Radix Rufescens (J. E. Gray, 1822) (Gastropoda: Lymnaeidae), A New Species For Oman And Arabian Peninsula

© Authors Published by the Hungarian Natural History Museum and the Biological Section of the Hungarian Academy of Sciences. The journal uses a CC BY-NC license Acta Zoologica Academiae Scientiarum Hungaricae 69(3), pp. 303–312, 2023 DOI: 10.17109/AZH.69.3.303.2023 RADIX RUFESCENS (J. E. GRAY, 1822) (GASTROPODA: LYMNAEIDAE), A NEW SPECIES FOR OMAN AND ARABIAN PENINSULA Andrzej Falniowski1, Aleksandra Jaszczyńska1,2 and Sebastian Hofman3 1Department of Malacology, Institute of Zoology and Biomedical Research, Jagiellonian University, ul. Gronostajowa 9, 30–387 Kraków, Poland E-mail: [email protected]; https://orcid.org/0000-0002-3899-6857 2 Department of Invertebrate Evolution, Institute of Zoology and Biomedical Research, Jagiellonian University, Gronostajowa 9, 30-387 Kraków, Poland E-mail: [email protected]; https://orcid.org/0000-0002-9395-9696 3Department of Comparative Anatomy, Institute of Zoology and Biomedical Research, Jagiellonian University, ul. Gronostajowa 9, 30–387 Kraków, Poland; E-mail: [email protected]; https://orcid.org/0000-0001-6044-3055 The fauna of freshwater gastropods of the Arabian Peninsula, a desert land, is neither rich nor extensively studied. Only five species of the family Lymnaeidae have been recorded from this subcontinent so far. In February 2023, a few specimens of Radix rufescens (J. E. Gray, 1822) have been collected from the stream at Wadi Tiwi, NE Oman. The shell, female part of the reproductive organs, prostate, paraeputium and phalloteca are presented. Cytochrome oxidase subunit I (COI) sequences confirmed the assignment of these specimens to R. rufescens, known from Nepal, Bangladesh, and Myanmar, not from the Arabian Peninsula. Either R. rufescens is the sixth lymnaeid species inhabiting Arabia, or at least some of the records of R. natalensis (Krauss, 1848), whose shells are hardly discernible from the ones of R. rufescens, should be interpreted as misidentified R. rufescens. Thousands of years of the trade relations between Oman and India suggest accidental transportation of this snail by trade vessels. Keywords: shell, reproductive organs, cytochrome oxidase subunit I (COI), mtDNA, molecular relationships INTRODUCTION Lymnaeidae Rafinesque, 1815, a globally distributed family of freshwater heterobranch snails, for more than century have been widely known as showing incredible variability of their shells. This was the reason why the list of synonyms in this group is extremely long. The classic research of Baker (1911) on the North American Lymnaeidae, and Roszkowski (1914) on the lymnaeids from the profundal of Lac Léman introduced the lymnaeid taxonomy based on the anatomical characters. Such studies were summarized and completed by Hubendick (1951), and continued (e.g., Falniowski 1980a, b, c, 1981). However, many ambiguities still remained in the lymnaeid taxonomy, most of them solved later with application of the molecular data (e.g., Correal et al. 2010, Vinarski et al. 2020, Saito et al. 2021). Acta Zool. Acad. Sci. Hung. 69, 2023 304 FALNIOWSKI, A., JASZCZYŃSKA, A. AND HOFMAN, S. The Arabian Peninsula inhabits a unique assemblage of animal species, the result of the geologic and climatic history of this region. Its fauna may originate from Eastern Africa, south-central Asia, or south-western Asia. The Arabian Peninsula, a desert land in general, inhabits a moderately rich freshwater fauna (Garcia et al. 2015). From among the Lymnaeidae, only five species have been recorded so far: Galba truncatula (O. F. Müller, 1774), Galba robusta Vinarski, 2018, Radix natalensis (Krauss, 1848), Radix auricularia (Linnaeus, 1758), and Stagnicola palustris (O. F. Müller, 1774) (Smith 1894, Brown & Gallagher 1985, Neubert 1998, Vinarski 2018). Lymnaea arabica E. A. Smith, 1894 from Saudi Arabia, studied by Bin Dajem (2012), was recently considered a synonym of Radix natalensis (Krauss, 1848). In February 2023, a few specimens of Radix Montfort, 1810 and Melanoides Olivier, 1804 were collected in Wadi Tiwi in Oman. MATERIAL AND METHODS The snails were collected 4th of February 2023 at the stream, from the stones and among the algae, at the lower part of Wadi Tiwi canyon in NE Oman, 22°48’15”N, 59°14’45”E, 20 m a.s.l. (Fig. 1). Wadi Tiwi is situated in Sharkiyah, Eastern Oman MounFig. 