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

2019·1.8 MB·

by  Du, Li-Na

ZOOLOGICAL RESEARCH
Systematic relationships of Chinese freshwater
semisulcospirids (Gastropoda, Cerithioidea) revealed
by mitochondrial sequences
Li-NaDu1,2,3,#,JunChen3,#,Guo-HuaYu2,*,#,Jun-XingYang1,4,*
1KunmingInstituteofZoology,ChineseAcademyofSciences,KunmingYunnan650223,China
2Key Laboratory of Ecology of Rare and Endangered Species and Environmental Protection (Guangxi Normal University), Ministry of
Education,GuilinGuangxi541004,China
3KeyLaboratoryoftheZoologicalSystematicsandEvolution,InstituteofZoology,ChineseAcademyofSciences,Beijing100101,China
4Yunnan Key Laboratory of Plateau Fish Breeding, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming Yunnan
650223,China
ABSTRACT INTRODUCTION
The systematics of Semisulcospiridae 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,andinwesternNorthAmerica(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
centuries (e.g., Dautzenberg & Fischer, 1906, 1908; Fulton,
family of Chinese Semisulcospiridae is represented
1914; Heude, 1888), with all cerithioidean freshwater
by three genera: i. e., viviparous Semisulcospira
Böttger, 1886, oviparous Hua Chen, 1943 and gastropods designated under the single genus Melania
Koreoleptoxis Burch and Jung, 1988. These genera Lamarck, 1799 (junior synonym 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
radula features. Species of Hua are mainly
Received:30January2019;Accepted:16July2019;Online:21August
distributed in southwest China and Guangxi,
2019
whereas Koreoleptoxis and Semisulcospira are
mainlydistributedinsouthandnortheastChina. 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 Taxonomy of the ChineseAcademy of Sciences (ZSBR-
Koreoleptoxis
011), Key Laboratory of the Zoological Systematics and Evolution of
the Chinese Academy of Sciences (Y229YX5105), Strategic Priority
OpenAccess 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 DiversityObservationNetwork
non-commercial use, distribution, and reproduction in any medium, #Authorscontributedequallytothiswork
providedtheoriginalworkisproperlycited. *Corresponding authors, E-mail: [email protected]; yugh2018@
Copyright ©2019 Editorial Office of Zoological Research, Kunming 126.com
InstituteofZoology,ChineseAcademyofSciences DOI:10.24272/j.issn.2095-8137.2019.033
SciencePress ZoologicalResearch 40(6):541-551,2019 541
MelaniainsouthChina,especiallyintheYangtzeRiverBasin. rotundata (Heude, 1888). Furthermore, most semisulcospirids
Yen (1939) subsequently identified Chinese freshwater snails were recorded in theYangtze River.Thus, based on previous
fromtheNaturmuseumSenckenberg,Germany,andrecorded literature, 52 species of semisulcospirids have been recorded
12 Chinese species of Thiaridae and a new genus, in China (Du & Yang, 2019; Du et al., 2019; Liu et al., 1993;
Senckenbergia Yen, 1939, for the type species Melania Xu, 2007). However, the systematic and phylogenetic
pleuroceroidesfromSichuan.Chen(1943)laterdescribedtwo relationshipsofsemisulcospiridsremainpoorlyunderstood.
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, Anhui, Jiangxi, Hunan, Zhejiang, Fujian,
respectively.Indispute,Morrison(1954)proposedWangaand Guangdong, Guangxi, Guizhou, Sichuan, Chongqing, and
Hua to be synonyms of Brotia H.Adams 1866 and Oxytrema Yunnan.Basedonmorphologicalandmolecularanalyses,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 systematicrelationshipsamongChinesesemisulcospirids.
subsequent authors (Du et al., 2019; Köhler, 2016, 2017;
Strong&Köhler,2009).Abbott(1948)describedanewgenus,
MATERIALSANDMETHODS
Namrutua, for the type species Melania ningpoensis.
However, Senckenbergia and Namrutua were primarily
Taxonsampling
ignoredbymostChineseresearchers,withallspeciesinstead
Specimenswerecollectedfrom2014to2017byL.N.Duetal.
placedinSemisulcospiraBöttger,1886(Liuetal.,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
topotypicspecimenphotos.
