ZOOLOGICAL RESEARCH

Systematic relationships of Chinese freshwater semisulcospirids (, ) revealed by mitochondrial sequences

Li-Na Du1,2,3,#, Jun Chen3,#, Guo- Yu2,*,#, Jun-Xing Yang1,4,*

1 Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming Yunnan 650223, China 2 Key Laboratory of Ecology of Rare and Endangered and Environmental Protection (Guangxi Normal University), Ministry of Education, Guilin Guangxi 541004, China 3 Key Laboratory of the Zoological Systematics and Evolution, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China 4 Yunnan Key Laboratory of Plateau Fish Breeding, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming Yunnan 650223, China

ABSTRACT INTRODUCTION

The systematics of in China is Semisulcospirids are an ecologically important and revised here based on morphological characters and taxonomically challenging family of freshwater snails mitochondrial phylogenetics. Phylogenetic distributed in lakes, rivers, and streams in eastern Asia, relationships within the Chinese semisulcospirids including Far East Russia, Japan, Korean peninsula, and were assessed via DNA sequences from China, and in western North America (Köhler, 2016, 2017; Liu mitochondrial analysis (cytochrome c oxidase I and et al., 1993; Strong & Köhler, 2009; Yen, 1939). Basic 16S rRNA). This research contains most knowledge on freshwater gastropods in China was first morphospecies of semisulcospirids previously established in the late nineteenth and early twentieth recorded in China. Based on these results, the family of Chinese Semisulcospiridae is represented centuries (e. g., Dautzenberg & Fischer, 1906, 1908; Fulton, by three genera: i. e., viviparous 1914; Heude, 1888), with all cerithioidean freshwater Böttger, 1886, oviparous Hua Chen, 1943 and gastropods designated under the single Melania Koreoleptoxis Burch and Jung, 1988. These genera Lamarck, 1799 (junior of Thiara Röding, 1798). can be distinguished from each other by Heude (1888, 1889 (1882 – 1890)) recorded 24 species of reproductive anatomy, reproductive mode, and features. Species of Hua are mainly Received: 30 January 2019; Accepted: 16 July 2019; Online: 21 August distributed in southwest China and Guangxi, whereas Koreoleptoxis and Semisulcospira are 2019 mainly distributed in south and northeast China. Foundation items: This research was funded by the National Natural Science Foundation of China (31301865), Special Funds for Young Keywords: Phylogeny; China; Semisulcospira; Hua; Scholars of of the Chinese Academy of Sciences (ZSBR- Koreoleptoxis 011), Key Laboratory of the Zoological Systematics and Evolution of the Chinese Academy of Sciences (Y229YX5105), Strategic Priority Open Access Research Program of the Chinese Academy of Sciences This is an open-access article distributed under the terms of the (XDA23080502), National Natural Science Foundation of China- Creative Commons Attribution Non-Commercial License (http:// Yunnan Joint Fund (U1702233) and Sino BON-Inland Water Fish creativecommons. org/licenses/by-nc/4.0/), which permits unrestricted Diversity Observation Network non-commercial use, distribution, and reproduction in any medium, #Authors contributed equally to this work provided the original work is properly cited. *Corresponding authors, E-mail: yangjx@mail. kiz. ac. cn; yugh2018@ Copyright ©2019 Editorial Office of Zoological Research, Kunming 126.com Institute of Zoology, Chinese Academy of Sciences DOI: 10.24272/j.issn.2095-8137.2019.033

