Zoosyst. Evol. 99 (2) 2023, 285-297 | DOI 10.3897/zse.99.102586 > PENSUFT. pg tuseum ror BERLIN A new genus of river snails, Dalipaludina (Gastropoda, Viviparidae), endemic to the Yunnan Plateau of SW China Le-Jia Zhang!, Li-Na Du*?, Thomas von Rintelen* 1 Museum fiir Naturkunde, Leibniz, Institut fiir Evolutions und Biodiversitatsforschung, Berlin 10115, Germany 2 Key Laboratory of Ecology of Rare and Endangered Species and Environmental Protection, Guangxi Normal University, Ministry of Education, Guilin, Guangxi 541004, China 3 Guangxi Key Laboratory of Rare and Endangered Animal Ecology, College of Life Science, Guangxi Normal University, Guilin, Guangxi 541004, China https://zoobank. org/6856760F-3F 9E-4B27-SAC2-B59D25845F 67 Corresponding author: Le-Jia Zhang (lejia.zhang@mfn.berlin) Academic editor: Frank Kohler # Received 23 February 2023 Accepted 29 March 2023 @ Published 11 May 2023 Abstract A new genus of viviparid snail, Dalipaludina gen. nov., from the Yunnan Plateau of China is described within an integrative taxo- nomic framework based on data from the mitochondrial COI marker and morphology. Dalipaludina can be distinguished from all other viviparid genera by a unique combination shell, operculum and radula characters. Four species are assigned here to the new genus, Dalipaludina delavayana comb. nov., Dalipaludina oxytropoides comb. nov., Dalipaludina occidentalis comb. nov., and Dalipaludina pyramidella comb. nov., and one species is newly assigned to Margarya, Margarya dianchiensis comb. nov. The four species of Dalipaludina are allopatrically distributed in shallow water lentic habitats at high altitude regions of the Yunnan Plateau. Key Words High altitude, lentic habitat, phylogeny, taxonomy Introduction River snails (Viviparidae) are widely distributed fresh- water gastropods that are found on all continents ex- cept for Antarctica and South America. The oldest fossil record of Viviparidae can be traced back to the mid Jurassic. About 28 extant genera and 125 to 150 extant species of this family worldwide are currently recognised (Van Bocxlaer and Strong 2019). China is the country with the highest biodiversity of Viviparidae, harbouring two subfamilies (Bellamyinae Rohrbach, 1937; Viviparinae J. E. Gray, 1847), 13 genera (Amur- opaludina Moskvicheva, 1979; Angulyagra Rao, 1931; Anularya Zhang & Chen, 2015; Cipangopaludina Han- nibal, 1912; Filopaludina Habe, 1964; Idiopoma Pilsb- ry, 1901; Margarya G. Nevill, 1877; Mekongia Crosse & P. Fischer, 1876 Rivularia Heude, 1890; Sinotaia F. Haas, 1939; Tchangmargarya He, 2013; Ussuripalu- dina Zatravkin & Bogatov, 1987; Viviparus Montfort, 1810), and at least 40 species (Liu et al 1995; Zhang et al. 2015; Van Bocxlaer et al. 2017). The Yunnan Province of China, which covers an area of only 394,000 km/?, harbours nine genera (Angulyagra; An- ularya, Cipangopaludina, Filopaludina, Idiopoma; Mar- garya, Mekongia; Sinotaia, Tchangmargarya) including three endemic ones (Anularya, Margarya;, Tchangmar- garya) and at least 24 species including 14 endemic ones of Viviparidae (Liu et al 1995; Zhang et al. 1997; Zhang et al. 2015). While it is the region with the highest diver- sity of Viviparidae at both the genus and species level not only in China but also worldwide, recent studies (Du et al. 2011; Zhang et al. 2015; Zhang 2017) have revealed that the diversity of Viviparidae in Yunnan is still underesti- mated. Based on an integrative study combining molecu- lar and morphological data, we here revise the taxonomy of several viviparid species from the Yunnan Plateau, and describe a new genus. The phylogeny and ecology of the new genus, as well as conservation aspects, are discussed. Copyright Zhang, L.-J. et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 286 Materials and methods Material Type specimens of three Viviparidae species from Yunann kept in the Institute of Zoology, Chinese Academy of Sci- ences, Beying (IZCAS), the Kunming Natural History Museum of Zoology, Kunming (KIZ), and the Museum of Comparative Zoology at Harvard University, Cam- bridge (MCZ), as well as other specimens in Museum fir Naturkunde, Berlin (ZMB), have been examined. From 2011 to 2022, altogether 19 lakes of Yunnan and the sur- rounding aquatic habitats, including rivers, creeks, springs ¢ Lashihay. séake Jianhu ri ake Haixi qf Lakd Cibj Lake Xihu : ake Qingshuihai Lake Erhai @ Lake Yangzonghai (ake Dianchi , y Lake Fuxian Lake Xing An Pravinse Lake Yilong Zhang, L.