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 reticulatus Delicado, Boulaassafer & Hauffe sp. nov. ( Figs 13 , 14 ) Pseudamnicola orsinii (Küster, 1852) — Delicado et al. 2015 Pseudamnicola sp. 5 — Delicado et al. 2018 Pseudamnicola sp. 5 — Boulaassafer et al. 2020 ZooBank registration: urn:lsid:zoobank.org:act: CD01E3A2- 61DA-4003-8950-F6743F7FCB01 Etymology In Latin, reticulatus means reticulated, describing the net-like shell sculptures found in the type population. Type material Holotype ( MNCN 15.05 /200503H ; dissected animal) and one paratype ( MNCN 15.05 /200503P) in the MNCN collection and six paratypes in the UGSB collection ( UGSB 5971 ). Type locality: a spring discharging to the Fiumefreddo River , Fiumefreddo di Sicilia , Sicily, Italy . Material studied A spring discharging to the Fiumefreddo River , Fiumefreddo Sicilia , Sicily, Italy , 37.7851°N , 15.2271°E , leg. M.B., January 2008 , MNCN 15.05 /200503 and UGSB 5971 (80% ethanol) . Figure 13. Shells, operculum, and radulae of Pseudamnicola reticulatus . A, holotype ( MNCN 15.05/200503H ). B–J, paratypes (MNCN 15.05/200503P; UGSB 5971). A–D, shells. E, F, operculum (E, inner side; F, outer side). G, protoconch. H, portion of radula ribbon. I, central radular teeth. J, inner and outer marginal teeth. Diagnosis Shell small, ovate-conic, with a fine net-like surface sculpture (in the type population); protoconch microsculpture pitted; central radular tooth formula (4)3-C-3(4)/1-1; bursa copulatrix medium-sized, 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, a moderately wide base, and a small patch of pigmentation on its distal region; nervous system darkly pigmented, elongate (mean RPG ratio =.52). Description Shell ovate-conic, whorls 3.5–4, height 2.00– 3.25 mm , with a fine net-like surface sculpture (in the type population) ( Fig. 13A–C ; Supporting Information, Table S3). Periostracum yellowish to whitish. Protoconch ~500 µm wide, eroded in the type population, whorls 1.5; nucleus ~150 µm wide; protoconch microsculpture pitted ( Fig.13G ). 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. 13D ). Umbilicus narrow, not covered by the inner lip. Holotype : SL = 3.28 mm , SW = 2.23 mm , AL = 1.85 mm , and AW = 1.40 mm . Operculum oval, yellowish, about two and a half whorls; muscle attachment area oval and located near the nucleus ( Fig. 13E, F ). Radular length intermediate, ~600 µm (~25% of total shell length), with ~55 rows of teeth ( Fig. 13H ). Central tooth formula (4)3-C-3(4)/1-1 ( Fig. 13I ); basal tongue V-shaped, length about equal to lateral margin. Lateral tooth formula 3-C-3. Inner marginal teeth having 15–20 tapered cusps, shortening towards the base. Outer marginal teeth with ~20 sharp cusps ( Fig. 13J ). Figure 14. Anatomy of Pseudamnicola reticulatus . A–H, paratypes (UGSB 5971). A, ctenidium and osphradium. B, stomach. C, partial nervous system. D, pallial oviduct. E, bursa copulatrix and seminal receptacle. F, head of male and penis. G, penis. H, prostate gland. Animal darkly pigmented, with pigmentation lighter on neck and tentacles ( Fig. 14F ). Snout as long as wide, with medium distal lobation; foot size intermediate, with dorsal pigmentation. Ctenidium with ~20 well-developed gill filaments, occupying ~50% of pallial cavity length and posteriorly positioned. Osphradium of intermediate width and opposite middle of ctenidium ( Fig. 14A ). 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. 14B ; Supporting Information, Table S4). Nervous system elongate (mean RPG ratio =.52), darkly pigmented, darker on ganglia than on connectives and commissures; cerebral ganglia approximately equal in size ( Fig. 14C ). Female glandular oviduct approximately three times as long as wide. Albumen gland shorter than capsule gland. Bursa copulatrix small, pyriform, longer than wide. Bursal duct slightly longer than bursal length. Renal oviduct black-pigmented until the area above the insertion of seminal receptacle, relatively straight. Seminal receptacle elongate, with a short duct, joining renal oviduct at the insertion point with bursal duct ( Fig. 14D, E ; Supporting Information, Table S5). Male genitalia with a prostate gland approximately twice longer than wide, bean-shaped; seminal duct entering the middle-posterior region; pallial vas deferens emerging close to its anterior edge ( Fig. 14H ). 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. 14F, G ; Supporting Information, Table S6); base moderately wide; attached well behind the right eye; penial duct narrow, curved, coursing close to outer edge. Habitat Endemic to a small coastal spring. Figure 15. Shells, operculum, and radulae of Pseudamnicola sardiniensis . A, holotype ( MNCN 15.05/200504H ). B–F, paratypes (UGSB 15501). A, B, shell. C, protoconch. D, portion of radula ribbon. E, central radular teeth. F, inner and outer marginal teeth. Remarks We dissected six of the eight specimens of P. reticulatus deposited in the UGSB collection and found only one female . All specimens had an eroded shell apex and a fine net-like shell sculpture, the latter feature atypical in Pseudamnicola . Delicado et al. (2015) identified the Pseudamnicola population occurring in the same spring near Fiumefreddo di Sicilia as P. orsinii . There is, however, a large genetic divergence (4% for COI ) between the P. reticulatus and the newly sequenced snail from the type locality region of P. orsinii , in addition to morphological differences detected in shells and other anatomical structures, which might disprove this identification. The phylogenetic studies of Delicado et al. (2018) and Boulaassafer et al. (2020) inferred that the P. reticulatus clade was phylogenetically distant from the P. moussonii clade. The two species differ according to a set of morphological characters: the shell spire and aperture are wider in P. reticulatus (for comparison, see Giusti 1976 ; Falkner and Boeters 2003 ); the bursal duct and seminal receptacle of the single dissected female from the type locality of P. moussonii are slightly longer than the ones measured in the P. reticulatus female, but this difference needs confirmation; and the penis is generally wider in P. reticulatus .