41
Mi ogenome and nuclea RNA gene clus e o Aus opeplea
subaqua ilis (Ta e, 1880) om Sou h Aus alia, wi h molecula
and mo phological compa ison o A. c . b azie i (Smi h, 1882)
om Vic o ia (Gas opoda, Hyg ophila, Lymnaeidae)
Zhe-Yu Chen1, Tanapan Sukee1, Anson V. Koehle 1, Bonnie L. Webs e 2, Robin B. Gasse 1,
Wins on F. Ponde 3, Neil D. Young1
1 Depa men o Ve e ina y Biosciences, Melbou ne Ve e ina y School, Facul y o Science, The Uni e si y o Melbou ne, Pa k ille, VIC 3010, Aus alia
2 Depa men o Li e Science, Na u al His o y Museum, London, SW7 5BD, UK
3 Aus alian Museum Resea ch Ins i u e, Aus alian Museum, Sydney, NSW 2010, Aus alia
Co esponding au ho s: Zhe-Yu Chen ([email p o ec ed]); Neil D. Young ([email p o ec ed])
Copy igh : © Zhe-Yu Chen e al.
This is an open access a icle dis ibu ed unde
e ms o he C ea i e Commons A ibu ion
License (A ibu ion 4.0 In e na ional – CC BY 4.0).
Resea ch A icle
Abs ac
Species o Aus opeplea a e lymnaeid snails endemic o Aus alia and New Zealand,
and mos a e in e media e hos s o pa asi ic ema odes. Thei axonomy has long been
unce ain due o he high pheno ypic plas ici y o mos species. In his s udy, we used
Ox o d Nanopo e sequencing echnology o cha ac e ise he mi ogenome and nuclea
ibosomal RNA ( RNA) gene clus e o Aus opeplea subaqua ilis om Sou h Aus alia
o suppo compa a i e axonomic in es iga ions o his species. Then, A. subaqua ilis
was compa ed wi h A. c . b azie i a bo h mo phological and molecula le els. Mo pho-
logically, A. subaqua ilis and A. c . b azie i can be dis inguished by shell mo phome ic
indices, man le edge mo phology, pigmen a ion, and ep oduc i e and neu al ana omy.
The wo axa di e ed by 1.9% in bo h he mi ogenome and nuclea RNA gene clus e .
Sequence di e gence was p onounced in he in e nal ansc ibed space (ITS) egions
o he la e gene clus e , wi h nucleo ide di e ences o 13.8% in ITS1 and 8.2% in ITS2.
Phylogene ic analyses o sequence da a o he mi ochond ial 16S gene and ITS2 placed
he wo axa in dis inc g oups. Taken oge he , he in eg a i e e idence p esen ed he e-
in suppo ed species-le el di e gence be ween A. subaqua ilis and A. c . b azie i.
Key wo ds: Aus alia, eshwa e snails, in eg a i e axonomy, mi ochond ial genome,
molecula phylogeny, nuclea RNA gene clus e
In oduc ion
Lymnaeidae Ra inesque, 1815 (commonly known as pond snails) a e a glob-
ally dis ibu ed g oup o eshwa e hyg ophilid snails, which ha e a ac ed
widesp ead a en ion as in e media e hos s o a ious species o pa asi ic
ema odes (e.g., Vina ski e al. 2019, 2023; Vázquez e al. 2023). The Aus a-
lian na i e lymnaeid genus Aus opeplea Co on, 1942, is one o he ec o s
o Fasciola hepa ica (li e luke) disease in Aus alia and New Zealand (Bo ay
and McMichael 1961; Bo ay 1964a, 1969, 1978; Ponde and Wa e house 1997).
Academic edi o : Thie y Backeljau
Recei ed:
4 July 2025
Accep ed:
12 Sep embe 2025
Published:
9 Oc obe 2025
ZooBank: h ps://zoobank.
o g/602AF124-3022-4B8C-BE69-
C9F9006DE8CF
Ci a ion: Chen Z-Y, Sukee T, Koehle
AV, Webs e BL, Gasse RB, Ponde
WF, Young ND (2025) Mi ogenome
and nuclea RNA gene clus e o
Aus opeplea subaqua ilis (Ta e, 1880)
om Sou h Aus alia, wi h molecula
and mo phological compa ison o A.
c . b azie i (Smi h, 1882) om Vic o ia
(Gas opoda, Hyg ophila, Lymnaeidae).
ZooKeys 1255: 41–62. h ps://doi.
o g/10.3897/zookeys.1255.164109
ZooKeys 1255: 41–62 (2025)
DOI: 10.3897/zookeys.1255.164109
42
ZooKeys 1255: 41–62 (2025), DOI: 10.3897/zookeys.1255.164109
Zhe-Yu Chen e al.: Molecula and mo phological compa ison o wo Aus opeplea spp.