1. Wadi Tiwi, stream in its lower part Acta Zool. Acad. Sci. Hung. 69, 2023 305 RADIX RUFESCENS (GASTROPODA: LYMNAEIDAE) NEW FOR OMAN AND ARABIA tains, rising to 2300 m altitude. Wadi (Arabic) means dry river valley. Wadi Tiwi is about eight km long. Its floor has eroded down through Eocene and Palaeocene to the top of the ophiolite which is composed of highly serpentinised mantle sequence harzburgite. The entire crustal sequence of the ophiolite has been eroded off, prior to deposition of the overlying Palaeocene limestones (Searle 2019).The lower part of the Wadi, where the snails have been found, has some small blue pools, lined with palms and reed-beds. The snails were collected by hand and with a small sieve and fixed in 80% analytically pure ethanol, changed twice. The shells were photographed with Canon EOS 50D camera with Canon Macro Lens. The snails were dissected under a Nikon SMZ18 microscope with a dark field, the anatomical structures photographed with Canon EOS 50D camera under a Nikon SMZ18 microscope with a dark field. Measurements of the praeputium and penis sheath were done on the photograph with curvimeter. DNA was extracted from whole specimens; tissues were hydrated in TE buffer (3 × 10 min); then, total genomic DNA was extracted with the SHERLOCK extraction kit (A&A Biotechnology), and the final product was dissolved in 20 μl of tris-EDTA (TE) buffer. The extracted DNA was stored at −80 °C at the Department of Malacology, Institute of Zoology and Biomedical Research, Jagiellonian University in Kraków (Poland). Mitochondrial cytochrome oxidase subunit I (COI) was sequenced. Details of PCR conditions, primers used, and sequencing were given by Szarowska et al. (2016). In the phylogenetic analysis, sequences from GenBank were used (Fig. 4). Sequences were initially aligned in the MUSCLE (Edgar 2004) Program in MEGA 7 (Kumar et al. 2016) and then checked in Bioedit 7.1.3.0 (Hall 1999). Uncorrected p-distances were calculated in MEGA 7. The estimation of the proportion of invariant sites and the saturation test for entire data sets (Xia 2000, Xia et al. 2003) were performed using DAMBE (Xia 2013). The data were analysed using approaches based on Bayesian inference (BI) and Maximum Likelihood (ml). For RAxML analysis, the jModelTest2 via the CIPRES Science Gateway (Miller et al. 2010) was used to find the best-fitting model, the model HKY+G was used. The ML analysis was conducted in RAxML-NG v. 0.8.0 (Kozlov et al. 2019) via web service available at https://raxml-ng.vital-it.ch/, with 10 random and 10 parsimony starting trees. In the BI analysis, the K81+G model of nucleotide substitution was applied in tree reconstruction. Model was selected using MrModelTest 2.4 (Nylander 2004). The analyses were run using MRBAYES v. 3.2.7a (Ronquist et al. 2012) with defaults of most priors. Two simultaneous analyses were performed, each with 10,000,000 generations, with one cold chain and three heated chains, starting from random trees and sampling the trees every 1,000 generations. The first 25% of the trees were discarded as burn-in. The analyses were summarized as a 50% majority-rule consensus tree. Convergence was checked in TRACER v.1.7.1 (Rambaut et al. 2018), in all cases Effective Sample Size exceeded 200. FigTree v. 1.4.4 (Rambaut 2010) was used to visualize the trees. RESULTS The shell (Fig. 2) with acute spire, high as for this genus, the aperture broad and variable in outline; the shell resembles the ones presented for R. rufescens in Vinarski et al. (2020). The albuminoid gland (Fig. 3A) flat and rather broad, the corpus pyriformis short and bulky, bursa copulatrix small and without a discernible duct, prostate narrow, slightly broadened in its distal part, the vas deferens outlet close to the margin of the terminal part of Acta Zool. Acad. Sci. Hung. 69, 2023 306 FALNIOWSKI, A., JASZCZYŃSKA, A. AND HOFMAN, S. the prostate. The praeputium (Fig. 3B) proximally cylindrical, distally gradually narrowing, with spots of black pigment, the