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.
including95sequencedsamplesfrom22speciesofHuafrom
qianensisLiuZhang&Duan1994andP.cinctusLiu,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(Table1).
species. The first systematic revision of semisulcospirids was
provided by Strong & Köhler (2009) based on morphological Morphology
charactersandmolecularphylogeneticanalysis.Theyshowed 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
(Pachychilidae 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
supportedNamrutuaandSenckenbergiaasviviparousandas (2009)andKöhler(2017).
synonyms of Semisulcospira. Additionally, the taxonomic
status of several species were discussed, in particular M. Molecularanalyses
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
andM.dulcisFulton1904,M.lautaFulton1904,andS.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)inthisstudy),PaludomusminensisChen1943 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
6, 6
3 – 8
5– 7, 4 2, 5, 2–
3 4 4 0 0 8
171,5– 17 1, 1– 90 90
544 5 2 0 6 6
277 2 7 1 9 9
K11 K 1 9 K K
M55 M 5 6 M M
22 2 9
07 34,MKMK 65 38, 68 98 MK26 61 14 100 9 16 MK05 05, 57 71, 30 81 13
6– 3–1,3, 2– 7– 6– 6– 0,3– 8– 2– 9– 69 4– 2,4– 1–7 5– 0– 7– 2– 2–
0 364 6 3 6 9 22 5 1 9 1 1 20 00 5 7 2 7 1
0 797 7 7 7 0 77 7 0 0 5 7 71 71 7 0 7 0 7
9 181 1 1 1 9 11 1 9 9 2 1 19 19 1 9 1 9 1
6 565 5 5 5 6 55 5 6 6 K 5 56 56 5 6 5 6 5
OI K9 K2K9K2 K2 K2 K2 K9 K2K2 K2 K9 K9 M K2 K2K9 K2K9 K2 K9 K2 K9 K2
C M MMM M M M M MM M M M ,– M MM MM M M M M M
0 1
4 2
0,6, 8– 4 7, 1, 7–
59 3 6 3 4 1
No. 48–44063,442 516 34, 36– 441 442
cession 43 MK251652,MK9MK251668,MK9 85 54,MK2 90 33 MK251639 82 50 35 09 27 MK9442243 16,MK9 78 06,MK9 44 16 24
nkac NA 142– 646,651–659,665– 683– 653– 689– 231– 632,636– 679– 148– 234– 608– 625– 635,242– 612–245 676– 205– 640– 207– 623–
a R 4 1111 1 1 1 4 11 1 4 4 1 1 14 14 1 4 1 4 1
ym GenB 16Sr MK94 MK25MK25MK25MK25 MK25 MK25 MK25 MK94 MK25MK25 MK25 MK94 MK94 MK25 MK25 MK25MK94 MK25MK94 MK25 MK94 MK25 MK94 MK25
n
o
kaccessionNos.andsyn Synonym Melaniaaristarchorum M.aubryana,Semisulcospiraaurbryana M.bailleti,Oxytremabailleti – M.praenotataintermedia,Paludomus(Hemimitra)kweichouensis,H.kwei-chouensis,H.praenotataintermedia – – Lithogryphuspallens,P.minensis M.rotundata M.scrupea,S.scrupea,Wangascrupea – M.telonaria,M.teloniaria,M.leprosa,S.telonaria,O.telonaria M.textrix,M.textoria,M.dulcis,M.lauta,S.dulcis,S.lauta,S.inflata M.vultuosa,S.vultuosa – – –
n
a 5,6,
B 64
numbers,localities,Gen ucherNo. 70,D872, 09,D201D203,D263D2––72,D274,D275,D281,D648D649,D1497,D1503,–505 99D901,D903– 68,D269,D282 19,D920,D922 471,D1472,D1475 36,D639,D641D644– 66D869– 048D1049,D1051– 481,D1486 89,D190 86D288– 40,D1492,D1496,D1526,528 94DD198,D860,D1578– 56,D858D859– 253D1257,D1268D1272– 48,D250,D252253– 258D1267– 56,D258
er Vo D8 D1D2D6D1 D8 D2 D9 D1 D6 D8 D1 D1 D1 D2 D6D1 D1 D8 D1 D2 D1 D2
h
c