Bo ay and McMichael (1961) synonymised all 23 p e iously named Aus ope-
plea species o jus one species, Aus opeplea omen osa (P ei e , 1885), a
name based on New Zealand specimens, al hough hey ecognised wo dis inc
pheno ypes A and B. The ype A snails we e da ke wi h a smalle man le bo de ,
and had a mo e obus and opaque shell wi h a dis inc spi e, while ype B snails
we e ligh e wi h a much la ge man le bo de , and wi h a agile and anspa -
en shell wi h a sho spi e (Bo ay and McMichael 1961; Bo ay 1964b). Despi e
he exis ence o wo pheno ypes, Bo ay and McMichael (1961) a gued ha hey
we e all one species, based on assumed en i onmen -media ed di e ences in
snail pheno ype and hey ci ed e idence o success ul hyb idisa ion o he wo
pheno ypes unde labo a o y condi ions (Bo ay 1964b). In con as , Pusled-
nik e al. (2009), using mo phological and molecula e idence, indica ed ha
Aus alian Aus opeplea ep esen ed a dis inc lineage om he New Zealand
A. omen osa, and could be di ided in o di e en ope a ional axonomic uni s
(OTUs). Based on hese OTUs, Ponde e al. (2024) di ided he Aus alian Aus-
opeplea in o ou species in wo subgene a, Aus opeplea (Aus opeplea) b a-
zie i (Smi h, 1882) which is widely dis ibu ed in eas e n Aus alia, Aus opeplea
(Aus opeplea) subaqua ilis (Ta e, 1880) in Sou h Aus alia, and Aus opeplea
(Aus opeplea) huonensis (Tenison Woods, 1876) and Aus opeplea (Ku ikina)
hispida (Ponde & Wa e house, 1997) in Tasmania. Ne e heless, due o limi ed
de ined mo phological cha ac e s and he low phylogene ic esolu ion o cu -
en molecula ma ke s, comp ehensi e species delimi a ion emains lacking
o he nominal species o he han A. (K.) hispida. As a esul , he cu en clas-
si ica ion o species (o OTUs) wi hin he Aus alian membe s o ypical Aus o-
peplea la gely elies on geog aphic o igin a he han diagnos ic ea u es o he
specimens hemsel es. The e o e, de ailed mo phological and molecula cha -
ac e isa ion is needed o imp o e he axonomic esolu ion wi hin his genus.
The aim o his s udy was o cha ac e ise A. subaqua ilis om Sou h Aus alia
mo phologically and molecula ly, and hen compa e his species wi h A. c . b azie i
om Vic o ia, Aus alia o es ablish hei axonomic and phylogene ic ela ionship.
Ma e ials and me hods
Sample collec ion and p ese a ion
Aus opeplea subaqua ilis we e collec ed om “D ain M” nea P inces High-
way in Tho nlea, Sou h Aus alia, Aus alia (la i ude −37.36891397, longi-
ude 140.2052126). Aus opeplea c . b azie i we e p e iously collec ed om
a oadside i iga ion channel in We ibee Sou h, Vic o ia, Aus alia (la i ude
−37.944706, longi ude 144.698857) (see Sukee e al. 2024).
Bo h species o Aus opeplea we e hen cul u ed in aqua ia wi hin a desig-
na ed labo a o y a The Uni e si y o Melbou ne, Vic o ia, Aus alia. Snails om
di e en sou ces we e main ained in s ic isola ion in sepa a e anks con ain-
ing clean a i icial pond wa e wi h ae a ion. Wa e was changed egula ly and
he snails we e ed a comme cial ish die . A small sec ion (~5 mm) o he oo
muscle was excised om adul specimens and p ese ed in RNAla e a 4 °C
o 24 h, -20 °C o one mon h and hen s o ed a -80 °C un il u he p ocessing.
The emainde o each adul specimen was hen placed in 70% e hanol o sub-
sequen dissec ion and collec ion o mo phological ea u es.
43
ZooKeys 1255: 41–62 (2025), DOI: 10.3897/zookeys.1255.164109
Zhe-Yu Chen e al.: Molecula and mo phological compa ison o wo Aus opeplea spp.
G oss mo phology o ma u e snails
Animal ex e nal ea u es in he li ing s a e we e obse ed in he ield and in indi-
iduals main ained in cul u e. Ten and 13 indi iduals o A. subaqua ilis and A. c .
b azie i we e dissec ed o in e nal mo phological obse a ion espec i ely, all
indi iduals had a ained a body size su icien o o iposi ion. Te minology o
he cha ac e s ollowed Ponde and Wa e house (1997) and Vina ski and Poin -
ie (2023). Images o li ing animals we e cap u ed using a Canon® 5D Ma k IV
came a wi h a Canon® EF 100 mm /2.8L Mac o IS USM lens. P ese ed spec-
imens we e gen ly cleaned wi h so b ushes o emo e he coagula ed mucus
and dissec ed unde an Olympus SZ30 s e eomic oscope. De ailed images o
ana omical ea u es we e aken wi h he same Canon® 5D Ma k IV came a wi h
a Laowa® 25 mm /2.8 2.5-5X Ul a Mac o lens. The inal high dep h-o - ield im-
ages we e p oduced by a WeMac o® Rail Sys em and s acked om 20–30 sin-
gle pho os using Ze ene S acke ® 1.04. Ana omical illus a ions we e p epa ed
using a Wild M5 s e eomic oscope a ached wi h a d awing ube. All images
we e modi ied and assembled using Adobe Pho oshop 2023.
Scanning elec on mic oscopy
The adula sac was emo ed om h ee adul snails o each species and he
so issue emo ed using a 10% po assium hyd oxide (KOH) solu ion. A e
comple e dissolu ion o so issues, each adula was washed ex ensi ely in
MilliQ wa e . While s ill so , each adula ibbon was ans e ed on o a ound
co e slip (Ø 13 mm), ai -d ied and ixed in place. The co e slip was hen moun -
ed on o an aluminium s ub using conduc i e ca bon ape (P oSciTech P y L d).
A 4-μm hick gold coa ing was deposi ed on he adula su ace using Sa eMa -
ic® CCU-010 coa e . Radulae we e hen scanned using a Hi achi® SU7000 scan-
ning elec on mic oscope unde a low acuum mode (5 kV). Images gene a ed
om he middle de ec o we e used in his s udy.
DNA isola ion
Foo issue o a specimen o A. subaqua ilis ( ouche numbe : AB291) was e-
mo ed om he RNAla e and washed ex ensi ely in nuclease- ee wa e (Qia-
gen). DNA was hen isola ed om each sec ion o issue using he E.Z.N.A.
Mollusc DNA Ki acco ding o manu ac u e ’s ins uc ions (Omega Bio- ek Inc.).
The quan i y o DNA isola ed om each issue was de e mined using he Qubi
1X dsDNA HS Assay and a Qubi 2.0 Fluo ome e 2 (In i ogen, The moFishe ).