Science Press Zoological Research 40(6): 541-551, 2019 541 Melania in south China, especially in the Yangtze River Basin. rotundata (Heude, 1888). Furthermore, most semisulcospirids Yen (1939) subsequently identified Chinese freshwater snails were recorded in the Yangtze River. Thus, based on previous from the Naturmuseum Senckenberg, Germany, and recorded literature, 52 species of semisulcospirids have been recorded 12 Chinese species of and a new genus, in China (Du & Yang, 2019; Du et al., 2019; Liu et al., 1993; Senckenbergia Yen, 1939, for the species Melania Xu, 2007). However, the systematic and phylogenetic pleuroceroides from Sichuan. Chen (1943) later described two relationships of semisulcospirids remain poorly understood. genera, Wanga (type species Melania henriettae Gray, 1833) From 2014 to 2017, specimens of semisulcospirids were and Hua (type species Melania telonaria Heude, 1888), into collected from 14 Chinese provinces, including Heilongjiang, which were placed eight and eleven nominal species, Jilin, Liaoning, , Jiangxi, Hunan, , , respectively. In dispute, Morrison (1954) proposed Wanga and , Guangxi, Guizhou, Sichuan, Chongqing, and Hua to be synonyms of Brotia H. Adams 1866 and Oxytrema Yunnan. Based on morphological and molecular analyses, the Rafinesque 1819, respectively, although the suggestion of specimens were used to clarify the phylogenetic and Hua being a synonym of Oxytrema was not accepted by systematic relationships among Chinese semisulcospirids. subsequent authors (Du et al., 2019; Köhler, 2016, 2017; Strong & Köhler, 2009). Abbott (1948) described a new genus, MATERIALS AND METHODS Namrutua, for the type species Melania ningpoensis. However, Senckenbergia and Namrutua were primarily Taxon sampling ignored by most Chinese researchers, with all species instead Specimens were collected from 2014 to 2017 by L.N. Du et al. placed in Semisulcospira Böttger, 1886 (Liu et al., 1979, 1993; in 14 Chinese provinces (Figure 1). The foot muscle tissues Xu, 2007; Zhang et al., 1997). Several species of were preserved in 99% ethanol. All voucher specimens were Semisulcospira were subsequently described in China, deposited at the Kunming Institute of Zoology, Chinese including S. inflata Tchang & Tsi 1949 recorded in Lake Academy of Sciences (KIZ, CAS) (Table 1). The identification Dianchi, Yunnan, and S. marica Li, Wang & Zhang 1994 and of specimens included verification of origin descriptions and S. crassicosta Liu, Wang & Zhang 1994 recorded in Guizhou topotypic specimen photos. Province (Liu et al., 1994; Tchang & Tsi, 1949). Liu et al. We examined 225 sequenced samples from 41 species, (1994) also descried two new species of Paludomus, P. including 95 sequenced samples from 22 species of Hua from qianensis Liu Zhang & Duan 1994 and P. cinctus Liu, Zhang & Yunnan, Chongqing, Guangxi, and Guizhou, 90 sequenced Duan 1994, in Guizhou Province. Xu (2007) stated that there samples from 12 species of Koreoleptoxis from Heilongjiang, were 53 species of Semisulcospiridae found in China, Jilin, Hunan, Jiangxi, Fujian, Zhejiang, and Anhui, and 40 including 49 species of Semisulcospira, two species of Hua, sequenced samples from seven species of Semisulcospira one species of Paludomus, and one species of Potodoma. from Hunan, Jiangxi, Guangxi, Chongqing, Zhejiang, and However, Xu (2007) did not revise the validity of these Liaoning (Table 1). species. The first systematic revision of semisulcospirids was provided by Strong & Köhler (2009) based on morphological Morphology characters and molecular phylogenetic analysis. They showed Measurements and descriptions, including shell dimensions, Hua to be a valid genus and distinguishable from radulae, and shell features, followed Strong & Köhler (2009) Semisulcospira by distinct features of the digestive, and Du et al. (2019). Radulae and embryonic shells were reproductive, and nervous systems. Du et al. (2017, 2019) cleaned enzymatically with proteinase K for 30 min, after designated Semisulcospira trivolvis Yen 1939 as synonym to which they were sonicated for 60 s, mounted on aluminum S. paludiformis Yen 1939 and further stated that it should be specimen stubs with adhesive tabs, and coated with gold assigned to the genus Sulcospira Troschel, 1858 palladium for analysis via scanning electron microscopy ( Fischer & Crosse, 1892). More recently, Du et (Hitachi S-3000, Japan) at 15 kV. Soft body anatomy was al. (2019) studied semisulcospirids of southwest China by observed using a microscope (Leica S6D, 6.3–40x) and was morpho-anatomy and mitochondrial phylogenetics and described following the terminology used by Strong & Köhler supported Namrutua and Senckenbergia as viviparous and as (2009) and Köhler (2017). synonyms of Semisulcospira. Additionally, the taxonomic status of several species were discussed, in particular M. Molecular analyses cancellata Benson 1842 and M. suifuensis Chen 1937 were Genomic DNA was purified via standard phenol-chloroform treated as junior synonyms to S. ningpoensis (I. Lea, 1856), isolation and ethanol precipitation. Partial sequences of and M. dulcis Fulton 1904, M. lauta Fulton 1904, and S. inflata mitochondrial cytochrome c oxidase subunit I (COI, 772 bp) Tchang 1949 were treated as junior synonyms to Hua textrix and 16S ribosomal RNA (16S, 856 bp) were amplified using (Heude, 1888). However, several semisulcospirid species primers F2 and R2 (Du et al., 2019) for COI, and 16S-F were not contained in Du et al. (2019), including H. (Wilson et al., 2004) and H3059 (Palumbi et al., 1991) for kweichouensis (Chen, 1937) (synonym to H. intermedia 16S. Homologous sequences of other semisulcospirids were (Gredler, 1885) in this study), Paludomus minensis Chen 1943 obtained from GenBank, with all new sequences deposited in (synonym to Lithogryphus pallens Bavay & Dautzenberg same under accession Nos. MK944096–MK944245, 1910, valid species name is H. pallens in this study), and P. MK968961–MK969107, MK991781–MK991783, MN013828–

