A holistic perspective on species delimitation outperforms all methods based on single data types in freshwater gastropods (Caenogastropoda: Hydrobiidae: Pseudamnicola) Author Delicado, Diana Department of Animal Ecology & Systematics, Justus Liebig University Giessen, Heinrich-Buff-Ring 26 - 32 IFZ, D- 35392 Giessen, Germany didelicado@gmail.com Author Boulaassafer, Khadija Muséum d’Histoire Naturelle de Marrakech, Cadi Ayyad University, Prince Moulay Abdellah Boulevard, B. P. 2390, 40000 Marrakesh, Morocco Author Khalloufi, Noureddine Environmental Biomonitoring Laboratory (LR 01 ES 14), Faculty of Sciences of Bizerte, University of Carthage, 7021 Jarzouna, Tunisia Author Hauffe, Torsten Department of Biology, University of Fribourg, Chemin du Musée 10, CH- 1700 Fribourg, Switzerland & Swiss Institute of Bioinformatics, Chemin du Musée 10, CH- 1700 Fribourg, Switzerland text Zoological Journal of the Linnean Society 2025 2024-03-05 203 1 1 31 https://doi.org/10.1093/zoolinnean/zlae010 journal article 307941 10.1093/zoolinnean/zlae010 5265b49c-637e-4b38-97cb-e0f7c234cf88 0024-4082 14774861 D1C88C6-B4B9-46D0-94AD-BA7439E6DF22 Pseudamnicola araujoi Delicado, Khalloufi & Hauffe sp. nov. ( Figs 5 , 6 ) ZooBank registration: urn:lsid:zoobank.org:act: D1CEB40E-0C7F-4FC1-A5D0-991A3CCF0B4A Etymology Named after Dr Rafael Araujo, curator of Malacology at Museo Nacional de Ciencias Naturales (MNCN-CSIC) (2008–2021), who contributed greatly to the knowledge of freshwater molluscs, including those of Tunisia . Type material Holotype ( MNCN 15.05 /200505H ) and three paratypes ( MNCN 15.05 /200505P) in the MNCN collection and 20 paratypes in the UGSB collection ( UGSB 17299 ). Type locality: Gnaa Spring , Jendouba , Tunisia . Material studied Gnaa Spring , Jendouba , Tunisia , 36.5653°N , 8.7895°E , leg. N.K. , August 2015 , MNCN 15.05 /200505 and UGSB 17299 (80% ethanol) . Figure 5. Shells, operculum, and radulae of Pseudamnicola araujoi . A, B, holotype ( MNCN 15.05/200505H ). C–J, paratypes (UGSB 17299). A–D, shells. E, F, operculum (E, inner side; F, outer side). G, protoconch. H, portion of radula ribbon. I, central radular teeth. J, outer marginal teeth. Diagnosis Shell small, ovate-conic; protoconch microsculpture pitted; central radular tooth formula 4-C-4/1-1; bursa copulatrix large, pyriform, with a duct longer than bursal length; SR1 long, elongate, with a short duct; penis gradually tapering, with many folds over the entire surface and a small patch of pigmentation on its distal region; nervous system darkly pigmented, elongate (mean RPG ratio =.60). Description Shell ovate-conic, whorls 4–4.5, height 2.5–3 mm ( Fig. 5A–C ; Supporting Information, Table S3). Periostracum yellowish. Protoconch ~450 µm wide, whorls 1.5; nucleus ~150 µm wide; protoconch microsculpture pitted ( Fig. 5G ). Teleoconch whorls moderately convex, with deep sutures; body whorl large, occupying about three-quarters of total shell length. Aperture longer than wide, slightly oval; inner lip thicker than outer lip; peristome margin simple, straight ( Fig. 5B ). Umbilicus narrow, not covered by the inner lip. Holotype : SL = 2.87 mm , SW = 2.08 mm , AL = 1.61 mm , AW = 1.10 mm . Operculum oval, yellowish, about two and a half whorls; muscle attachment area oval and located near the nucleus ( Fig. 5E, F ). Radular length intermediate, ~650 µm (~25% of total shell length), with ~55 rows of teeth ( Fig. 5H ). Central tooth formula 4-C-4/1-1 ( Fig. 5I ); basal tongue V-shaped, length about equal to lateral margin. Lateral tooth formula 3-C-3. Inner marginal teeth having ~20 tapered cusps, shortening towards the base. Outer marginal teeth with ~25 sharp cusps ( Fig. 5J ). Animal darkly pigmented, except for neck and tentacles ( Fig. 6F, G ). Snout as long as wide, with medium distal lobation; foot size intermediate, with dorsal pigmentation. Ctenidium with 17–19 well-developed gill filaments, occupying ~50% of pallial cavity length and posteriorly positioned. Osphradium of intermediate width and opposite middle of ctenidium ( Fig. 6A ). Stomach slightly longer than wide, with two chambers almost equal in size and a medium-sized posterior caecum; style sac longer than wide, surrounded by an unpigmented intestine ( Fig. 6B ; Supporting Information, Table S4). Nervous system elongate (mean RPG ratio =.60), darkly pigmented, darker on ganglia than on connectives and commissures; cerebral ganglia approximately equal in size ( Fig. 6C ). Figure 6. Anatomy of Pseudamnicola araujoi . A–H, paratypes (UGSB 17299). A, ctenidium and osphradium. B, stomach. C, partial nervous system. D, pallial oviduct. E, bursa copulatrix and seminal receptacle. F, G, head of male and penis. H, prostate gland. Female glandular oviduct approximately three times as long as wide. Albumen gland shorter than capsule gland. Bursa copulatrix large, pyriform, almost as long as wide. Bursal duct longer than bursal length. Renal oviduct black-pigmented until the insertion of seminal receptacle, making one or two loops. Seminal receptacle elongate, with a short duct, joining renal oviduct at the insertion point with bursal duct ( Fig. 6D, E ; Supporting Information, Table S5). Male genitalia with a prostate gland approximately twice as long as wide, bean-shaped; seminal duct entering the middle-posterior region; pallial vas deferens emerging close to its anterior edge ( Fig. 6H ). Penis as long as head, gradually tapering, with many folds over the entire surface and a small patch of pigmentation on its distal region ( Fig. 6F, G ; Supporting Information, Table S6); base medium wide; attached well behind the right eye; penial duct narrow, curved, coursing close to outer edge. Habitat Endemic to a small spring, in which the species lives in high abundance. The substrate is composed of silt and gravel. The average water temperature varies between 14°C in winter and 25°C in summer. The conductivity varies between 0.53 and 0.78 mS. Co-occurring mollusc species are Bullaregia tunisiensis Khalloufi, Béjaoui & Delicado, 2017 , Pisidium sp. , Galba truncatula (O. F. Müller, 1774) , and Physella acuta (Draparnaud, 1805) . Remarks According to our DNA analyses, this species is more closely related to its congeners distributed in north-western Africa ( Morocco ) than those distributed in north-eastern Africa ( Tunisia ). It resembles Pseudamnicola conovula from Tunisia in shell and penis features (Supporting Information, Figs S7F–L, S 8O–Q ). Otherwise, it differs in having a larger bursa copulatrix and a COI average sequence divergence of 7.4%. The shell and aperture shapes of P. araujoi are more similar to those of P. calamensis and P. linae (most probably a synonym of P. calamensis ) than any of the 15 Pseudamnicola species described in Algeria ( Glöer et al. 2010 : figs 6–20), which are mostly known from their shells. The morphology and morphometry of its female genitalia are, however, different from the other two species ( Glöer et al. 2010 : figs 6–20, for comparison; Fig. 2 ).