Ta ge -en iched ampli ica ion o mi ochond ial DNA
Ampli ica ion o he A. subaqua ilis mi ochond ial DNA was pe o med using he
REPLI-g mi ochond ial DNA ki (Qiagen) ollowing he manu ac u e ’s p o ocol us-
ing a cus om p ime mix ha was designed o ma ch conse ed egions o he lym-
naeid 12S and 16S mi ochond ial ibosomal subuni s and he cox1 gene. P ime s
we e 11–14 n in leng h and inco po a ed phospho o hioa e links be ween he las
h ee bases a he 3’ end o he p ime s (Table 1). A o al o 8 p ime s we e com-
bined in o a 100 µM s ock. Templa e DNA sample was dilu ed wi h wa e (supplied
44
ZooKeys 1255: 41–62 (2025), DOI: 10.3897/zookeys.1255.164109
Zhe-Yu Chen e al.: Molecula and mo phological compa ison o wo Aus opeplea spp.
om he ki ) o 150 ng in a o al olume o 20 µL. A esh ampli ica ion mix con-
aining he cus om p ime s was p epa ed as pe manu ac u e ’s ins uc ions (Qia-
gen). In b ie , 29 µL o DNA empla e and p ime mix was dena u ed a 75 °C o
5 min hen cooled o oom empe a u e. Nex , 1 µL o REPLI-g Midi Polyme ase
was added, and he sample was incuba ed a 33 °C o 8 h. Finally, he polyme ase
was deac i a ed by aising he empe a u e o 65 °C o 3 min. Ampli ied DNA was
quan i ied as desc ibed abo e and s o ed a 4 °C un il u he p ocessing.
Lib a y p epa a ion and long- ead sequencing
Fo A. subaqua ilis, a ba code was assigned o ampli ied DNA empla e using
he RAPID 24 (SQK-RBK114) ba coding lib a y ki (Ox o d Nanopo e Technolo-
gies) and loaded on o a R10.4.1 low cell and sequenced o 4 h on he P ome-
hION 2 Solo (Ox o d Nanopo e Technologies) sequencing pla o m. Pos -se-
quencing base-calling o POD5 da a was pe o med using he p og am Do ado
. 0.7.2 (Ox o d Nanopo e Technologies) in supe -accu a e mode and eads
we e s o ed in FASTQ o ma . Nanopo e long ead sequence da a o A. c . b a-
zie i was a ailable om a p e ious s udy (Sukee e al. 2024).
Clus e ing analyses, mi ochond ial genome assembly, and gene
anno a ions
Long eads wi h homology o e e ence mi ochond ial genomes o ibosomal
RNA subuni o eshwa e molluscs we e iden i ied using pbla . 2.5.1 (Wang
and Kong 2019) and ex ac ed om he aw long ead da a ile using seq k
. 1.4- 122 (h ps://gi hub.com/lh3/seq k/). A consensus mi ochond ial se-
quence o nuclea RNA gene clus e sequence was assembled using canu .
2.3 (h ps://gi hub.com/ma bl/canu) wi h minimum ead leng h se o 1,000 bp
and genome size se o 20,000 bp (mi ochond ial genome) o 8,000 bp (nu-
clea RNA gene clus e ). The A. subaqua ilis mi ochond ial genome was pe -
lima ly anno a ed using Geneious P ime . 2024.0.7 and MITOS . 2.0.2 (Be n
e al. 2013), hen manully cu a ed ollowing he sugges ed oles o molluscs
(Fou d ilis e al. 2018; Ghiselli e al. 2021). The published A. c . b azie i mi o-
chond ial genome (GenBank accession numbe PP100270) we e also u he
cu a ed acco ding o he same c i e ia. All sequence anno a ions and GC con-
en plo s we e isualised using he P oksee online se e (G an e al. 2023).
Table 1. Modi ied p ime s used o ampli y lymnaeid mi ochond ial DNA using he
REPLI-g mi ochond ial DNA ampli ica ion ki (Qiagen). As e isks ep esen he inco po-
a ed phospho o hioa e links.
P ime name P ime sequence Ta ge ed egion Di ec ion
RepGS_Aus 16F TACCTGTTTATC*A*A 16S Fo wa d
RepGS_16SBRis AACTCAGATCAT*G*T 16S Re e se
RepGS_12sF CAACGGCAATAT*A*T 12S Fo wa d
RepGS_12sR CTAGGATTAGAT*A*C 12S Re e se
RepGT_JB3 ATCCT GAGGTTT*A*T cox1 Fo wa d
RepGT_JB4.5 ACATAATGAAAA*T*G cox1 Re e se
RepGT_16sF CCTTTTGCATCA*T*G 16S Fo wa d
RepGT_16sR CGGTCTTAACTC*A*A 16S Re e se
45
ZooKeys 1255: 41–62 (2025), DOI: 10.3897/zookeys.1255.164109
Zhe-Yu Chen e al.: Molecula and mo phological compa ison o wo Aus opeplea spp.
Nucleo ide di e si y compa ison
Nucleo ide pai wise dis ances (p-dis ance) o comple e mi ochond ial genome,
he nuclea RNA gene clus e and each gene we e calcula ed in Geneious P ime
. 2024.0.7 a e alignmen wi h Clus al Omega . 1.2.3. Sliding window analy-
ses o nucleo ide di e si y (300-bp windows wi h 10-bp s eps o mi ochond ial
genome and 50-bp windows wi h 10-bp s eps o nuclea RNA gene clus e )
we e pe o med on he aligned mi ogenomes and nuclea RNA gene clus e o
A. c . b azie i and A. subaqua ilis using he PopGenome package (P ei e e al.
2014) in R. Fo each compa ison, nucleo ide di e si y alues we e plo ed using
he R package ggplo 2 (Wickham 2016). The GC/AT con en and skew alues
we e calcula ed using a cus om Py hon . 3.9.13 sc ip .