542 www.zoores.ac.cn , – 36 86 – , , , – 47 02 , – , – 251735 251744 969005 969082 MK MK MK MK 251741 251745 251721 969101 , , , , MK MK MK MK 07 34 65 38 68 98 26 61 14 100 16 05 05 57 71 30 81 13 – – , , – – – – , – – – – – , – – – – – – – 251699 969006 251733 968961 251743 251762 251737 251766 969096 251720 251723 251758 969012 969099 251714 251722 969104 251701 969107 251755 969070 251727 969072 251712 MK , COI MK MK MK MK MK MK MK MK MK MK MK MK MK – MK MK MK MK MK MK MK MK MK MK 40 21 , , – , , – 50 37 . – , , – 944096 944238 251664 944141 944217 MK MK MK MK MK 251648 251663 251634 944236 , , , , , MK MK MK MK 43 52 68 85 54 90 33 39 82 50 35 09 27 243 16 78 06 44 16 24 – , – , – – – – – , – – – – – – , – – – – – – – 944142 251646 251651 251659 251665 251683 251653 251689 944231 251632 251636 251679 944148 944234 251608 251625 251635 944242 251612 944245 251676 944205 251640 944207 251623 rRNA S eBn ceso No accession GenBank 16 MK MK MK MK MK MK MK MK MK MK MK MK MK MK MK MK MK MK MK MK MK MK MK MK MK , , , . - , ) M , , dulcis kwei . . n synonym and S . teloniaria H vultuosa , . , telonaria praenotata . textoria . . M S Hemimitra , . ( , S inflata , H , , , . M , lauta , , S . , M , aubryana bailleti intermedia praenotata rotundata scrupea telonaria leprosa textrix vultuosa telonaria scrupea lauta minensis ...... Synonym aristarchorum Melania M aurbryana Semisulcospira M bailleti Oxytrema – M Paludomus kweichouensis chouensis intermedia – – pallens Lithogryphus P M M S scrupea Wanga – M M O M dulcis S M – – – , , , 646 265 , D D , – eBn ceso Nos accession GenBank 1272 1526 , 1578 D 1503 D – 281 263 644 D , D , D , D D 253 , – – , 1475 1051 1268 860 1496 D D D 275 282 922 641 252 203 1497 903 D 859 , D , localities , , , D D D D D D D D D , , – , , , , – , , , . 198 1472 1486 1049 1267 1257 872 201 274 269 920 639 190 1492 858 250 258 649 901 869 288 D D D D D , – , – D D D D D D D D D D D D D D D DD , , , – – , , , – , – , – , , , 870 109 272 648 1505 899 268 919 1471 636 866 1048 1481 189 286 640 1528 194 856 1253 248 1258 256 oce No Voucher D D D D D D D D D D D D D D D D D D D D D D D , , Yunnan Yunnan Yunnan , , , , Yunnan Guangxi Guangxi Kunming Yunnan , Yunnan Guizhou Guangxi , , , , Yunnan , , , , Guizhou Guizhou Guizhou , , , Yunnan , County e ’ Locality County Huize County Songming County Libo County Xundian County Funing City Anshun County Songming City Chongqing City Anshun County Chenggong Yunnan County Songming County Songming Kunming County Jinning Yunnan County Jinning County Lingyun Tian County Lingyun County Zhunyi aeil sdi hssuywt oce numbers voucher with study this in used Materials 1 . 2 2 3 1 sp sp sp sp aubryana bailleti funingensis intermedia kunmingensis liuii pallens rotundata srupea tchangsii telonaria textrix vultuosa ...... ao name Taxon aristarchorum Hua H H H H H H H H H H H H H Hua Hua Hua Hua Table