-J. et al.: Anew genus Dalipaludina and wetlands, have been surveyed (Fig. 1A). Newly collected fresh samples of the species belonging to the new genus were collected by Le-Jia Zhang and Jiao-Wei Ning from 2018 to 2022 in northwest Yunnan (Fig. 1B—D). The newly sampled material is stored in ZMB and the private collection of Le-Jia Zhang (ZLJ) Jiao-Wei Ning (NJW). Examination of morphology Shell height (H) and width (W) of only mature and com- plete specimens were measured with a caliper to a pre- cision of 0.1 mm (Table 1). The morphometric data of Altitude (m) 5000 4000 =| 3000 2000 1000 rake Ohangqiaoha ake Batunhai fate Figure 1. Collecting sites and habitats of Dalipaludina gen. nov. in Yunnan, China. A. Map of Yunnan Province showing 19 surveyed lakes (adapted from Zhang et al. 2015), study area in northwest Yunnan highlighted in blue, where Dalipaludina species were found during sur- veys; B. Map of study area in northwest Yunnan, star—Dalipaludina oxytropoides, triangle—Dalipaludina occidentalis, square—Dalipalu- dina delavayana, citcle—Dalipaludina pyramidella, altitude of each collecting site is given; C. Environment of one collecting site of Dali- paludina oxytropoides in Lake Lugu; D. Environment of one collecting site of Dalipaludina occidentalis in an unnamed pond in Lijiang. zse.pensoft.net Zoosyst. Evol. 99 (2) 2023, 285-297 Dalipaludina oxytropoides kept in ZMB are from Wiese et al. (2022). The specimens were photographed in a consis- tent orientation using a Sony alpha 7R 4 camera. Radulae and embryonic shells were extracted through dissection; radulae were cleaned by boiling in 1% NaOH solution for half an hour and rinsed with distilled water. Radulae and embryonic shells were coated with gold before scanning electron microscopy with a Zeiss EVO LS/0 scanning elec- tronic microscope. The terms used for describing the oper- culum are according to Zhang and von Rintelen (2021). Table 1. Shell measurements of Dalipaludina species. Values are arithmetic means (mm). Species W/H Dalipaludina delavayana (n=25) | 58.30 + 8.30 | 38.30 + 5.27 | 0.70 + 0.04 Dalipaludina oxytropoides (n=91)| 44.60 + 5.78 | 32.30 + 3.82 | 0.70 +0.04 Dalipaludina occidentalis (n=27) | 50.17 + 4.17 | 41.00 + 2.38 | 0.82 +0.05 Dalipaludina pyramidella (n=30) | 47.86 + 7.06 | 41.34 + 5.48 | 0.87 + 0.05 DNA extraction and amplification DNA was extracted from the foot tissue of eight individu- als (10-20 mg of each individual), using a mollusc-specific CTAB/chloroform extraction protocol (Winnepenninckx et al. 1993). A fragment of the mitochondrial cytochrome c oxidase subunit I (COT) gene was amplified through polymerase chain reaction (PCR) with the following primer pair: LCO1490, 5'-GGTCAACAAATCATAAAGATATTGG-3' and COX- B7R, 5'-ACCACCAGCTGGATCAAAAA-3". (SchultheiB et al. 2011) PCR amplifications were conducted in 25 ul volumes under the following cycling conditions: initial denaturing step at 94 °C for 10 min, followed by 30 cycles of 94 °C for 1 min, 50 °C for 1 min, and 72 °C for 1 min, with a final extension step of 10 min at 72 °C. The purification and sequencing were conducted by Macrogen Europe, Amsterdam, Netherlands. Phylogenetic analysis Eight new DNA sequences (from eight individuals of two Dalipaludina species) have been uploaded to GenBank (accession numbers and museum voucher numbers, see Suppl. material 1). Additionally, three sequences of Dali- paludina oxytropoides published by Wiese et al. (2022) and sequences of 17 different genera of the subfamily Bellamy- inae (Suppl. material 1) published by Stelbrink et al. (2020) and Hirano et al. (2019a) were downloaded from Gen- Bank; Viviparus viviparus of the subfamily Viviparinae was selected as outgroup based on Stelbrink et al. (2020). Sequences were aligned using MUSCLE as implement- ed in Geneious Prime 2020 (https://www.geneious.com). Genetic distances were calculated using MEGA X (Ku- mar et al. 2018). The dataset was tested in MEGA X for the best-fit model of sequence evolution by means of the Akaike and Bayesian information criteria, GTR+G+I was suggested as the best-fitting nucleotide substitution mod- el. This model was employed in a maximum