Phylogene ic analyses
To compa e A. c . b azie i and A. subaqua ilis samples wi h a ailable molec-
ula da a o Aus opeplea spp. (see Puslednik e al. 2009), phylogenies we e
econs uc ed using only he mi ochond ial 16S and nuclea ITS2 egions
(Suppl. ma e ial 1: able S1). O ien ogalba i idis was used as ou g oup (Liu
e al. 2012; Suwancha oen e al. 2023). The 16S and ITS2 sequences we e
aligned sepa a ely using MUSCLE . 3.7 in AliView . 1.28 and conca ena ed
using ca as a2phyml (h ps://gi hub.com/nylande /ca as a2phyml). Num-
be s o a iable and pa simony in o ma i e si es o he nucleo ide we e calcu-
la ed using MEGA . 11.0.13. Pa i ioned Maximum Likelihood (ML) analyses
wi h he majo i y- ule consensus ee we e pe o med in IQ-TREE . 1.6.12
(Minh e al. 2013) using Ul a as as boo s ap app oach (Minh e al. 2013)
wi h 10,000 ei e a ions. Bayesian in e ence (BI) was conduc ed in M Bayes
. 3.2.6 (Ronquis e al. 2012), wi h ou independen uns, each o which was
pe o med o 1,000,000 gene a ions and sampled e e y 1000 gene a ions
wi h he i s 25% samples disca ded as bu n-in. Con e gence o he Ma -
ko chain Mon e Ca lo simula ions was assessed o ensu e ha he a e age
s anda d de ia ion o spli equencies was < 0.01 and he po en ial scale e-
duc ion ac o s (PSRFs) we e ~1. Addi ionally, T ace . 1.7.2 (Rambau e al.
2018) was used o e i y ha all e ec i e sample size (ESS) alues exceeded
200. The mos app op ia e model o sequence e olu ion (ML: K2P+G4 o
ITS2 and K3Pu+F+G4 o 16S; BI: K2P+G4 o ITS2 and HKY+F+G4 o 16S)
was selec ed using ModelFinde (Kalyaanamoo hy e al. 2017).
Resul s
Mi ochond ial genome composi ion and compa ison
The mi ochond ial genomes (mi ogenome) o A. subaqua ilis (GenBank ac-
cession numbe PV749633) and A. c . b azie i (GenBank accession num-
be PP100270) a e 13,768 and 13,757 bp, espec i ely (Fig. 1A, B). Bo h
genomes con ain 37 genes: 13 p o ein-coding genes (PCGs), 2 ibosomal
RNA genes ( RNAs), and 22 ans e RNA genes ( RNAs) (Fig. 1A, B, Table
2). Eigh (nad4L, cy b, cox1, cox2, a p8, a p6, nad3, nad2) o he PCGs e -
mina e wi h a unca ed s op codon, which is assumed o be comple ed as
46
ZooKeys 1255: 41–62 (2025), DOI: 10.3897/zookeys.1255.164109
Zhe-Yu Chen e al.: Molecula and mo phological compa ison o wo Aus opeplea spp.
TAA by he addi ion o 3’ A esidues o he mRNA du ing ansc ip ion. The
a angemen o genes wi hin bo h mi ogenomes a e iden ical wi h only mi-
no di e ences in in e ed leng hs o non-coding RNA ( RNA-D, F, W, C, G,
H, Q, L2, M, T and K) and 12S genes (Table 2). The mi ogenome nucleo ide
composi ion o A. subaqua ilis is 32.8% A, 40.7% T, 12.4% C and 14.1% G,
esul ing in an AT con en o 73.4%, which is nea ly same as he 73.3% ob-
se ed in A. c . b azie i (wi h 32.8% A, 40.5% T, 12.5% C and 14.2% G). The
AT/GC skew alues o A. subaqua ilis and A. c . b azie i a e -0.1072/0.0624
and -0.1051/0.0612, espec i ely. The o e all plo s o GC con en in A. sub-
aqua ilis closely esembles hose obse ed in A. c . b azie i, wi h no subs an-
ial di e ences in con en o dis ibu ion pa e ns ac oss he mi ogenomes
(Fig. 1A, B). Bo h species exhibi highe GC con en in he cox3 and cox1
egions and educed GC con en in nad2, nad6, and 16S RNA. The o e all
mi ochond ial genome p-dis ance be ween A. subaqua ilis and A. c . b azie i
is 1.9%. The p-dis ance o each o he PCGs ange om 3.1% (nad5) o 0.6%
(16S) (Fig. 2A, Table 2).
Figu e 1. Re e ence mi ochond ial genome and nuclea RNA gene clus e o subaqua ilis (A, C) and Aus opeplea c .
b azie i (B, D). The di ec ion o gene ansc ip ion is shown wi h an a ow. In each panel GC con en is displayed ia he
obse ed skew pa e ns.
47
ZooKeys 1255: 41–62 (2025), DOI: 10.3897/zookeys.1255.164109
Zhe-Yu Chen e al.: Molecula and mo phological compa ison o wo Aus opeplea spp.
Nuclea RNA gene clus e composi ion and compa ison
The comple ed nuclea RNA gene clus e o A. subaqua ilis (GenBank acces-
sion no. PV593739) and A. c . b azie i (GenBank accession no. PV593740) span
6,712 bp and 6,747 bp espec i ely (Fig. 1C, D). The wo ibosomal DNA sequenc-
es exhibi simila o e all s uc u es, comp ising he 18S RNA, 5.8S RNA, 28S
RNA genes and wo in e nal ansc ibed space egions (ITS1 and ITS2) (Fig.
