fairly constant rates of morphological differentiation among subterranean taxa, but mosaics of character states in closely related groups indicate that different mechanisms act at different rates in each population. In addition, differences in population sizes affect rates of divergence.

It is noteworthy that three of the seven Potamolithus spp. that form troglophilic populations in the Alto Ribeira are characterized by a depigmented body (sp. 2 from Ouro Grosso, 4 from the Água Suja, and sp. 5 from Roncador systems; the periostracum is also depigmented in spp. 4 and 5), indicating relaxed selection for dark pigmentation. On the other hand, all epigean and troglophilic populations have eyes, indicating a stabilizing selective pressure for maintenance of eyes in the epigean habitat.

Degradation of water quality resulting, for instance, from quarrying and mining activities, poorly controlled tourism, and deforestation, especially upstream of the subterranean systems, may severely impact cave dwelling Potamolithus populations, since they show a strong preference for clean, well-oxygenated waters. Eberhard (1992) described the negative impacts of mining activities on cave snails from Tasmania.

The Alto Ribeira karst area in São Paulo State is mostly situated within the limits of State Parks, which should warrant protection for the aquatic gastropods endemic to this region. Unfortunately, in practice, this is not the case because Brazilian law allows for the exclusion of areas for any extension or social or economic reasons considered strategic for the State. This has happened recently with the area including the Areias resurgence in the PETAR, the locality of a differentiating population of the blind catfish, Pimelodella kronei (Miranda Ribeiro, 1907), a troglobitic catfish included on the IUCN Brazilian Red List (Endangered - EN category) (ICMBio 2016). Therefore, even protection by conservation units is not definitive. Moreover, the headwaters of several micro-basins (e.g., Pilões and Iporanga rivers) and the sources of the Betari river are not protected and are subject to pollution. Pollution by pesticides has been reported for the Betari river (Moraes et al. 2003)

Another important threat to the subterranean fauna is over-collection (Trajano et al. 2012). Potamolithus troglobius and P. karsticus are listed as Critically Endangered (CR) on the IUCN Brazilian Red List (ICMBio 2016) and collections must be controlled and responsible. P. troglobius is endemic to a single cave system (Areias) and it is an important component of the diet of P. kronei.

Regrettably, a loose ethical attitude of many modern researchers imbued by the “publish or perish” philosophy (Trajano 2017), allied with poor control of collections by the licensing authorities, have resulted in instances of over-collection. An illustrative example is the collection of a total of 3,325 specimens of P. troglobius from 12 sites during four sampling occasions using the Surber technique (Silva et al. 2015). This sampling technique is non-selective (with respect to benthic organisms) and disturbs the streambed in the area inside the sampling frame—where the rocky substrate must be cleaned and all fauna dislodged—and can result in significant impacts on the ecosystem when used without proper criteria; the huge number of gastropods collected (+ 3,000) indicates that this was the case in the Areias system. It is noteworthy that such collections were done for a study authorized by the Park authorities, in a cave where another similarly authorized study—on the troglobitic catfish population using mark-recapture—was in progress for almost five years (Guil and Trajano 2013), in the context of one of the longest monitoring projects of a subterranean species worldwide, which started in the 1980s (Trajano 1991). Indeed, P. troglobius is part of the diet of the catfish Pimelodella kronei, the first troglobite described for Brazil collected in the late 1890s, and Areias system is the type-locality of P. kronei. There is no justification for such an enormous number of specimens to be collected. This cave has been studied biologically since the 1940s and was recognized as the first high-diversity spot for troglobites in 2005 (Trajano et al. 2016).

It is clear that any experimental design, and its associated sampling techniques, that result in the collection of such a huge number of individuals is a misconception, and a threat to be avoided. No scientific study, even focusing on a completely unknown species (not the case), would require such a sample size.

On the other hand, collections are indispensable because effective conservation policies are based on good biological data, and good biological data depend on careful examination of specimens by experts. There are no a priori rules regarding sample sizes, which depend not only on the objectives of the study but also on the taxon under consideration.

Only ethically oriented, experienced researchers are qualified to assess the need for collections and the scientifically acceptable minimum and maximum sample sizes. Therefore, the best way to ensure protection of biodiversity is to rely on the scientific community’s self-regulation, initiatives such as the São Francisco Declaration on Research Assessment (DORA) as guidelines for refereeing processes for funding agencies, editorial policies for scientific journals, and selection of positions at universities and research centers, etc.