penis sheath pigmentless, with moderately broadened proximal end. The length proportion between the praeputium and penis sheath was 1.25, compared with 1.09–1.27 recorded for. R. rufescens by Vinarski et al. (2020). The maximum likelihood tree for COI (Fig. 4) clusters our Omanian specimen (bootstrap support 94%, Bayesian probability 0.99) within R. rufescens, all the other specimens of this species are from Nepal, Bangladesh, and Myanmar. The p-distance within this group was 0.017. The Omanian specimen differed from the other R. rufescens by p-distance 0.007–0.036. The sister species of R. rufescens is R. natalensis. p-distance between these two species equals 0.092. Fig. 2. Shells and mantle pigmentation of Radix rufescens. Scale bar: 5 mm Acta Zool. Acad. Sci. Hung. 69, 2023 307 RADIX RUFESCENS (GASTROPODA: LYMNAEIDAE) NEW FOR OMAN AND ARABIA DISCUSSION Our data clearly identified our Omanian specimens as belonging to Radix rufescens. Genetic differences between this specimen and the other sequences R. rufescens were much smaller than between R. rufescens and its sister species, R. natalensis. Also length proportion between the praeputium and penis sheath confirmed this species assignment. This is the first record of Radix rufescens not only in Oman, but in all Arabian Peninsula. Only five species of the Lymnaeidae, two of them representing the genus Radix, have been recorded (Smith 1894, Brown & Gallagher 1985, Neubert 1998): R. natalensis, molecuFig. 3. Reproductive organs of Radix rufescens: A = ventrally, B–C = praeputium and phallotheca (ag – albuminoid gland, cp – corpus pyriformis, hg – haermaphrodite gland, ng – nidamental gland, ph – phallotheca (penis sheath), pr – prostate, prae – praeputium, ut – uterus, v – vagina, vd – vas deferens). Scale bar: 1 mm Acta Zool. Acad. Sci. Hung. 69, 2023 308 FALNIOWSKI, A., JASZCZYŃSKA, A. AND HOFMAN, S. larly identified so far only in Africa (Saito et al. 2021), and R. auricularia, with a wide Eurasian range. Surprisingly, R. euphratica (Mousson, 1874), inhabiting Iran separated from Oman only by relatively narrow Ormuz Straight, has Fig. 4. Maximum likelihood tree computed for COI; bootstrap supports/Bayesian probabilities and GB numbers are given Fig. 5. Hypothetical way of invasion of R. rufescens, after Saito et al. (2021), modified Acta Zool. Acad. Sci. Hung. 69, 2023 309 RADIX RUFESCENS (GASTROPODA: LYMNAEIDAE) NEW FOR OMAN AND ARABIA not been recorded from the Arabian Peninsula. Thus R. rufescens would be the sixth lymnaeid species in the Arabia. However, the shells of R. rufescens are practically indistinguishable from the ones of R. natalensis (e.g., Vinarski et al. 2020). Thus, either R. rufescens inhabits Wadi Tiwi (and some other still unstudied localities), or simply this is the first properly identified specimen of this species, and the records of R. natalensis (at least some of them) refer to R. rufescens. On the other hand, the supposed occurrence of R. natalensis in Oman may be the result of migration from the Horn of Africa Hotspot and the Eastern Afromontane Hotspot (Mittermeier et al. 2004). The evolutionary history of Radix and main colonization routes are well known (Saito et al. 2021). The first diversification area was situated in east India, the rufescens/natalensis group split then into two lines. The ancestor of R. natalensis probably moved into western India, and later to Africa, R. rufescens remained in Nepal and Bangladesh both NE of the Indian Subcontinent, as well as in Myanmar at Malay peninsula. How R. rufescens colonized Wadi Tiwi remains enigmatic. One of the excellent maps of Saito et al. (2021) we have completed with our finding (Fig. 5). The known area of occurrence of R. rufescens may be much wider than the molecularly identified individuals confirm, thus the population discovered by us may be a part of the range covering perhaps the entire Indian Peninsula, southern Pakistan and Iran, and a large part of the Arabian Peninsula. Obviously, migration from the western coast of India would be more probable. Some older migrations may have been possible at the time when the Persian Gulf was reduced to a series of freshwater lakes (Lambeck 1996). However, small