Zoological Research 40(6): 541-551, 2019 543 , 10 97 37 969045 – , – – , , , MK 41 55 72 – , , , – – Continued 251706 968996 969058 969028 969068 968982 MK MK MK MK MK MK 969105 969040 969046 969043 969047 969051 969057 969056 968971 , , , , , , 30 MK MK MK MK MK MK MK MK MK 64 67 69 50 26 63 80 22 94 04 75 18 – , – , , – , – , – – , – – , – , – – , – – 013828 969103 968962 969038 969044 969064 969039 968984 968965 969042 969049 969023 969048 969059 969069 968976 968995 969092 968983 969019 969027 968981 968986 969002 969095 968974 969015 COI MK MK MK MK MK MK MK MK MK MK MK MK MK MK MK MK MK MK MK MK MK MK MK MN MK MK MK , – 21 33 73 18 – , – – , , , – 77 91 07 . – , , , – – 251617 944132 944194 944164 944204 944117 MK MK MK MK MK MK 944243 944176 944182 944179 944183 944187 944193 944192 944106 , , , , , , MK MK MK MK MK MK MK MK MK 99 03 04 16 62 99 58 30 40 83 10 54 15 , – , , – , – , – – , – , – , – – – , – – – 944241 944097 944174 944180 944200 944175 944181 944120 944100 944178 944185 944159 944184 944195 944111 944131 944227 944119 944155 944163 944116 944122 944138 991781 944230 944109 944151 rRNA S eBn ceso No accession GenBank 16 MK MK MK MK MK MK MK MK MK MK MK MK MK MK MK MK MK MK MK MK MK MK MK MK MK MK MK paucicincta friniana . . , . , , davidi , , M . H , , S , amurensis davidi friniana pacificans resinacea schmackeri theaepotes terminalis calculus var gredleri gredleri bicintus amurensis dolichostoma pacificans terminalis minbeiensis ...... Synonym – M S amurensis Parajuga H M S M M S P M M M M S M M cinnamomea M , , , , , – , , 665 553 466 777 720 D , D D D D – , – , 1149 1178 1155 1167 1128 1140 1116 D D D D D D 664 D , , , – – , 462 552 774 , 718 D , D D D D , , , , 1145 1173 1150 1154 1196 1127 1137 1161 1115 662 D D D D D D D D D 461 551 771 717 D , , , , – , , , , , D D D , D , , – , . 1527 661 1594 1055 1144 1172 1147 1152 1157 1193 1162 1125 1135 1113 460 549 820 726 1163 768 840 715 D D D D D D D D D D D D D D , – , – D D D D D D D , , , , – – , , , D , , , , , , , – , 1522 456 1142 1151 1179 1143 729 546 816 1146 1164 1122 1132 1153 1171 711 723 810 1403 728 1112 1126 721 766 838 1592 1455 1052 oce No Voucher D D D D D D D D D D D D D D D D D D D D D D D D D D D D Hunan , Liaoning , Jiangxi Jiangxi Jiangxi Jiangxi Jiangxi Jiangxi Anhui Yunnan , , , , , , , , , Anhui Anhui Anhui Anhui Guizhou Guangxi , , , , Zhejiang , , , Zhejiang Zhejiang , , Locality County Luquan City Mudanjiang Heilongjiang County Wuyuan County Wuyuan City Ningguo City Lishui County Wuyuan County Wuyuan City Ningguo City Lishui County Daweishan City Ningguo County Wuyuan County Wuyuan City Ningguo City Huangshan City Ningpo County Kuandian City Tongren City Hezhou 4 sp diminuta . bicintus davidi dolichostoma friniana pacificans pacificans praenotata terminalis terminalis calculus cinnamomea gredleri ...... ao name Taxon Hua Koreoleptoxis amurensis K K Ko K K K K K K K S S S