edinthisstudywithvou Locality HuizeCounty,Yunnan SongmingCounty,Yunnan LiboCounty,Guizhou XundianCounty,Yunnan FuningCounty,Yunnan AnshunCity,Guizhou SongmingCounty,Yunnan ChongqingCity AnshunCity,Guizhou ChenggongCounty,Yunnan SongmingCounty,Yunnan SongmingCounty,Kunming,Yunnan JinningCounty,Kunming,Yunnan JinningCounty,Yunnan LingyunCounty,Guangxi TianeCounty,Guangxi’ LingyunCounty,Guangxi ZhunyiCounty,Guizhou
s
u
s m
Table1Material Taxonname Huaaristarchoru H.aubryana H.bailleti H.funingensis H.intermedia H.kunmingensis H.liuii H.pallens H.rotundata H.srupea H.tchangsii H.telonaria H.textrix H.vultuosa Huasp.1 Huasp2 Huasp2 Huasp3
ZoologicalResearch 40(6):541-551,2019 543
5
4
0
Continued 05 5170610– 4041,–46, 43,MK969 6899697– 47,69058 6902837,–5155,–69068, 68982 57 56 7172,–
1 2 00 0 9 09 909 9 0 0 9
9 K 99 9 K 9K K9K K 9 9 8
6 M 66 6 M 6M M6M M 6 6 6
9 99 9 9 9 9 9 9
MK 64, MKMK67 MK 69, MK50, 26,MK63, 80, MK 22 MK 94 04 30 MK75 18
3, 2– 8,4,4– 9, 4 5– 2,9– 3–8,9–9 6– 5, 2 3 9– 7, 1 6– 2– 8– 5,4– 5–
0 6 346 3 8 6 44 2456 7 9 9 8 1 2 8 8 0 2 97 1
1 9 000 0 9 9 00 0000 9 9 0 9 0 0 9 9 0 8 09 0
9 8 999 9 8 8 99 9999 8 8 9 8 9 9 8 8 9 3 98 9
6 6 666 6 6 6 66 6666 6 6 6 6 6 6 6 6 6 1 66 6
OI K9 K9 K9K9K9 K9 K9 K9 K9K9 K9K9K9K9 K9 K9 K9 K9 K9 K9 K9 K9 K9 N0 K9K9 K9
C M M MMM M M M MM MMMM M M M M M M M M M M MM M
nkaccessionNo. NA 241,MK944243 09799,MK25161721–– 174,MK94417677,–180,MK944182,20003– 175,MK944179,181 120 10004,MK94413233–– 178,MK944183,18516,MK944194– 15962,MK94416473,––184,MK94418791,–19599,MK944204,–– 11115,MK94411718–– 131,MK944193 227 119 15558– 163,MK944192 116 12230– 13840– 78183– 230,MK94410607,–10910– 15154–
a R 4 4 444 44 4 4 44 444 4 4 4 4 4 4 4 4 4 1 44 4
GenB 16Sr MK94 MK94 MK94MK94MK94 MK94MK94 MK94 MK94 MK94MK94 MK94MK94MK94 MK94 MK94 MK94 MK94 MK94 MK94 MK94 MK94 MK94 MK99 MK94MK94 MK94
a
ct
n
a ci
Synonym – M.amurensis,S.amurensis,Parajugaamurensis H.bicintus M.davidi,S.davidi S.dolichostoma M.friniana,H.frinian M.pacificans,S.pacificans,P.minbeiensis M.resinacea M.schmackeri M.theaepotes M.terminalis,S.terminalis M.calculus,M.pauci M.gredlerivar.cinnamomea M.gredleri
6 3, 0, 7 65
No. 1527 60,D461,D462D46– 1144,D1145,D1149,1172,D1173,D1178, 1147,D1150 49,D551,D552,D5520 1152,D1154,D1155, 1125,D1127D1128,–1135,D1137D1140,–1157D1161,D1167––1193,D1196 15,D717,D718,D7226 163, 1113,D1115,D1116 1162 68D771,D774D77–– 40 1594 661,D662,D664,D6 1055
D 4 DD D 58 D DDDD 77 1 D D 7 8 D D D
ucher 522, 56,D 142,151,179 143, 29 46,D16,D 146,164 122–132–153,171, 11,D23,D 10,D 403 28 112, 126, 21 66,D 38D– 592– 455, 052–
o 1 4 111 1 7 58 11 1111 77 8 1 7 1 1 7 7 8 1 1 1
V D D DDD D D DD DD DDDD DD D D D D D D D D D D D
n g
a n
Locality LuquanCounty,Yunnan MudanjiangCity,Heilongjiang WuyuanCounty,Jiangxi WuyuanCounty,Jiangxi NingguoCity,Anhui LishuiCity,Zhejiang WuyuanCounty,Jiangxi WuyuanCounty,Jiangxi NingguoCity,Anhui LishuiCity,Zhejiang DaweishanCounty,Hun NingguoCity,Anhui WuyuanCounty,Jiangxi WuyuanCounty,Jiangxi NingguoCity,Anhui HuangshanCity,Anhui NingpoCity,Zhejiang KuandianCounty,Liaoni TongrenCity,Guizhou HezhouCity,Guangxi