likelihood 287 (ML) analysis conducted by RAxML as implemented in Geneious Prime 2020, with support estimated by 1,000 bootstrap replicates, and a Bayesian inference (BI) analysis performed with MrBayes 3.2.6 (Ronquist et al. 2012) as implemented in Geneious Prime 2020 with four indepen- dent chains for 5,000,000 generations, samplefreq = 1,000, burnin = 25%, and confirmed that convergence was reached based on the trace plots generated in Geneious Prime 2020. Results Genetic differentiation and phylogeny COI p-distances between species of Dalipaludina are 1.81% to 3.98%; the p-distances between Dalipaludina and its closest relative, viz. the Cipangopaludina/Margarya complex, are 9.22%—14.98%, while p-distances within the Cipangopaludina/Margarya complex are 0-9.31%. The phylogenetic trees reconstructed by BI and ML based on COI are highly congruent, therefore only the BI tree is shown (Fig. 2). The new genus Dalipaludina is monophyletic and distinct from all other included gen- era of Viviparidae. The three sequenced species of Dali- paludina are each recovered as monophyletic as well. Dalipaludina occidentalis is the sister species of Dalipa- ludina delavayana, and Dalipaludina oxytropoides is sister to both. Dalipaludina is the sister clade of a clade formed by the Cipangopaludina/Margarya complex, Celetaia, Us- suripaludina, Hetergen, Torotaia, Anularya and Sinotaia. Systematics Family Viviparidae J.E. Gray, 1847 Subfamily Bellamyinae Rohrbach, 1937 Dalipaludina Thang, gen. nov. https://zoobank.org/6817CC4A-D98A-4D33-B890-49C91F64A 1E5 Type species. Paludina delavayana Heude, 1889. Etymology. “Dali” refers to the ancient Dali Kingdom (K##£[E]) mostly situated in modern Yunnan, China; “pa- ludina” refers the assignment to Viviparidae. The recom- mended Chinese name is “J FBR”. Diagnosis. Shell large, thin but solid; apex acute; te- leoconch whorls with strong keel at suture, above suture relatively smooth or with several weak spiral threads or strong spiral cords, many thin and weak spiral threads on base; umbilicus narrow, sometimes bordered by a keel; exterior surface of operculum rather smooth, inner oper- cular region relatively small, nuclear region of opercu- lum smooth, sometimes with small grains; outer marginal tooth with 9 to 11 small sharp cusps. Comparative remarks. Dalipaludina gen. nov. resem- bles Cipangopaludina (widely distributed in East Asia), Ussuripaludina Zatravkin & Bogatov, 1987 (endemic to Far East), Heterogen Annandale, 1921 (endemic to Japan zse.pensoft.net 288 32/0.51 Zhang, L.-J. et al.: Anew genus Dalipaludina 68/0.51 Cipangopaludina chinensis chinensis 100/1]- Cipangopaludina chinensis chinensis Cipangopaludina chinensis 85/0.92L Cjpangopaludina chinensis chinensis 99/0.99 “ Margarya melanioides 23/0.66} | wargarya francheti a Margarya dianchiensis Margarya dianchiensis 26/0.93 Cipangopaludina haasi 384/0.gf Cipangopaludina chinensis laeta ar Cipangopaludina chinensis laeta ; Cipangopaludina wisseli 94/1 L Cipangopaludina chinensis laeta Cipangopaludina chinensis laeta Cipangopaludina lecythis 59/0.72 Celetaia persculpta 74/0.94 94/1 91/0.7 84/0.79 82/1 Dalipaludina occidentalis 100/1[ Dalipaludina occidentalis Ussuripaludina ussuriensis Heterogen japonica Torotaia cf. mainitensis Anularya mansuyi Sinotaia tricinctus i ponds in Lijiang 93/0.67—- Datipaludina occidentalis Dalipaludina occidentalis Lake Jianhu 92/0.99|* Dalipaludina occidentalis 0.79 Dalipaiudina delavayana 100/4 Dalipaludina delavayana j Lake Cibi Dalipaludina gen.nov. Dalipaiudina delavayana Dalipaludina oxytropoides 56/0.53 Dalipaludina oxytropoides Lake Lugu Dalipaludina oxytropoides 70/0.94 75/0.99 37/0.94 52/0.98 o6/0.99 Anulotaia sp. Trochotaia trochoides Bellamya crawshayi Angulyagra mutica Filopaludina sumatrensis peninsularis Taia sp. 0,06 Viviparus viviparus Figure 2. The BI tree based on cytochrome c oxidase subunit I (COI) showing the phylogenetic position of Dalipaludina gen. nov. within Viviparidae. Numbers above branches are ML bootstrap values/BI posterior probabilities. and Korea), and Jorotaia Haas, 1939 (endemic to Phil- ippines, Sulawesi and New Guinea). It differs from Ci- pangopaludina, Ussuripaludina, Heterogen and Torotaia by having one strong keel at the suture, a shell base with many thin and weak spiral threads, a narrow umbilicus