genetic differences between R. rufescens from Oman and from Nepal, Bangladesh, and Myanmar suggest much younger episodes of migration. Arabian Peninsula, especially Oman, since about 2,200 BC (Paine 2013) was in trade relations with India. Arabian dhow sailed with monsoon winds (Miles 1919, LeBaron Bowen 1949, Villiers 2018), conveying food, timber, etc., thus the accidental transportation of eggs or adult specimens of aquatic snails was probable. Especially the barrels and cans with drinkable water for the crew, rinsed before refilling in the stream in the outlet of Wadi Tiwi (situated not far from Sur, for centuries the main port at this region) were suitable for such transportation. The anthropogenic transportation of snails is extremely common (Preston et al. 2022). Also transportation by birds cannot be excluded (Charalambidou & Santamaria 2002, Figuerola & Green 2002). * Acknowledgments – The study was supported by the grant N18/DBS/000012 from the Institute of Zoology and Biomedical Research of Jagiellonian University. We are grateful to Dr M. V. Vinarski for his valuable corrections and additions to the former version of our manuscript. Acta Zool. Acad. Sci. Hung. 69, 2023 310 FALNIOWSKI, A., JASZCZYŃSKA, A. AND HOFMAN, S. REFERENCES Baker, F. C. (1911): The Lymnaeidae of North and Middle America. Recent and fossil. – The Chicago Academy of Sciences, special publication 3, Chicago. Bin Dajem, S. M. (2012): Molecular typing of the freshwater snail Lymnaea arabica, the possible intermediate host of Fasciola hepatica, collected from Saudi Arabia, by RAPDPCR. – Egyptian Academy Journal of Biological Science, B, Zoology 4(1): 173–181. Brown, D. S. & Gallagher, M. D. (1985): Freshwater snails of Oman, South Eastern Arabia. – Hydrobiologia 127: 125–149. Charalambidou, I. & Santamaria, L. (2002): Waterbirds as endozoochorous dispersers of aquatic organisms: a review of experimental evidence. – Acta Oecologica 21: 165–176. Correal, A. C., Escobar, J. S., Durand, P., Renaud, F., David, P., Jarne, P., Pointier, J.- P. & Hurtrez-Boussès, S. (2010): Bridging gaps in the molecular phylogeny of the Lymnaeidae (Gastropoda: Pulmonata), vectors of Fascioliasis. – BMC Evolutionary Biology 10: 381. Edgar, R. C. (2004): MUSCLE: multiple sequence alignment with high accuracy and high throughput. – Nucleic Acids Research 32: 1792–1797. https://doi.org/10.1093/nar/gkh340 Falniowski, A. (1980a): Podrodzaj Radix s. str. (Gastropoda, Basommatophora) w Polsce. I. Pigmentacja i anatomia. Opis Lymnaea peregra roszkowskiana subsp. nov. – Zeszyty naukowe UJ, DLXXII, Prace zoologiczne 26: 67–108. Falniowski, A. (1980b): The anatomical determination of Polish Lymnaeidae (Mollusca, Gastropoda). – Acta Hydrobiologica 22: 327–335. Falniowski, A. (1980c): Pigmentation of the mantle border in Polish representatives of the subgenus Radix (Lymnaeidae, Basommatophora, Gastropoda). – Basteria 44: 3–8. Falniowski, A. (1981): Podrodzaj Radix s. str. (Gastropoda, Basommatophora) w Polsce. II. Muszla i jej zmienność. – Zeszyty naukowe UJ DCXXXI, Prace zoologiczne 27: 113–141. Figuerola, J. & Green, A. J. (2002): Dispersal of aquatic organisms by waterbirds: a review of past research and priorities for future studies. – Freshwater Biology 47: 483–494. Garcia, N., Harrison, I., Cox, N. & Tognelli, M. F. (compilers) (2015): The status and distribution of freshwater biodiversity in the Arabian Peninsula. – IUCN, Gland. Hall, T. A. (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. – Nucleic Acids Symposium 41: 95–98. Hubendick, B. (1951): Recent Lymnaeidae, their variation, morphology, taxonomy, nomenclature, and distribution. – Almqvist Publisher, Sweden, 3: 1–223. Kozlov, A. M., Darriba, D., Flouri, T., Morel, B. & Stamatakis, A. (2019): RAxML-NG: a fast, scalable and user-friendly tool for maximum likelihood phylogenetic inference. – Bioinformatics 35: 4453–4455. https://doi.org/10.1093/bioinformatics/btz305 Krauss, F. (1848): Die südafrikanischen Mollusken. Ein Beitrag zur Kenntniss der Mollusken des Kapund Natallandes und zur Geographischen Verbreitung derselben mit Beschreibung und Abbildung der neuen Arten. – Ebner and Seubert, Stuttgart, 140 pp., 6 pls, available