Taxonname Huasp4 Koreoleptoxisamurensis K.bicintus K.davidi Ko.diminuta K.dolichostoma K.friniana K.pacificans K.pacificans K.praenotata K.terminalis K.terminalis S.calculus S.cinnamomea S.gredleri
544 www.zoores.ac.cn
e
u
n
nti
o
C 4 8,
9 9
– –
3 7
9 9
6 6
1 1
5 5
2 2 1
K K 0
91 94 M 11 M96 51 90
7– 3– 3, 5 9– 0,5– 8– 8–
8 9 7 8 0 79 4 9
0 0 9 9 0 96 7 9
9 9 8 8 9 81 1 8
6 6 6 6 6 65 5 6
OI K9 K9 K9 K9 K9 K9K2 K2 K9
C M M M M M MM M M
3 7,
0 0
No. 02– 06–
cession 26 29 MK2516 47 MK251605 72 37
nkac NA 222– 228– 108, 121 145– 105,604– 669– 134–
a R 4 4 4 4 4 41 1 4
GenB 16Sr MK94 MK94 MK94 MK94 MK94 MK94MK25 MK25 MK94
s
Synonym M.cancellata,M.ning-poensis,M.bensoni,M.fortune,M.suifuensis,S.cancellata,Melanoidesningpoensis,H.(Namrutua)ningpoensi M.pleuroceroides –
7
D22 046
4, D1 6
D22 45– D83
7 2, 10 4,
6 8 D 3
6 6 8
5 4 D 4 D 3, D
o. 38 45 6– 02 2, 04 1,
N 1 1 6 1 7 1 3
D D 6 D 6 D 8
ucher 381– 453– 63,D 34 022– 56,D 042– 28,D
o 1 1 6 7 1 6 1 8
V D D D D D D D D
Locality HengyangCity,Hunan TongrenCity,Guizhou ZhangshuCity,Jiangxi NingguoCity,Anhui NeijiangCity,Sichuan ZhangshuCity,Jiangxi Chongqing NingpoCity,Zhejiang e. FCiogluorrsei1ndSicaamtepcloinllgecltoiocnalsititieessooffssaemmpilseuslbceolsopnigriindgstioncClahdinesadelimited
bl
Taxonname S.ningpoensis S.pleuroceroides Semisulcospirasp. :Notavaila– iafn●pnruieon:FbrmgeKripgygooDuarriernnieeanaoons;2rlceui▼shapm;int:o▲;dHBxa.i:Fs:sfSiuKgipn.nu.a.girdcnerRoigefil3eedcic.dnalheB:snorisli●ssa;;;tc★o■♦k:m::S::a●SKeH;.m.:.ctHsieis:npuruKnm3al.ac;iamonp★msaaopl:luilmseiHrr;naee.▲snaip;ns;:l▲ti♦♦eseK.ur:m.:roHpSecr..daejciaeraoacn;ilqodc◄utueaets:tuta;Sis;;■p.■★eBc:l::uieHSKes..:.
▼ ♦
ZoologicalResearch 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),andthirteenHuaspecies(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 Vietnam, and two
kunmingensis Du, Köhler,Yu, Chen &Yang, 2019, H. liuii Du, undescribedspecies(Figure2).CladeBcouldbedividedinto
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.
werealsoobtainedfromGenBankastheoutgrouptorootthe 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)andKöhler(2017). structure, which roughly adhered to the major drainage
Sequence alignment of 16S rRNAand COI was performed systems in China (Figure 1). Results indicated that clade A
withdefaultparametersusingMafftv7.305(Katohetal.,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 correctedAkaike Information Criterion collectedfromnortheastChina(Figure1).
using jModeltest v2.1.10 (Posada, 2008). MRBAYES v3.2.6
Comparativemorphology
(Ronquistetal.,2012)andRAxML-HPCv.8.2.10(Stamatakis,
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
andmaturefemalesdonotpossessanovipositorporeoregg-
(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,
rapidbootstrapreplicates.