sometimes bordered by a keel, and operculum characters. The molecular phylogeny supports that Dalipaludina is zse.pensoft.net distinct from Cipangopaludina, Ussuripaludina, Het- erogen and Torotaia. The location of the testis in male Dalipaludina specimens in the mantle cavity supports the classification of this genus in the subfamily Bellamyinae. There are four known species of Dalipaludina. Distribution. This genus is endemic to the lakes or ponds in northwest to northeast Yunnan, China. Zoosyst. Evol. 99 (2) 2023, 285-297 Dalipaludina delavayana (Heude, 1889), comb. nov. Paludina delavayana Heude, 1889: 47 (“lacu Ta-li fou”, Lake Erhai). Sinotaia delavayana — Qian et al. 2014. Material examined. 2 syntypes, IZCAS-FG-492553, IZCAS-FG-492554; 1 paratype, MCZ-Mala-167342; 8 specimens in ZMB, collected by Le-Jia Zhang from around 0.5 to 1 metres depth in the north shore of Lake Cibi in Augest 2018, 2 specimens in ZMB, collected by Jiao-Wei Ning from around | to 2 metres depth in Lake Haixihai; 17 specimens in NJW, collected by Jiao-Wei Ning from around | to 2 metres depth in Lake Cibi. Description. Shell (Fig. 3A—H) large, conical, thin, greenish yellow to dark olive in colour; up to six whorls at adulthood, including one relatively smooth protoconch whorl, apex acute, spire high; each teleoconch whorls with one strong keel at suture, above suture smooth or with many weak spiral threads, sometimes with addition- al two to three stronger cords, base of shell with many thin weak spiral threads, weak spiral cords and threads with short periostracal hairs; aperture ovate, less than half of shell in height, lip thin and simple, umbilicus narrow, sometimes bordered by a keel. Operculum (Fig. 31) corneous, ovate, rather thin, yel- low, with reddish brown nuclear region, exterior surface of operculum rather smooth, inner opercular region rela- tively small, nuclear region smooth. Radular (Fig. 7A, B) central tooth with one broad central denticle and with four small sharp cusps on el- ther side; lateral tooth with one broad central denticle and four small sharp cusps on either side; inner margin- al tooth with one broad central denticle and three small sharp cusps on either side; outer marginal tooth with 10 to 11 small sharp cusps. Remarks. This species differs from the other Dalipalu- dina species by having a larger shell with a higher spiral. It may be difficult to distinguish from the smooth form of Dalipaludina oxytropoides from Lake Lugu based on morphology. However, this species 1s not recorded from Lake Lugu, and can be distinguished from D. oxytropoides based on differences in COI gene sequences. The recom- mended Chinese name of this species is “(#417 2: HH A HR”. Habitat and distribution. shallow water area with sandy and muddy bottoms in Erhai, Cibi and Hatxthai, Yunnan, China. Dalipaludina oxytropoides (Heude, 1889), comb. nov. Paludina oxytropoides Heude, 1889: 176 (‘lacu prope Tchao-tong’ = lake near Zhaotong City, Yunnan Province, China). Vivipara oxytropoides — Kobelt 1909: 128. Margarya sp. — Du et al. 2012: 45. Margarya oxytropoides — Zhang et al. 2015: 777; Wiese et al. 2022. Material examined. 2 syntypes, IZCAS-FG-492586, IZCAS-FG-492587;, 1 paratype, MCZ-Mala-167343; 289 91 specimens in ZMB, collected by Frank Riedel from around | to 6 metres depth in all over the Lake Lugu in September and October 2014; 5 specimens in ZMB, col- lected by Le-Jia Zhang from 1 metre depth in the east shore of Lake Lugu in April 2022. Description. Shell (Fig. 4A—H) large, conical, thin, greenish yellow to dark brown in colour; up to six whorls at adulthood, including one relatively smooth protoconch whorl, apex acute; each teleoconch whorls with one strong keel at suture, above suture with many rather weak spiral threads, sometimes with additional two to three strong cords, base of shell with many thin weak spiral threads, weak spiral cords and threads with short perio- stracum setae; aperture ovate, less than half of shell in height, lip thin and simple, umbilicus narrow, sometimes bordered by a keel. Operculum (Fig. 41) corneous, ovate, thin to relatively thick, yellow to reddish brown, with darker coloured nu- clear region, exterior surface of operculum rather smooth, inner opercular region relatively small, nuclear region smooth or with small grains. Radular (Fig. 7C, D) central tooth with one broad central denticle and three to four small sharp cusps on either side; lateral tooth with one broad central denticle and three to four small sharp cusps on either side; inner marginal tooth with one broad central denticle and four small sharp cusps on either side; each outer marginal tooth with 10 to 11 small sharp cusps. Remarks. This species has the most variable shell within Dalipaludina. The form with strong cords of this species can be easily distinguished from the other Dalipaludina species. It used to be considered as a spe- cies of Margarya with a distribution in Lake Dianchi as well (Zhang et al. 2015). However, the COI-based phylogeny showed that “Margarya oxytropides” from Dianchi is not a species of Dalipaludina, but indeed a member of the Cipangopaludina/Margarya complex. The syntypes of Paludina oxytropoides Heude, 1889 from Zhaotong in Northeast Yunnan show a strong keel at the suture, two strong keels above the suture, a relatively smooth shell base with many weak spiral keels, and a narrow umbilicus with a keel around it. It is distinct from the “Margarya oxytropides” from Lake Dianchi but quite similar to the strongly keeled form of D. oxytropoides from Lake Lugu. Meanwhile, the shell morphology of “Margarya oxytropides” from Lake Dianchi matches the description and figure of Cipangopaludina dianchiensis Zhang, 1990 endemic to Lake Dianchi (Zhang 1990). Therefore, instead of D. oxytropoides, “Margarya oxytropides” from Lake Dianchi is here recognised as Margarya dianchiensis (Zhang, 1990) comb. nov. Paludina oxytropoides Heu- de, 1889 is reclassified as a member of Dalipaludina. coZIN The recommended Chinese name of this species 1s “Sec Tey BE ER” Habitat and distribution. shallow to medium-depth water area with sandy bottoms in Lake Lugu, Yunnan/ Sichuan, China; lakes in Zhaotong, Yunnan, China. zse.pensoft.net 290 Zhang, L.-J. et al.: Anew genus Dalipaludina Figure 3. Shell and operculum of Dalipaludina delavayana. A—C. Specimen collected from Lake Cibi, ZMB Moll. 122707; D, E. Varieties collected from Lake Cibi, ZMB Moll. 122707; F. specimen collected from Lake Haixihai,LJZ; G. Paratype MCZ-Ma- la-167342, photo taken by Alana Rivera, Museum of Comparative Zoology, Harvard University, President and Fellows of Harvard College; H. Syntype IZCAS-FG-492554, photo taken by Kaibaryer Meng, Institute of Zoology, Chinese Academy of Sciences; I. Exterior (left) and interior (right) surface of operculum. Scale bar: 1 cm, B—H with the same scale bar of A. Dalipaludina occidentalis (Annandale, 1924), comb. nov. —_“Viviparus occidentalis — Yen 1942. Lecythoconcha malleata f. occidentalis Annandale, 1924: 415 (“Ho- | Material examined. 8 specimens in ZMB, collected by ching and Shihku”, Heging County and Shigu town, Lijiang City, local fishermen from 3 to 5 metres depth in the south Yunnan, China). shore of Lake Jianhu. February 2020; 4 specimens in zse.pensoft.net Zoosyst. Evol. 99 (2) 2023, 285-297 A 291 Figure 4. Shell and operculum of Dalipaludina oxytropoides. A—C. Specimen collected from Lake Lugu, ZMB.Moll. 250844; D-F, Varieties collected from Lake Lugu,ZLJ; G. Syntype IZCAS-FG-492587 from a lake in Zhaotong, Yunann, photo taken by Kaibaryer Meng, Institute of Zoology, Chinese Academy of Sciences; H. Paratype MCZ-Mala-167343 from a lake in Zhaotong, Yunann, photo taken by Alana Rivera, Museum of Comparative Zoology, Harvard University, President and Fellows of Harvard College; I. Exterior (left) and interior (right) surface of operculum. Scale bar: 1 cm, B—F, H—I with the same scale bar of A. ZMB, collected by Le-Jia Zhang from 0.5 metres depth in an unnamed pond on the Sheshan hill in Lijiang City in April 2022; 19 specimens in NJW, collected by Jiao-Wei Ning from Lake Jianhu. Description. Shell (Fig. 5A—G) large, broadly conical or sub globose, thin, olive to dark brown in colour; up to six whorls at adulthood, including one relatively smooth protoconch whorl, apex acute; teleoconch whorls in- flated, occasionally shouldered, with one strong keel at suture, above suture with many weak spiral threads, sometimes with additional two to three stronger cords, base of shell with many thin weak spiral threads, weak spiral threads and cords with short periostracum setae; aperture ovate, slightly less than half of shell in height, zse.pensoft.net 202 Zhang, L.