1C, D, Table 2). Bo h sequences sha e iden ical leng hs o he 5.8S RNA (158
bp). The 18S RNA and 28S RNA egions measu e 1,865 bp and 3,815 bp in A.
subaqua ilis, and 1,866 bp and 3,832 in A. c . b azie i, espec i ely. No able di e -
ences a e obse ed in he ITS egions: A. subaqua ilis con ained a 509 bp ITS1
and a 368 bp ITS2, whe eas A. c . b azie i possesses a 504 bp ITS1 and a 388
bp ITS2 (Table 2). The nuclea RNA gene clus e o A. subaqua ilis and A. c .
b azie i show simila nucleo ide composi ions: 22.8% A, 21.8% T, 25.7% C, and
29.7% G in A. subaqua ilis; 22.5% A, 22.0% T, 25.7% C, and 29.8% G in A. c . b a-
zie i. Bo h sequences ha e consis en GC con en (55.4% and 55.5%, espec i e-
ly) and simila GC skew alues, while A. subaqua ilis shows sligh ly highe AT
skew han A. c . b azie i (AT/GC skew: 0.0227/0.0715 and 0.0123/0.0737). The
GC con en plo s o he nuclea RNA gene clus e in Aus opeplea subaqua ilis
and A. c . b azie i a e simila (Fig. 1C, D). In bo h species, he GC con en plo s
exhibi se e al local maxima in GC p opo ion wi hin he cen al egion o he
28S RNA gene, while p onounced dec eases in GC p opo ion we e obse ed a
he bounda ies o he in e nal ansc ibed space s (ITS1 and ITS2), pa icula ly
a hei junc ions wi h he adjacen 5.8S and 28S RNA genes. The o e all nucle-
a RNA gene clus e p-dis ance be ween A. subaqua ilis and A. c . b azie i was
1.9%. While he RNA genes a e la gely iden ical (99%–100%), he ITS egions
exhibi subs an ial sequence di e gence, wi h he p-dis ance o ITS1 and ITS2
egions being 13.8% and 8.2%, espec i ely (Fig. 2B, Table 2).
Figu e 2. Sliding window analysis o he pai wise di e ences in he nucleo ide iden i y o Aus opeplea subaqua ilis and
Aus opeplea c . b azie i mi ochond ial genomes (A) and nuclea RNA gene clus e (B). Gene bounda ies a e indica ed
by e ical do ed lines. The ho izon al do ed line indica es he a e age nucleo ide di e si y be ween he wo sequences.
48
ZooKeys 1255: 41–62 (2025), DOI: 10.3897/zookeys.1255.164109
Zhe-Yu Chen e al.: Molecula and mo phological compa ison o wo Aus opeplea spp.
Table 2. Loca ion, leng hs, and di ec ions o anno a ed genes wi hin he mi ochond ial and nuclea RNA gene clus e s o
Aus opeplea subaqua ilis, wi h compa isons o Aus opeplea c . b azie i ( alues a e slash). Nucleo ide pai wise iden i y
o each gene be ween he wo species is shown.
Gene designa ions Loca ion s a Loca ion end Leng h (bp) Di ec ion Pai wise Iden i y p-dis ance
Mi ochond ial genes
16S 1/1 986/986 986/986 o wa d 99.4% 0.6%
RNA-L1( ag) 988/988 1051/1051 64/64 o wa d N/A N/A
RNA-P( gg) 1047/1047 1106/1106 60/60 o wa d N/A N/A
RNA-A( gc) 1107/1107 1170/1170 64/64 o wa d N/A N/A
nad6 1171/1171 1629/1629 459/459 o wa d 97.6% 2.4%
nad51631/1631 3277/3277 1647/1647 o wa d 96.9% 3.1%
nad1 3279/3279 4154/4154 876/876 o wa d 98.3% 1.7%
nad4L 4155/4155 4452/4452 298/298 o wa d 98.3% 1.7%
cy b 4453/4453 5533/5533 1081/1081 o wa d 98.3% 1.7%
RNA-D(g c) 5536/5536 5588/5587 53/52 o wa d N/A N/A
RNA-F(gaa) 5589/5588 5651/5650 63/63 o wa d N/A N/A
cox25652/5651 6294/6293 643/643 o wa d 98.3% 1.7%
RNA-Y(g a) 6297/6296 6346/6345 50/50 o wa d N/A N/A
RNA-W( ca) 6347/6346 6405/6405 59/60 o wa d N/A N/A
RNA-C(gca) 6410/6410 6468/6468 59/59 o wa d N/A N/A
RNA-G( cc) 6471/6470 6524/6522 54/53 o wa d N/A N/A
RNA-H(g g) 6527/6525 6583/6582 57/58 o wa d N/A N/A
RNA-Q( g) 6592/6591 6650/6649 59/59 e e se N/A N/A
RNA-L2( aa) 6651/6650 6703/6701 53/52 e e se N/A N/A
a p86705/6703 6855/6853 151/151 e e se 98.2% 1.8%
RNA-N(g ) 6857/6854 6920/6917 64/64 e e se N/A N/A
a p6 6921/6918 7560/7557 640/640 e e se 98.6% 1.4%
RNA-R( cg) 7561/7558 7623/7620 63/63 e e se N/A N/A
RNA-E(gaa) 7624/7621 7675/7672 52/52 e e se N/A N/A
12S 7676/7673 8393/8388 718/716 e e se 98.2% 1.8%
RNA-M(ca ) 8394/8389 8466/8457 73/69 e e se N/A N/A
nad38467/8458 8806/8797 340/340 e e se 99.1% 0.9%
RNA-S2( ga) 8817/8808 8871/8862 55/55 e e se N/A N/A
RNA-S1(gc ) 8872/8863 8926/8917 55/55 e e se N/A N/A
nad4 8927/8918 10252/10243 1326/1326 o wa d 98% 2%
RNA-T( g ) 10253/10244 10319/10311 67/68 e e se N/A N/A
cox3 10321/10313 11100/11092 780/780 e e se 98.8% 1.2%
RNA-I(ga ) 11141/11133 11205/11197 65/65 o wa d N/A N/A
nad2 11206/11198 12109/12101 904/904 o wa d 97.8% 2.2%
RNA-K( ) 12110/12102 12191/12180 82/79 o wa d N/A N/A
cox1 12203/12192 13694/13683 1492/1492 o wa d 97.7% 2.3%
RNA-V( ac) 13695/13684 13755/13744 61/61 o wa d N/A N/A
Nuclea RNA gene clus e
18S 1/1 1865/1865 1865/1865 o wa d 100% 0%
ITS1 1866/1866 2374/2369 509/504 o wa d 86.2% 13.8%
5.8S 2375/2370 2532/2527 158/158 o wa d 100% 0%
ITS2 2533/2528 2900/2915 368/388 o wa d 91.8% 8.2%
28S 2901/2916 6731/6747 3831/3832 o wa d 99.3% 0.7%
49
ZooKeys 1255: 41–62 (2025), DOI: 10.3897/zookeys.1255.164109
Zhe-Yu Chen e al.: Molecula and mo phological compa ison o wo Aus opeplea spp.