544 www.zoores.ac.cn , Continue 94 98 – – 251693 251697 MK MK 91 94 11 96 51 9001 – – , – , – – – 969087 969093 968973 968985 969009 968970 251695 251748 968998 COI MK MK MK MK MK MK MK MK MK , 03 07 . – – 251602 251606 MK MK 26 29 47 05 72 37 – – , – , – – – 944222 944228 944108 944121 944145 944105 251604 251669 944134 rRNA S eBn ceso No accession GenBank 16 MK MK MK MK MK MK MK MK MK , - , ning . ningpoensis M suifuensis Melanoides ) , . , bensoni , M . , M , Namrutua cancellata fortune pleuroceroides ( cancellata . . . . . Synonym M poensis M S ningpoensis H M – 227 D , 1046 D – 224 836 D D , , 1045 D 682 834 667 , D D D – , , . 1385 1454 1024 1043 666 672 831 D D D D – – – – D D D , , , 1381 1453 663 734 1022 656 1042 828 oce No Voucher D D D D D D D D Hunan Jiangxi Jiangxi , , , Anhui Guizhou Sichuan , Zhejiang , , ,

Figure 1 Sampling localities of semisulcospirids in China . Locality City Hengyang City Tongren City Zhangshu City Ningguo City Neijiang City Zhangshu Chongqing City Ningpo Colors indicate collection sites of samples belonging to clades delimited . in Figure 2 and Figure 3. Black: : Hua pallens; : H. jacqueti; : H. sp ● ▲ ■ aubryana; : H. funingensis; : H. sp3; : H. intermedia; ◄: Species ▼ ♦ ★ from Dianchi Basin. Red: : Semisulcospira pleuroceroides; : S. ● ■

o available Not ningpoensis; : S. gredleri; : S. cinnamomea; : S. calcutus. Blue: : ▲ ★ ♦ – : Koreoleptoxis pacificans; : K. terminalis; : K. praenotata; : K. ningpoensis pleuroceroides

. . ● ■ ▲ ★

ao name Taxon S S Semisulcospira peregrinorum; : K. dolichostoma; : K. amurensis. ▼ ♦