-J. et al.: Anew genus Dalipaludina Figure 5. Shell and operculum of Dalipaludina occidentalis. A-C. Specimen collected from Lake Jianhu, ZMB.Moll. 122712; D, E. Varieties collected from Lake Jianhu, ZMB.Moll. 122713; F. Sub-adult collected from Lake Jianhu, ZMB.Moll. 122713; G. Original figure of the type specimen in Annandale, 1922; H. Exterior (left) and interior (right) surface of operculum. Scale bar: 1 cm, B-F with the same scale bar of A. lip thin and simple, umbilicus narrow, sometimes bor- dered by akeel. Operculum (Fig. 5H) corneous, ovate, rather thin, yellow, with red nuclear region, exterior surface of oper- culum rather smooth, inner opercular region relatively small, nuclear region smooth. Radular (Fig. 7E, F) central tooth with one broad cen- tral denticle and four small sharp cusps on either side; lat- zse.pensoft.net eral tooth with one broad central denticle and four small sharp cusps on either side; inner marginal tooth with one broad central denticle and four small sharp cusps on either side; outer marginal tooth with 9 to 10 small sharp cusps. Remarks. This species differs from the other species of Dalipaludina by having rather inflated, shouldered whorls. Annandale (1924) presented a photo of the type specimen. The shell morphology and COI phylogeny of the speci- Zoosyst. Evol. 99 (2) 2023, 285-297 mens collected from an unnamed pond in Lijiang City, which is very close to the type locality, confirm that this species should be assigned to Dalipaludina. The recom- mended Chinese name of this species is “YA PU HE HAW”. Habitat and distribution. Shallow ponds with mud- dy bottoms and abundant aquatic plants in Lijiang City, Yunnan, China; Shallow to medium-depth water area with muddy bottoms in Lake Jianhu, Jianchuan County, Yunnan, China. Dalipaludina pyramidella (Du, Yang & Chen, 2011), comb. nov. Trochotaia pyramidella Du, Yang & Chen, 2011: 85—89 (Yousuo village of Er- Yuan County, Yunnan, China). Material examined. Holotype, KIZ-DLN20100035; 19 paratypes, KIZ-DLN20100036 to KIZ-DLN20100054; 3 specimens in ZMB, 7 specimens in NJW, collected by Jiao-Wei Ning from | metre depth in the ponds of Yousuo Village, Eryuan County. Description. Shell (Fig. 6A—D) large, conical, thin, greenish brown to brown in colour; up to six whorls at adulthood, including one relatively smooth protoconch whorl, apex acute, spire low; each teleoconch whorls with one strong keel at suture, above suture smooth, with two rings of short periostracum setae, base of shell smooth, with many rather thin weak spiral threads; aperture ovate, almost half of shell in height, lip thin and simple, umbili- cus narrow, sometimes bordered by a keel. Operculum (Fig. 6E) corneous, ovate, rather thin, yel- low or orange, with red nuclear region, exterior surface of operculum rather smooth, inner opercular region relative- ly small, nuclear region smooth. Radular (according to Du et al. 2011) central tooth with one broad central denticle and three to four small sharp cusps on either side; lateral tooth with one broad central denticle and four small sharp cusps on either; inner marginal tooth with one broad central denticle and two small sharp cusps on either side; outer marginal tooth with 9 small sharp cusps. Remarks. This species differs from the other spe- cies of Dalipaludina by having a relatively smooth shell with lower spire. Du et al. (2011) assigned this species to Trochotaia mainly based on the strongly keeled shell with lower spire. However, characters of the opercu- lum, embryonic shells, the reduced number of cusps on the outer marginal teeth (9 in D. pyramidella vs 14-16 in Trochotaia trochoides) and the distribution in Yunnan strongly support its assignment to Dalipaludina. Species of Trochotaia have a very different operculum according to Zhang and von Rintelen (2021). This species is also quite similar to Cipangopaludina miyagii Kuroda, 1941 endemic to Kaohsiung, S Taiwan, China. It can be distin- guished from C. miyagii by having a thinner shell, a shell base with many rather thin weak spiral threads, and a nar- rowly open umbilicus with a keel around it. The recom- mended Chinese name of this species is “18722 HAW”. 