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
Sequenceanalysisandmitochondrialphylogeny central teeth is w-shaped in clade A, but triangular in
Thefinalconcatenateddatasetcontained22116Ssequences subclade B3.
and 220 COI sequences. The 16S alignment contained 567 Regarding operculum 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 subcladeA1, 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. loweronesixthoftheoperculum(Figure4C–D);insubclades
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 growthlineandthenucleusislocatedintheloweronethirdof
from previously published studies, which were obtained from theoperculum(Figure4E–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 Juga, type species of funingensis,possesssmoothshells,whereasallotherspecies
the genera Semisulcospira libertina and Koreoleptoxis globus possess a sculptured shell. In P. minensis, L. pallens, H. liuii,
ovalis, and other published sequences of K. nofifila and S. andH.funingensis,thelateralteethexhibitasingleprominent
reiniana from GenBank were included to check the squarish cusp without small denticles at either side (Figure
phylogeneticpositionsoftheChinesesemisulcospirids. 5B),whereas,inallotherspecies,thelateralteethexhibittwo
Bothphylogeneticanalysesproducedidentical,usuallywell- tofoursmalldenticlesateachside(Figure5A).InP.minensis,
supportedtopologiesconsistingoftwomainclades(AandB), 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 cladeAbased on analysis of concatenated dataset of mitochondrial cytochrome c oxidase subunit I
(COI)and16SrRNAsequences
ColoredcirclesindicateoriginofsamplesasindicatedonmapinFigure1.
ZoologicalResearch 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)and16SrRNAsequences.
ColoredcirclesindicateoriginofsamplesasindicatedonmapinFigure1.
548 www.zoores.ac.cn
Figure4 OperculaofsemisulcospiridsfromChina
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.Scalebar:1mm.
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. smoothshell.
Figure5 RadulaeofsemisulcospiridsfromChina
A:Huatelonaria;B:H.funingensis;C:Koreoleptoxis;D:Semisulcospira.Scalebar:100μm.
Mitochondrialdifferentiation DISCUSSION
Pairwise comparisons of COI distances revealed that cladeA
andsubcladesB2andB3differedfromeachotherbyaverage 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 cladeAdiffered 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
subcladeB2differedbyanaverageuncorrectedP-distanceof intergeneric divergence of 14.7%. Hence, clade A, together
10.0%to14.9%(average12.0%).SpecieswithinsubcladeB3 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%(average6.6%). Morphologically, the three genera can be distinguished from
ZoologicalResearch 40(6):541-551,2019 549
each other by reproductive mode, radula, and female fourundescribedspecies(Duetal.,2019);otherspeciesfrom
reproductivefeatures. 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,butdidnotprovideareasonforthisdecision. 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 andK.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)fromYangtzeRiver,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,itistreatedasavalidspeciesinthisstudyasitcan 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 whorl 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, body whorl inflated or
phylogenies do not accurately reflect the relationships of
not,numberofwhorls)andradulae.
their host 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,andP.cinctus(Chen,1937,1943;Liuetal.,1994).
Although the Chinese Paludomus species are similar to P.
COMPETINGINTERESTS
conica Gray (type species of Paludomus, type location in
India) in operculum features, Abbott (1948) stated that the
Theauthorsdeclarethattheyhavenocompetinginterests.
mantlemargininP.conicacontains20to25papillae,whereas
the mantle margin in Chinese Paludomus is smooth.
AUTHORS'CONTRIBUTIONS
Furthermore, in the current study, phylogenetic evidence
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
samelocality,arevirtuallyidentical,exceptthatthebodywhorl 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 DNAand wrote the manuscript with the other authors'
here, they are deemed to be synonyms, with Hua pallens input.L.N.D.andG.H.Y.sequencedmitochondrialDNAandsubmitted to
considered as the valid species name as the description of L. GenBank. L.N.D. and G.H.Y. revised the manuscript. All authorsread
pallens predates that of P. minensis. Furthermore, P. andapprovedthefinalversionofthemanuscript.
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
nameduetothedescriptionofM.intermediapredatingthatof WearegratefultoG.H.Cui,S.S.Shu,S.W.Liu,X.A.Wang,Y.E.Jiang,C.
P.kweichouensis. Yuan,X.F.Pan,T.Qin(KunmingInstituteofZoology,ChineseAcademyof
Chen (1943) originally described Wanga (containing eight Sciences),J.Yang(NanningNormalUniversity,China)andJ.H.Lan(Du'an
species) and Hua (containing eleven species). In this study, FisheryTechniquePopularizationStation)forspecimenscollection.Special
however, the species of Wanga and Hua from southwest thanksareduetoDr.V.Héros(MuséumNationald'HistoireNaturelle,Pairs,
China and Guangxi are placed in Hua, which therefore France),Dr.A.Hänggi(NaturhistorischesMuseum,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 polishingthismanuscript.
550 www.zoores.ac.cn