Zoological Research 40(6): 541-551, 2019 545 MN013830 (Table 1). We also acquired various sequences four subclades: subclade A1 contained species P. rotundata, from GenBank for use as the ingroup following Du et al. P. minensis, and L. pallens; subclade A2 contained one (2019), including one Koreoleptoxis species (K. amurensis), undescribed species from Nanpanjiang River, Hua sp. 3, H. three Semisulcospira species (S. pleuroceroides, S. gredleri aristarchorum, and species of Wanga as orphans without (Böttger, 1886), S. ningpoensis), and thirteen Hua species (H. genus affiliation from southwest China and Guangxi Zhuang aristarchorum (Heude, 1888), H. telonaria (Heude, 1888), H. Autonomous Region; subclade A3 contained H. funingensis aubryana, H. bailleti (Bavay & Dautzenberg, 1910), H. textrix, and an undescribed species, Hua sp. 2, from Nanpanjiang H. vultuosa (Fulton, 1914), H. scrupea (Fulton, 1914), H. River; subclade A4 contained H. telonaria (type species), funingensis Du, Köhler, Yu, Chen & Yang, 2019, H. species of Hua from southwest China and , and two kunmingensis Du, Köhler, Yu, Chen & Yang, 2019, H. liuii Du, undescribed species (Figure 2). Clade B could be divided into Köhler, Yu, Chen & Yang, 2019, H. tchangsii Du, Köhler, Yu, three subclades; subclades B1 and B2 contained species of Chen & Yang, 2019, and two undescribed) (MK251600 – viviparous Semisulcospira from Japan and China; subclade MK251768)); Elimia clenchi, E. catenaria, and Melanopsis sp. B3 contained oviparous K. globus ovalis, K. nofifila, and K. were also obtained from GenBank as the outgroup to root the amurensis, and species from the lower Yangtze River (Figure phylogeny in accordance with the trees in Strong & Köhler 3). These clades revealed an approximate phylogeographic (2009) and Köhler (2017). structure, which roughly adhered to the major drainage Sequence alignment of 16S rRNA and COI was performed systems in China (Figure 1). Results indicated that clade A with default parameters using Mafft v7.305 (Katoh et al., 2002) (black) occurred predominantly in southwest China and and the CIPRES Science Gateway (Miller et al., 2010). drainages of the upper Yangtze and Pearl rivers, whereas Pairwise distances between species were determined using subclades B2 (red) and B3 (blue) mainly occurred in the MEGA 7 (Kumar et al., 2016) and the optimal substitution middle and lower Yangtze River, with a few specimens model was selected by corrected Akaike Information Criterion collected from northeast China (Figure 1). using jModeltest v2.1.10 (Posada, 2008). MRBAYES v3.2.6 (Ronquist et al., 2012) and RAxML-HPC v.8.2.10 (Stamatakis, Comparative morphology We identified several patterns largely consistent with the 2014) were used for Bayesian inference and maximum phylogenetic clustering in the mitochondrial trees shown in likelihood analyses, respectively, in reference to the selected Figure 2 and Figure 3. Subclades B1 and B2 are viviparous model of sequence evolution. Bayesian posterior probabilities and mature females do not possess an ovipositor pore or egg- (BPPs) of nodes were determined using Metropolis-coupled laying groove below the right cephalic tentacle; furthermore, Markov chains (one cold chain) for five million generations, the lower margin of the central teeth is straight in both with sampling every 100 generations. Node support for subclades. Clade A and subclade B3 are both oviparous, maximum likelihood analysis was determined using 1 000 but show differences in reproductive anatomy; specifically, rapid bootstrap replicates. mature females in clade A possess an ovipositor pore or egg-laying groove below the right cephalic tentacle, whereas RESULTS mature females in subclade B3 possess both an ovipositor and egg-laying groove. In addition, the lower margin of the Sequence analysis and mitochondrial phylogeny central teeth is w-shaped in clade A, but triangular in The final concatenated dataset contained 221 16S sequences subclade B3. and 220 COI sequences. The 16S alignment contained 567 Regarding features, the nucleus is obvious and nucleotide positions after pruning alignment ends and located in the lower base of the operculum in all subclades, removing poorly aligned portions. The COI dataset had 762 except subclade A1, in which it is not obvious (Figure 4A–B). nucleotide positions and a total length of 1 339 bp, including In subclade A3, the nucleus is very small and located in the 553 variable sites and 497 phylogenetically informative sites. lower one sixth of the operculum (Figure 4C–D); in subclades The GTR+I+G model was selected as the best-fit model of A2 and A4 and clade B, the operculum shows a clear spiral nucleotide substitution. The dataset contained sequences growth line and the nucleus is located in the lower one third of from previously published studies, which were obtained from the operculum (Figure 4E–H). GenBank, as well as the new sequences, which were In clade A, species of subclades A1 and A4 (except H. deposited in GenBank (accession Nos. shown in Table 1). aubryana and H. jacqueti), as well as H. sp. 3, and H. Sequences of Western North America , type species of funingensis, possess smooth shells, whereas all other species the genera and Koreoleptoxis globus possess a sculptured shell. In P. minensis, L. pallens, H. liuii, ovalis, and other published sequences of K. nofifila and S. and H. funingensis, the lateral teeth exhibit a single prominent reiniana from GenBank were included to check the squarish cusp without small denticles at either side (Figure phylogenetic positions of the Chinese semisulcospirids. 5B), whereas, in all other species, the lateral teeth exhibit two Both phylogenetic analyses produced identical, usually well- to four small denticles at each side (Figure 5A). In P. minensis, supported topologies consisting of two main clades (A and B), P. rotundata, L. pallens, H. sp. 3, and H. textrix, the mature which contained sequences of various nominal species females possess an egg-laying groove, whereas mature previously recorded in China. Clade A could be divided into females in all other species possess an oviparous pore. In

546 www.zoores.ac.cn Figure 2 Bayesian phylogram of clade A based on analysis of concatenated dataset of mitochondrial cytochrome c oxidase subunit I (COI) and 16S rRNA sequences Colored circles indicate origin of samples as indicated on map in Figure 1.