Phylogene ic analysis
Phylogene ic ees we e cons uc ed om a da ase consis ing o 29 16S +
ITS2 sequences o Aus opeplea, 27 o hem we e ob ained om p e ious s udy
(Puslednik e al. 2009), wo we e gene a ed o his s udy and one a ailable ou -
g oup axon (Liu e al. 2012; Suwancha oen e al. 2023). The aligned leng hs o
16S and ITS2 genes we e 433 and 452 nucleo ides, espec i ely. Wi hin hese
sequences, 79 and 87 si es we e a iable, while 69 and 46 si es we e pa si-
mony in o ma i e. The Bayesian-in e ed (BI) and maximum likelihood (ML)
phylogene ic ees o Aus opeplea we e no ully esol ed. Ne e heless, bo h
Bayesian pos e io p obabili ies (BPP) and ML boo s ap (BS) alues suppo ed
sepa a ion be ween Aus alian and New Zealand species. Aus opeplea hispida
was eco e ed as sis e o he emaining Aus alian species, which oge he
o med a icho omy in he in e ed opology (Fig. 3). The sequences gene a ed
he e o Aus opeplea subaqua ilis and A. c . b azie i g oup wi h he sou he n
Aus alian and eas e n Aus alian samples om p e ious s udy, espec i ely.
Taxonomic accoun
Aus opeplea subaqua ilis (Ta e, 1880)
Figs 4A, C, E, 5A, C, 6A, C, 7A
Ma e ial examined. Specimens om “D ain M” nea P inces Highway in Tho n-
lea, Sou h Aus alia, Aus alia, and hei a i icially b ed o sp ing.
Desc ip ion. Shell (Fig. 4A) medium in size (up o 12.5 mm in heigh ), o a e,
wi h low, na ow conical spi e and s ongly in la ed las who l. Shell wall hin,
agile in some specimens. Who ls (4.0–4.5 in numbe ) ounded, sligh ly con-
ex, sepa a ed by a shallow, sligh ly oblique o nea ly s aigh su u e. Las who l
comp ises ~0.9 o shell heigh . Shell su ace smoo h, somewha shiny, ligh
b own o nea ly colou less, co e ed by collab al g ow h lines. Ape u e py i-
o m, wi h e enly ounded basal and pala al ma gins, pos e io co ne o ming
angle wi h las who l. Pe is ome sha p, no expanded bu columella lip e lexed
and a ached o back o las who l. Pa ie al callus hin bu dis inc , ex ending
o las who l a beyond inne lip. Columella old weakly de eloped. Umbilicus
co e ed by inne lip, closed o e y na ow (slo -like).
Head- oo (Fig. 4C, E) ypical o amily. Foo b oad, eaching 1.5–2× shell
heigh when ully ex ended, ligh g ey wi h spa se whi e eckles (obse ed
when li ing). When s imula ed, conside able quan i ies o mucus p oduced and
co e s en i e body. Ten acles shield-shaped (Fig. 4E), wice as long as wide
(obse ed when li ing). Man le ligh g ey wi h la ge black blo ches on pallial
oo . Man le colla (Fig. 4E) e lexed and a ached o shell, ex ended as hin
lap on bo h sides o enclose shell ully o la gely in ma u e indi iduals. Closed
edge o man le colla si ua ed along midline o animal, o ming ma ginal old
nea shell apex. Man le co e ing isce al coil wi h band-like black pigmen in
ma u e indi iduals (Fig. 4C); disconnec ed om pigmen a ion on pallial oo ,
and eadily los in p ese ed specimens.
Cen al ne ous sys em ypical o amily (Fig. 5A). Ce eb al ganglia wi h eg-
ula bo de s, pale yellow ( esh). Commissu al lobule dis inc , whi e, app oxi-
ma ely equal o ce eb al ganglia in size.
56
ZooKeys 1255: 41–62 (2025), DOI: 10.3897/zookeys.1255.164109
Zhe-Yu Chen e al.: Molecula and mo phological compa ison o wo Aus opeplea spp.
The man le pigmen on he isce al coil o ma u e indi iduals o A. subaqua i-
lis does no occu in A. c . b azie i. The in e nal pigmen a ion o A. subaqua ilis
is gene ally ligh e han ha o A. c . b azie i. The di e ence in he ela i e size
o he bulbous e mina ion o he p aepu ium is a consis en dis inc ion in he
male ep oduc i e sys em o he wo species, as a e he shape o he spe ma-
heca and he wid h o he spe ma hecal duc in he emale sys em. Apa om
he la ge commissu al lobule in A. c . b azie i, ma u e A. subaqua ilis o med a
dis inc ly dema ca ed and ellipsoid lobe om each ce eb al ganglion si ua ed
opposi e he commissu al lobule (namely adjacen o he buccal mass).