293 Habitat and distribution. shallow ponds with muddy bottoms and aquatic plant Ottelia acuminata (Gagnep.) in Yousuo Village, Eryuan County, Yunnan, China. Discussion The molecular and morphological data strongly support that four endemic viviparid species from Yunnan, for- merly classified as species of Cipangopaludina, Sinotaia, Margarya or Trochotaia, should be assigned to a new ge- nus described here: Dalipaludina. The former genus-lev- el classification of these species was primarily based on shell morphology (Du et al. 2011; Qian et al. 2014). However, the shell morphology of freshwater snails can be quite similar due to convergence even between differ- ent genera. One remarkable example is the case of three highly sculptured but phylogenetically distinct viviparid genera (Margarya, Tchangmargarya and Anularya) en- demic to the plateau lakes of Yunnan (Zhang et al. 2015). Our study has confirmed once again the importance of using an integrative approach in generic classification. In additional to shell characters, the operculum and radula, especially the cusps on the outer marginal teeth, provide important data for genus-level taxonomy of Viviparidae as well. The species-level phylogeny solely based on COI has been considered problematic in Viviparidae (Hirano et al. 2019b). However, at the genus level, it does not seem to be a big problem here (Fig. 2). Our BI tree based on partial COI sequences is broadly consistent with the genus-level topology for the Bellamyinae derived from an analysis of multiple genes in Stelbrink et al. (2020), suggesting that Dalipaludina likely falls into clade A of Bellamyinae proposed by Stelbrink et al. (2020). Four species of Dalipaludina can be differentiated based on shell morphology. The scatter plot (Fig. 8) shows that D. delavayana has a larger and taller shell; D. pyra- midella and D. occidentalis both have a lower spire, and D. pyramidella can be distinguished from D. occidenta- lis based on its less inflated whorls and comparatively smooth shell surface. The COI phylogeny has supported the reciprocal monophyly of the three sequenced species. The populations of D. occidentalis from two localities roughly 50 km apart cluster togther in the tree, without obvious geographic structuring (Fig. 2). The keeled, shouldered form of D. occidentalis (Fig. SE) cannot be differentiated from the smooth form based on p-distance or phylogeny of COI as well. Moreover, the four species of Dalipaludina have allopatric distributions (Fig. 1B), and we have yet to find any lake or pond with more than one Dalipaludina species. Based on these results derived from morphological, molecular, and distribution data, we believe that there are four valid species of Dalipaludina. Intraspecific diversity of shell morphology varies among the four species. Dalipaludina delavayana and D. pyramidella both display a relatively low intraspecif- ic diversity; morphological diversity within D. occiden- talis is higher; and that of D. oxytropoides is by far the zse.pensoft.net 294 Zhang, L.-J. et al.: Anew genus Dalipaludina Figure 6. Shell and operculum of Dalipaludina pyramidella. A—C. Specimen collected from Eryuan County, ZMB.Moll. 122714; D. Varieties collected from Eryuan County, ZMB Moll. 122714; E. Exterior (left) and interior (right) surface of operculum. Scale bar: 1 cm, B—D with the same scale bar of A. highest within the genus. An integrative study combining morphometrics and a COI phylogeny of D. oxytropoides from Lake Lugu (“Margarya oxytropoides” in Wiese et al. 2022) showed that it should be considered a single, but highly variable species. We have also observed many intermediate forms in this species. In contrast to the other three endemic genera of Vi- viparidae from Yunnan (Margarya, Tchangmargarya and Anularya) mostly found in medium-depth to deep water (Zhang et al. 2015), Dalipaludina displays a preference for shallow water. In addition to large lakes like Lake Lugu or Jianhu, Dalipaludina also can be found in small shallow ponds, such as the artificial ponds for farming the aquatic vegetable Oftelia acuminata in Eryuan County (D. pyramidella) and an unnamed small pond on the top of Sheshan Hill in Lijiang (D. occidentalis). D. oxytropoi- des and D. delavayana are also more common in shallow water areas than in deeper water. However, Dalipaludina is only found in lentic environments. It has never been found in