Zoological Research 40(6): 541-551, 2019 547 Figure 3 Bayesian phylogram of clade B based on analysis of concatenated dataset of mitochondrial cytochrome c oxidase subunit I (COI) and 16S rRNA sequences. Colored circles indicate origin of samples as indicated on map in Figure 1.

548 www.zoores.ac.cn Figure 4 Opercula of semisulcospirids from China A: Hua pallens; B: H. rotundata; C: H. funingensis; D: H. sp. 2; E: H. telonaria; F: Semisulcospira pleuroceroides; G: N. ningpoensis; H: K. amurensis. Scale bar: 1 mm. subclade B2, S. ningpoensis, S. gredleri, and S. cinnamomea amurensis and Melania pacificans Heude, 1888 have well- (Gredler, 1887) have well-sculptured shells, whereas all other sculptured shells, whereas all other Chinese species have a Chinese species possess a smooth shell. In subclade B3, K. smooth shell.

Figure 5 Radulae of semisulcospirids from China A: Hua telonaria; B: H. funingensis; C: Koreoleptoxis; D: Semisulcospira. Scale bar: 100 μm.

Mitochondrial differentiation DISCUSSION Pairwise comparisons of COI distances revealed that clade A and subclades B2 and B3 differed from each other by average In this study, the Chinese semisulcospirids were divided into uncorrected P-distances of 14.6% to 14.7% (average 14.6%). two main clades, i. e., clades A and B. Minton & Lydeard Species within clade A differed by an average uncorrected P- (2003) reported maximum intergeneric differences of 15.5% distance of 0.8% to 14.5% (average 11.2%). Species within for COI in Semisulcospiridae. Here, we observed a maximum subclade B2 differed by an average uncorrected P-distance of intergeneric divergence of 14.7%. Hence, clade A, together 10.0% to 14.9% (average 12.0%). Species within subclade B3 with subclades B2 and B3, represented the genera of Hua, differed by an average uncorrected P-distance of 1.2% to Koreoleptoxis, and Semisulcospira, respectively. 11.2% (average 6.6%). Morphologically, the three genera can be distinguished from