Discussion
This s udy used an in eg a i e app oach, combining mo phological da a, mi og-
enome and nuclea RNA gene clus e compa isons and phylogene ic analyses
o assess he ela ionship be ween A. subaqua ilis and A. c . b azie i. Based on
mo phological ea u es, hese wo snail axa could be eadily dis inguished.
The comple e mi ochond ial and nuclea RNA gene clus e s sequenced and
compa ed he ein a e some o he i s such da a se s o he amily Lymnaeidae
and a e among he e y ew a ailable o he o de Hyg ophila (McQui k e al.
2025). Analysis o mi ochond ial and nuclea ma ke s showed no signi ican
di e ence in gene o de o s uc u e, al hough su icien phylogene ically in o -
ma i e si es we e epo ed o sugges ha hei di e gence co esponded o
species-le el di e en ia ion, and hus suppo ed ou mo phological indings.
The pa e ns o nucleo ide di e gence in mi ochond ial genes and he nuclea
RNA clus e s o A. subaqua ilis and A. c . b azie i a e dis inc . Mos mi ochon-
d ial genes exhibi low pai wise nucleo ide di e gence be ween he wo species.
Fo ins ance, he nucleo ide p-dis ance o he cox1 gene is 2.3%, a alue ha was
a he h eshold be ween in a- and in e speci ic a ia ion in o he lymnaeid ax-
onomic s udies (Bolo o e al. 2014; Vina ski e al. 2016, 2022; Akseno a e al.
2017, 2024; Lounnas e al. 2018; Fe ei a e al. 2021; Falniowski e al. 2023), and
is compa able o he minimal gene ic di e gence (p-dis ance = 2.4%) obse ed be-
ween Ladisla ella um okensis and L. elodes, and be ween Galba cubensis and G.
neo opica (see Vina ski e al. 2016; Fe ei a e al. 2021). Mo eo e , he mi ochon-
d ial 16S RNA gene shows he lowes p-dis ance (0.6%) among all mi ochond ial
genes be ween he wo species, u he suppo ing p e ious indings ha mi o-
chond ial 16S RNA alone lacked su icien phylogene ic esolu ion o delimi Aus-
alian Aus opeplea species (Puslednik e al. 2009; Sukee e al. 2024). In con as ,
al hough he sequences o he h ee nuclea RNA genes (18S, 5.8S, 28S) o he wo
species a e nea ly iden ical, he obse ed di e ences in ITS egions (p-dis ance
= 13.8% o ITS1; p-dis ance = 8.2% o ITS2) all wi hin he ange o in e speci ic
di e gence commonly used in cu en lymnaeid axonomic p ac ices (see Vina ski
e al. 2016; Fe ei a e al. 2021; Akseno a e al. 2024). The highe di e gence in ITS
egions compa ed wi h mi ochond ial genes may sugges he p esence o in o-
g ession and incomple e lineage so ing wi hin Aus alian Aus opeplea (see Da is
and Nixon 1992; Doyle 1992; Ha ison and La son 2014). Labo a o y expe imen s
showed ha ma ing be ween species o Aus opeplea was possible (Bo ay 1964b),
al hough he e ili y o hyb id o sp ing had no been demons a ed.
Despi e he limi a ions o using 16S gene and ITS2 ma ke s alone o his
g oup, we p oceeded o use only hese egions o make a phylogene ic compa i-
57
ZooKeys 1255: 41–62 (2025), DOI: 10.3897/zookeys.1255.164109
Zhe-Yu Chen e al.: Molecula and mo phological compa ison o wo Aus opeplea spp.
son wi h da a a ailable o Aus opeplea. The opology o he 16S + ITS2 phyloge-
ne ic ees in his s udy and ha o Puslednik e al. (2009) we e la gely consis en .
The newly sequenced A. subaqua ilis and A. c . b azie i we e placed in wo
geog aphically s uc u ed lineages wi hin Aus opeplea (sou he n e sus eas -
e n Aus alia), consis en wi h egional s uc u ing a he han cons i u ing de-
ini i e e idence o species le el di e gence. Ne e heless, he limi a ions o he
cu en ma ke s we e e iden , as he main Aus alian Aus opeplea lineage and
se e al wi hin g oup ela ionships showed poly omies, indica ing un esol ed
ela ionships. These un esol ed nodes may ha e e lec ed limi ed phylogene ic
signal, apid adia ion, o incomple e lineage so ing, and hus wa an u he
in es iga ion using addi ional gene ic ma ke s. Fu u e s udies employing com-
ple e mi ochond ial genome sequences and nuclea RNA gene clus e s a e
wa an ed o add ess he un esol ed ela ionships wi hin he genus.
The mo phological ea u es o A. subaqua ilis and A. c . b azie i gene ally co -
esponded o he ype B and ype A mo phs, espec i ely, as desc ibed by Bo ay
and McMichael (1961). Mo phologically, he di e ences be ween he wo spe-
cies we e p ima ily ocused on he shell, man le ex ension, ne ous sys em, and
he ep oduc i e sys em. The di e ences in he ep oduc i e sys em be ween
A. subaqua ilis and A. c . b azie i we e dis inc . The mo phological di e ence in
he bulbous e mina ion o he p aepu ium may sugges ed unde lying in e nal
s uc u al di e ences, which wa an u he in es iga ion in u u e s udies. The
ne ous sys ems o A. subaqua ilis and A. c . b azie i di e ed, pa icula ly in
he size o he commissu al lobule and he p esence o addi ional lobes a ising
om he ce eb al ganglia in A. subaqua ilis. These di e ences we e no epo -
ed in p e ious s udies. The lack o compa a i e neu oana omical s udies on
his g oup cu en ly limi ed ou abili y o assess whe he hese ea u es we e
s uc u ally signi ican o how hey ela ed o o he membe s o he Lymnaei-
dae. Fu he esea ch is needed o cla i y hei na u e and axonomic ele ance.