online at https://www.biodiversitylibrary.org/page/13801704, page(s): 85, pl. V, fig. 15. Kumar, S., Stecher, G. & Tamura, K. (2016): MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. – Molecular Biology and Evolution 33: 1870– 1874. https://doi.org/10.1093/molbev/msw054 Lambec, K. (1996): Shoreline reconstructions for the Persian Gulf since the last glacial maximum. – Earth and Planetary Science Letters 142: 43–57. https://doi.org/10.1016/0012-821X(96)00069-6 Acta Zool. Acad. Sci. Hung. 69, 2023 311 RADIX RUFESCENS (GASTROPODA: LYMNAEIDAE) NEW FOR OMAN AND ARABIA LeBaron Bowen, R. (1949): Arab dhows of Eastern Arabia: A dissertation on the sailing. characteristics of the Lateen-rigged Arab watercraft of the Western Persian Gulf. Privately printed, University of Michigan, Ann Arbor. Miles, S. B. (1919): The countries and tribes of the Persian Gulf. – Harrison and Sons, London. Miller, M. A., Pfeiffer, W. & Schwartz, T. (2010): Creating the CIPRES Science Gateway for inference of large phylogenetic trees. – In: Proceedings of the Gateway Computing Environments Workshop (GCE). New Orleans, L.A. pp. 1–8. https://doi.org/10.1109/GCE.2010.5676129 Mittermeier, R. A., Robles-Gil, P., Hoffmann, M., Pilgrim, J., Brooks, T., Mittermeier, C. G., Lamoreux, J. & Da Fonseca, G. A. B. (2004): Hotspots revisited: Earth’s biologically richest and most endangered terrestrial ecoregions. – CEMEX, Mexico City. Mousson, A. (1874): Coquilles terrestres et fluviatiles recueillies par M. le Dr Alex. Schlaefli en Orient. – Journal de Conchyliologie 22(1): 5–60. Paris [21 January]. Neubert, E. (1998): Annotated checklist of the terrestrial and freshwater molluscs of the Arabian Peninsula with descriptions of new species. – Fauna of Arabia 17: 333–461. Nylander, J. A. A. (2004): MrModeltest v.2. Program distributed by the author. – Evolutionary Biology Centre, Uppsala University, Uppsala. Paine, L. (2013): The sea and civilization. A maritime history of the World. – Knopf Doubleday Group, PenguinRndom House LLC, New York. Preston, D. L., Crone, E. R., Miller-ter Kuile, A., Lewis, C. D., Sauer, E. L. & Trovillion, D. C. (2022): Non-native freshwater snails: a global synthesis of invasion status, mechanisms of introduction, and interactions with natural enemies – Freshwater Biology 67: 227–239. https://doi.org/10.1111/fwb.13848 Rambaut, A. (2010): FigTree v1.3.1. http://tree.bio.ed.ac.uk/software/figtree Rambaut, A., Drummond, A. J., Xie, D., Baele, G. & Suchard, M. A. (2018): Posterior summarisation in Bayesian phylogenetics using Tracer 1.7. – Systems Biology 67: 901–904. https://doi.org/10.1093/sysbio/syy032 Ronquist, F., Teslenko, M., van der Mark, P., Ayres, D. L., Darling, A., Höhna, S., Larget, B., Liu, L., Suchard, M. A. & Huelsenbeck, J. P. (2012): Efficient Bayesian phylogenetic inference and model choice across a large model space. – Systematic Biology 61: 539–542. https://doi.org/10.1093/sysbio/sys029 Roszkowski, W. (1914): Contribution à l’etude des Limnées du lac Léman. – Revue Suisse Zoologie 22: 453–539. Saito, T., Hirano, T., Ye, B., Prozorova, L., Shovon, M. S., Do, T. V., Kimura, K., Surenkhorloo, P., Kameda, Y., Morii, Y., Fukuda, H. & Chiba, S. (2021): A comprehensive phylogeography of the widespread pond snail genus Radix revealed restricted colonization due to niche conservatism. – Ecology and Evolution 11: 18446–18459. https://doi.org/10.1002/ece3.8434 Searle, M. (2019): Geology of the Oman mountains, Eastern Arabia. – Springer Nature Switzerland AG, Cham. Sowerby, G. B. I. (1821–1834): The genera of recent and fossil shells, for the use of students, in conchology and geology. Publ. in 42 numbers. Vol. 1, pls 1–126 [1821–1825]; vol. 2, pls 127–262 + text (unpaginated) [1825–1834]. Sowerby, London. Smith, E. A. (1894): On a small collection of land and fresh-water shells from Oman, Arabia. – Proceedings of the Malacological Society of London. 1(3): 141–142, fig. 1-3. London. Szarowska, M., Osikowski, A., Hofman, S. & Falniowski, A. (2016): Pseudamnicola Paulucci, 1878 (Caenogastropoda: Truncatelloidea) from the Aegean Islands: a long or short story? – Organisms Diversity and Evolution 16: 121–139. https://doi.org/10.1007/s13127-015-0235-5