Zoological Research 40(6): 541-551, 2019 549 each other by reproductive mode, radula, and female four undescribed species (Du et al., 2019); other species from reproductive features. south of the middle and lower Yangtze River are placed into Liu et al. (1993) and Xu (2007) assigned Senckenbergia Koreoleptoxis, which therefore contains K. diminuta (Boettger, pleuroceroides and Namrutua ningpoensis into 1887), K. friniana (Heude, 1888), K. joretiana (Heude, 1890), Semisulcospira, but did not provide a reason for this decision. K. naiadarum (Heude, 1890), K. oreadarum (Heude, 1888), K. Du et al. (2019) treated Namrutua and Senckenbergia as parenotata (Gredler, 1884), K. schmackeri (Boettger, 1886), junior synonyms to Semisulcospira based on morphological and K. toucheana (Heude, 1888). and phylogenetic evidence. In addition to three Chinese Within Hua, species can be distinguished from each other species, i. e., S. gredleri, S. ningpoensis, and S. by the shell (sculptured or smooth, elongated or ovate), pleuroceroides, four other species, i. e., S. cinnamomea operculum, radula, and female reproductive features. (Gredler, 1887) from Yangtze River, S. calculus (Reeve, 1860) Additionally, within Koreoleptoxis, species can be from Liaoning Province in northeast China, and two distinguished from each other by the shell (sculptured or undescribed species from Zhejiang Province were contained smooth, elongated or ovate) and radula features. Although K. in this study. Semisulcospira cinnamomea was initially pacificans and K. terminalis are not monophyletic in the described as a subspecies of S. gredleri (Gredler, 1887); phylogenetic tree, K. pacificans can be distinguished from K. however, it is treated as a valid species in this study as it can terminalis by morphological characteristics: i. e., shell solid be distinguished from S. gredleri by its six to nine whorls (vs. and sculptured (vs. thin and smooth) and same number of four to five) and body not inflated (vs. inflated). inner and outer margin teeth (vs. different). Hence, K. Furthermore, M. calculus and M. paucicincta Martens 1894 pacificans and K. terminalis are treated as valid species in are virtually identical (shell size, proportion, rate of whorl this study. Incongruence between mtDNA phylogeny and expansion, and color bands), and are here treated as morphospecies is not a rare phenomenon in synonyms, with Semisulcospira calculus considered as the semisulcospirids (Köhler, 2016, 2017). Retention of ancestral valid species name due to the description of M. calculus polymorphisms, introgression, heteroplasmy, recombination, predating that of M. paucicincta. The Chinese species of and the presence of morphologically cryptic species or Semisulcospira can be distinguished from each other by their taxonomic misidentification may explain why mtDNA shell features (sculptured or smooth, inflated or phylogenies do not accurately reflect the relationships of not, number of whorls) and radulae. their organisms (Ballard & Whitlock, 2004; Edwards & Several species were originally described as members of Beerli, 2000; Funk & Omland, 2003; Köhler & Deein, 2010; Paludomus, including P. kweichouensis, P. minensis, P. Köhler, 2016; White et al., 2008). qianensis, and P. cinctus (Chen, 1937, 1943; Liu et al., 1994). Although the Chinese Paludomus species are similar to P. COMPETING INTERESTS conica Gray (type species of Paludomus, type location in India) in operculum features, Abbott (1948) stated that the The authors declare that they have no competing interests. mantle margin in P. conica contains 20 to 25 papillae, whereas the mantle margin in Chinese Paludomus is smooth. Furthermore, in the current study, phylogenetic evidence AUTHORS' CONTRIBUTIONS indicated that Chinese Paludomus species recorded in southwest China could belong to Hua (Semisulcospiridae). In L.N.D. and J.X.Y. designed the study. J.C. checked the specimens of addition, P. minensis and L. pallens, which come from the Koreoleptoxis and Semisulcospira that preserved in Institute of same locality, are virtually identical, except that the body whorl Zoology, CAS. G. H. Y. supervised the molecular analyses. L. N. D. in L. pallens is more inflated than that in P. minensis; thus, extracted genomic DNA and wrote the manuscript with the other authors' here, they are deemed to be synonyms, with Hua pallens input. L.N.D. and G.H.Y. sequenced mitochondrial DNA and submitted to considered as the valid species name as the description of L. GenBank. L.N.D. and G.H.Y. revised the manuscript. All authors read pallens predates that of P. minensis. Furthermore, P. and approved the final version of the manuscript. kweichouensis is treated as a synonym of M. intermedia Gredler, 1884 due to their virtually identical shells and same ACKNOWLEDGEMENTS locality, with Hua intermedia considered as the valid species name due to the description of M. intermedia predating that of We are grateful to G.H. Cui, S.S. Shu, S.W. Liu, X.A. Wang, Y.E. Jiang, C. P. kweichouensis. Yuan, X.F. Pan, T. Qin (Kunming Institute of Zoology, Chinese Academy of Chen (1943) originally described Wanga (containing eight Sciences), J. Yang (Nanning Normal University, China) and J.H. Lan (Du'an species) and Hua (containing eleven species). In this study, Fishery Technique Popularization Station) for specimens collection. Special however, the species of Wanga and Hua from southwest thanks are due to Dr. V. Héros (Muséum National d'Histoire Naturelle, Pairs, China and Guangxi are placed in Hua, which therefore France), Dr. A. Hänggi (Naturhistorisches Museum, Basel, Switzerland), Dr. contains 19 species in China, i. e., H. aristarchorum, H. E. Strong (National Museum of Natural History in Washington DC, USA), aubryana, H. bailleti, H. funingensis, H. intermedia, H. and Dr. J. Ablett (Natural History Museum, London, UK) for providing jacqueti, H. kunmingensis, H. liuii, H. pallens, H. rotundata, H. photographs of type specimens. We are grateful to Dr. C. Watts for scrupea, H. textrix, H. telonaria, H. tchangsii, H. vultuosa, and polishing this manuscript.

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