Bulimus ater, I ALS AND M ETHODS

Rosa, Rafael M., Salvador, Rodrigo B. & Cavallari, Daniel C., 2025, Te disappearing act of the magician tree snail: anatomy, distribution, and phylogenetic relationships of Drymaeus magus (Gastropoda: Bulimulidae), a long-lost species hidden in plain sight, Zoological Journal of the Linnean Society 203 : -

M ATER I ALS AND M ETHODS

Te specimens of D. magus analysed in this study were either live individuals observed in situ or dry shells and ethanol-preserved snails deposited in the following natural history collections: Coleção Malacológica de Ribeirão Preto , Faculdade de Filosofia , Ciências e Letras de Ribeirão Preto, Universidade de São Paulo ( CMRP, Ribeirão Preto, Brazil) ; Museu de Zoologia da Universidade de São Paulo ( MZSP, São Paulo, Brazil) ; and Zoologische Staatssammlung München ( ZSM, Munich, Germany) . Specimens of other species (including type material) from these collections were analysed for comparative purposes. Photographs of type specimens from other species were also consulted using published literature.

Anatomy

A few specimens from the aforementioned collections were selected for anatomical analysis. Shells were inspected visually with the assistance of a Leica M205C stereomicroscope equipped with a Leica MC170 HD digital camera at the Centro para Documentação da Biodiversidade (FFCLRP-USP, Ribeirão Preto, Brazil). Shell measurements were taken using callipers and LEICA APPLICATION SUITE X 4.12 sofware. Other anatomical structures were examined on ethanol-preserved specimens using stereomicroscopes, scanning electron microscopy, and computed tomography (CT), following the methods outlined below.

Anatomical structures were identified and described following previous descriptions of Drymaeus species (e.g. Breure 1979, Breure and Eskens 1981, Simone and Amaral 2018, Simone et al. 2020). Te anatomical nomenclature used herein aligns with Simone and Amaral (2018) and Simone et al. (2020).

Abbreviations: aa, anterior aorta; ac, albumen chamber; ag, albumen gland; an, anus; au, auricle; bc, bursa copulatrix; bd, bursa copulatrix duct; bm, buccal mass; ce, cerebral ganglion; cs, circulatory system; cv, pulmonary (efferent) vein; da, digestive gland anterior lobe; dd, duct to digestive gland; df, dorsal folds of buccal mass; dg, digestive gland posterior lobe; ds, digestive system; eh, epiphallus; es, oesophagus; fo, free oviduct; fp, genital pore; F, foot; go, gonad; hd, hermaphrodite duct; in, intestine; jw, jaw; ki, kidney; mb, mantle edge; mo, mouth; ne, nephrostome; ns, central nervous system; oc, odontophore cartilage; om, ommatophore; pe, penis; pm, penis muscle; pn, pneumostome; pp, pedal ganglion; ps, penis sheath; pt, prostate; pu, pulmonary cavity; pv, pneumostome right flap; ra, radula; rc, renopericardial canal; re, reproductive system; rn, radular nucleus; rt, rectum; sd, salivary duct; sr, seminal receptacle; st, stomach; ut, uterus; ve, ventricle; vg, vagina; vm, visceral mass.

Computed tomography

Specimens preserved in 70% ethanol were immersed individually in 30 mL of a contrasting solution consisting of 1% phosphotungstic acid and 1% dimethyl sulphoxide diluted in 70% ethanol for 7–15 days to enhance the contrast of sof tissues. CT scanning was conducted at the Centro para Documentação da Biodiversidade using a Phoenix v|tome|x S240 Industrial High-Resolution CT & X-Ray System (General Electric, USA). Tis system is equipped with a digital high-contrast detector DXR250RT and a 180 kV high-power nanofocus source. High-resolution X-ray computed tomographies were captured with the following setings: source at 70 kV and 200 µA, 1000 projections, binning of 1 × 1, averaging three frames with one frame skipped, exposure time of 333.09 ms, default offset and gain correction, and no filter applied. Te resulting 16-bit greyscale images measured 990 × 1000 pixels. Tree-dimensional (3D) reconstructions were processed using GE Phoenix Datos X2 sofware, and visualization and editing of the 3D models were performed with VGStudio Max® 3.0 (Volume Graphics, Germany).

To reconstruct specific organs and structures in 3D, CT images were segmented manually using AMIRA 5.3.2 (Visage Imaging Inc.). Interpolation was applied across intervals of up to five slices to optimize processing time. Afer segmentation, models were saved and exported in.stl format for further visualization in various sofware platforms, including VGStudio Max® 3.0. Te final 3D models are available at MorphoMuseuM (Rosa et al. 2025).

Scanning electron microscopy

Scanning electron microscopy was used to aid in the visualization and characterization of radular morphology. Te radula was extracted using standard dissection methods and coated with gold using a Quorum Q150R ES sputer coater at the Centro para Documentação da Biodiversidade. Imaging was conducted with a JEOL JSM-6610LV scanning electron microscope at the Laboratório Multiusuário de Microscopia Eletrônica (FMRP-USP, Ribeirão Preto, Brazil).

DNA extraction and sequencing

Tissue clips were obtained from selected specimens of D. magus and other potentially related species for DNA extraction ( Table 1). Four specimens of D. magus from different localities were used, representing two conchological morphotypes. DNA extraction was done using the QIAGEN DNEasy® Blood & Tissue Kit mostly following the manufacturer’s standard protocol, but modifying the final step to increase yield: one-quarter of the suggested amount of buffer AE was used, and a repetition of the step was added.