rivers or any other lotic environment. The lowest altitude where we have found Dalipalu- dina species is in Eryuan County, around 1974 m a.s.L., and one historical record of D. oxytropoides in Zhaotong zse.pensoft.net County from ~1920 m as.l. (Heude 1886); Lake Lugu harbouring D. oxytropoides is ~2698 m a.s.1., which is the highest altitude record of Viviparidae (Fig. 1B). Among the other three viviparid genera endemic to Yunnan Pla- teau, only Margarya is found within a similar but smaller altitudinal range (1886-2192 m a.s.1), while 7changmar- garya (1770-1907 m a.s.1.) and Anularya (1284-1721 m a.s.l1.) inhabit a relatively lower altitude with no overlap with that of Dalipaludina. Viviparids from elsewhere in the world occur at much lower altitudes. In conclusion, Dalipaludina displays an adaptation to the shallow lentic environment in high altitude regions, and occurs at the highest altitude of any viviparid. With the discovery of the new genus Dalipaludina, the Yunnan Plateau now harbours four endemic genera of Viviparidae, confirming again that it 1s the most im- portant diversity hotspot of Viviparidae in the world. However, almost all of these species are threatened by human activities such as overharvesting, pollution, and habitat destruction (Yang et al. 2004; Zhang et al. 2015). D. pyramidella nearly disappeared from its only known site, the ponds in Eryuan County, during our recent sur- veys in 2021; the present population of D. oxytropoides Zoosyst. Evol. 99 (2) 2023, 285-297 A _ ” nh TTVULL, N Fy , S ' VA Vj r Y, a, A omt un * A f / 7) i | | fp UN) Figure 7. SEM photo of the radula and the protoconch of Dalipaludina species. A, B. Radula of Dalipaludina delavayana, A-ZMB. Moll. 181719, B-ZMB.Moll. 122707; C, D. Radula of Dalipaludina oxytropoides, ZMB.Moll. 250844; E, KF. Radula of Dalipaludi- na occidentalis, ZMB.Moll. 122713. Abbreviations: ct—central teeth; It—lateral teeth; imt—inner marginal teeth; omt—outer marginal teeth. Scale bars: 100 um. in Lake Lugu has declined dramatically compared to that —u/arya are on the national Red List of China (Wang and in the 1990s according to local fishermen. Most endemic Xie 2005), and one species Margarya melanioides Nevill, viviparid species of Margarya, Tchangmargarya and An- 1877 is a Class 2 species under state protection in the zse.pensoft.net 296 Zhang, L.-J. et al.: Anew genus Dalipaludina Dalipaludina delavayana e Dalipaludina oxytropoides @ Dalipaludina occidentalis e Dalipaludina pyramidella Figure 8. Comparison of Dalipaludina species with respect to parameters H (in mm) and W/H. latest List of State Key Protected Wild Animals of China in 2021. Since all four Dalipaludina species are restricted to just one or two lakes or several small ponds, specif- ic conservation measures should be applied urgently for each species based on more detailed population biology and ecology studies. The phylogeny based on genomic data, such as data from exon capture method, can also improve our understanding of the evolution of these rare species endemic to the plateau lentic environment. Acknowledgments The authors thank Jiao-Wei Ning from Yuxi, Yunnan and Hong-Quan Xiang (Yuxi Agriculture Agricultural Technology College) for collecting specimens, Bernhard Schurian (Museum fiir Naturkunde, Berlin) for help with photography, Kaibaryer Meng (Institute of Zoology, Chi- nese Academy of Sciences, Beijing), Adam Baldinger and Alana Rivera (Museum of Comparative Zoology at Harvard University, Cambridge) for providing the 1m- portant type specimens for study. 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[In Chinese] Zhang LJ, Chen SC, Yang LT, Jin L, Kohler F (2015) Systematic re- vision of the freshwater snail Margarya Nevill, 1877 (Mollusca: Viviparidae) endemic to the ancient lakes of Yunnan, China, with description of new taxa. Zoological Journal of the Linnean Society 174(4): 760-800. https://doi.org/10.1111/zoj.12260 Supplementary material | Accession numbers and museum voucher numbers Authors: Le-Jia Zhang, Li-Na Du, Thomas von Rintelen Data type: table (excel document) Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons. org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow us- ers to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://do1.org/10.3897/zse.99.102586.suppl1 zse.pensoft.net