Te combination of genetic markers used in this study was the same as in the studies by Breure and Romero (2012) and Salvador et al. (2023), to allow the inclusion of our data in their phylogenetic framework of the subfamily Peltellinae . Te markers (and primers) used were as follows: the barcoding fragment of the mitochondrial COI gene ( Folmer et al. 1994; primers LCO/HCO); a fragment of the nuclear H3 (histone 3) gene (Uit de Weerd and Gitenberger 2013; primers H3pulF/ H3pul3); and a fragment of nuclear DNA including the 3 ′ end of the 5.8S rRNA gene, the ITS2 region, and the 5 ′ end of the 28S rRNA gene ( Wade and Mordan 2000, Wade et al. 2006; amplified in two parts using the primer pairs LSU-1/LSU-3 and LSU-2/LSU-5).

Te PCR amplification protocols were the same as those used by Salvador et al. (2023), starting with initial denaturation at 95°C (3 min), then having specific cycles as follows: for COI, 35 cycles of denaturation at 95°C (30 s), annealing at 48°C (1 min), and extension at 72°C (2 min); for H3, 40 cycles of denaturation at 95°C (30 s), annealing at 57°C (30 s), and extension at 72°C (40 s); and for ITS2 + 28S, 40 cycles of denaturation at 95°C (30 s), annealing at either 50°C (ITS2 section) or 45°C (28S section) (1 min), and extension at 72°C for either 5 min (ITS2 section) or 2 min (28S section). Te PCR ended with a final extension at 72°C (5 min) for all markers.

Te success of amplification was assessed visually via agarose gel electrophoresis. Te successful PCR products were then cleaned with ExoSAP-IT™ (Affymetrix Inc.) following the manufacturer’s standard protocol. Samples were then prepared for sequencing and sent to Macrogen Europe ( Amsterdam , Te Netherlands) for Sanger sequencing. Te resulting sequences were quality checked and de novo assembled in GENEIOUS PRIME (v. 2023.2.1, Biomaters Ltd.). Te consensus sequences were extracted and uploaded to GenBank (for accession numbers, see Table 1) .

Phylogenetic analysis

We have included our new samples in the phylogenetic framework recently published by Salvador et al. (2023). To that end, we used their entire dataset of subfamily Peltellinae , using one Bulimulinae and one Strophocheilidae as outgroups. Te taxa used in the phylogenetic analysis, alongside their GenBank accession numbers and locality data, can be found in Table 1.

Te alignment of genetic sequences of each marker was done in GENEIOUS PRIME using the MAFFT plugin ( Katoh et al. 2002, Katoh and Standley 2013) with the default setings. Te resulting alignment of each marker was proofed visually for inconsistencies. Alignment of the ITS/28S marker was run through GBLOCKS ( Castresana 2000, Talavera and Castresana 2007) to eliminate poorly aligned or data-deficient positions that could impact the analysis. Te alignments were then concatenated, resulting in a total of 2033 bp (COI = 654 bp, H3 = 267 bp, and ITS2/28S = 1112 bp).

Te Bayesian inference analysis was conducted with MRBAYES (v.3.2.7; Ronquist et al. 2012), via the CIPRES Science Gateway ( Miller et al. 2015). It consisted of two concurrent runs, each with four Markov chains of 100 million generations (the first 20% discarded as ‘burn-in’), the default priors, nst = 6, rates = invgamma, temperature parameter = 0.1, sampling every 1000 generations, and with substitution model parameters unlinked across the markers (COI, H3, and ITS/28S). Markov chain Monte Carlo convergence was assessed by examining the SD of split frequencies (~0.001) and the potential scale reduction factor (PSRF ~1.0), in addition to the trace plots (Ronquist et al. 2009).

Occurrence records

Te distribution of D. magus was reviewed by combining data from the aforementioned natural history collections, field observations, published literature, and the iNaturalist citizen science platform (htps://www.inaturalist.org/), which contains geolocated records accompanied by photographs of living animals or empty shells. All specimens had their identifications reviewed, and misidentified specimens were excluded from the dataset. Likewise, all iNaturalist records were checked by us, following the same verification protocol applied by Rosa et al. (2022). Data on the reviewed iNaturalist records are available as Supporting Information File S1.

In the text, iNaturalist records are reported using their observation number in the platform. To visualize the record on iNaturalist, simply add that number to the end of the command ‘htps://www.inaturalist.org/observations/’, which will become a functioning URL.

Field observations

Field observations of live specimens of D. magus were carried out to gather data on life appearance, ecology, and natural history. Observations were made in two localities in northern São Paulo state: (i) the University of São Paulo (USP) campus in the municipality of Ribeirão Preto; and (ii) a forested area surrounding the reservoir of the Caconde hydroelectric power plant near the municipality of Caconde. Te localities were chosen owing to both being relatively accessible to the authors and both having previous records of D. magus in the form of specimens in the CMRP and MZSP collections. Fieldwork was conducted mostly at night (~19.00–00.00 h) during the rainy season (October–February) in 2023 and 2024, but additional searches were carried out at other times and periods when possible. Te specimens found were photographed, and notes on their behaviour were made. Some of the photographs were uploaded to iNaturalist by the authors and other people who helped with the fieldwork.

Additional natural history information was also gathered from iNaturalist observations, whenever possible. Te resulting data were compared with the few published accounts on the ecology and natural history of other Drymaeus species (e.g. Breure 1979).