The wo unique ea u es men ioned by Puslednik e al. (2009) o he Sou h
Aus alian samples (A. subaqua ilis in his s udy), namely he longe cephalic
en acles ( wice as long as wide) and he p os a e ( ube) being much longe
han he emale ep oduc i e sys em, we e also obse ed in his s udy. Howe -
e , he ecogni ion o hese ai s is la gely depended on how much con ac ion
had occu ed due o p ese a ion, as hese ea u es we e less p onounced in
con ac ed ma e ial compa ed o he o he dis inguishing ea u es no ed abo e.
The ex e nal ea u es o he head- oo and man le o A. subaqua ilis includ-
ed a b oad oo wi h he shell en eloped by he man le. In i s na u al habi a ,
A. subaqua ilis was obse ed li ing in ex emely dense wa e mil oil (My iophyl-
lum spp.) en i onmen s, which p esumably necessi a ed mo ing be ween igh ly
packed b anches. The educ ion o shell- ela ed hind ance may ha e enhanced
he animal’s abili y o su i e in such en i onmen s. The abno mally high mucus
p oduc ion in A. subaqua ilis when s imula ed may ha e se ed as an al e na i e
de ence mechanism agains p eda o s, pe haps compensa ing o he agile
shell. This ai was commonly obse ed in land slugs and semi-slugs (Luch el
and Dey up-Olsen 2001). The man le ex ension may also ha e unc ioned o en-
hance cu aneous espi a ion, he eby educing he equency o su acing o ai
o adap ing o hypoxic condi ions caused by in ensi ied espi a ion o aqua ic
plan s a nigh (Russell-Hun e 1978). The deg ee o man le ex ension has no
been well cha ac e ised ac oss species in his amily, wi h only he Eu ope-
58
ZooKeys 1255: 41–62 (2025), DOI: 10.3897/zookeys.1255.164109
Zhe-Yu Chen e al.: Molecula and mo phological compa ison o wo Aus opeplea spp.
an Myxas glu inosa (O. F. Mülle , 1774) explici ly documen ed as ha ing a ully
shell-en eloping man le, simila o wha was obse ed in A. subaqua ilis (Huben-
dick 1951; S adnichenko 2004). Howe e , based on he phylogene ic ela ion-
ships o Aus opeplea om Tasmania and Sou h Aus alia econs uc ed in his
s udy and p e ious esea ch (Puslednik e al. 2009), i appea ed ha he de el-
opmen o man le ex ensions in Aus opeplea was no an isola ed occu ence.
Bo ay and McMichael (1961) no ed ha man le ex ension in hei ype B mo phs
ended o diminish o disappea o e successi e labo a o y gene a ions. This ob-
se a ion closely esembled he dwa o ms we obse ed. Howe e , hese dwa
indi iduals egained man le ex ension once placed in a ou able en i onmen al
condi ions, in con as o A. c . b azie i, in which his ai appea ed o be pe ma-
nen ly absen . Fu he s udies a e equi ed o cla i y he mechanism o man le
ex ension and o de e mine i s biological o ecological ele ance.
Taken oge he , he in eg a i e e idence p esen ed he ein suppo ed spe-
cies-le el di e gence be ween A. subaqua ilis and A. c . b azie i. While mi o-
chond ial di e gence alone app oached he h eshold o in e speci ic sepa a-
ion, he p onounced di e ences in ITS egions, along wi h he mo phological
ai s, p o ided a cohe en amewo k o species delimi a ion. Ne e heless,
he phylogeny o his g oup emains poo ly esol ed. Con inued e o s in ol -
ing addi ional phylogene ically in o ma i e gene ic da ase s and compa a i e
ana omical s udies will be essen ial o esol ing ou s anding axonomic un-
ce ain ies and unde s anding he e olu iona y dynamics o his g oup.
Acknowledgemen s
We a e g a e ul o Ms Ch is ine Ande sen, P o . Ian Be e idge, and M Kasem
D i e o hei in aluable suppo du ing he ieldwo k.
Addi ional in o ma ion
Con lic o in e es
The au ho s ha e decla ed ha no compe ing in e es s exis .
E hical s a emen
No e hical s a emen was epo ed.
Use o AI
No use o AI was epo ed.
Funding
This s udy was suppo ed by he Aus alian Resea ch Council Disco e y P ojec no.
DP230100270.
Au ho con ibu ions
All au ho s ha e con ibu ed equally.
Au ho ORCIDs
Zhe-Yu Chen h ps://o cid.o g/0000-0002-4150-8906
Tanapan Sukee h ps://o cid.o g/0000-0003-3181-5045
59
ZooKeys 1255: 41–62 (2025), DOI: 10.3897/zookeys.1255.164109
Zhe-Yu Chen e al.: Molecula and mo phological compa ison o wo Aus opeplea spp.
Anson V. Koehle h ps://o cid.o g/0000-0001-8330-6416
Bonnie L. Webs e h ps://o cid.o g/0000-0003-0930-9314
Robin B. Gasse h ps://o cid.o g/0000-0002-4423-1690
Wins on F. Ponde h ps://o cid.o g/0000-0002-8600-3952
Neil D. Young h ps://o cid.o g/0000-0001-8756-229X
Da a a ailabili y
All o he da a ha suppo he indings o his s udy a e a ailable in he main ex o
Supplemen a y In o ma ion.
Re e ences
Akseno a O, Vina ski M, Bolo o I, Kondako A, Bespalaya Y, Tomilo a A, Pal se I, Go-
a o M (2017) Two Radix spp. (Gas opoda: Lymnaeidae) endemic o he mal sp ings
a ound Lake Baikal ep esen eco ypes o he widesp ead Radix au icula ia. Jou nal
o Zoological Sys ema ics and E olu iona y Resea ch 55(4): 298–309. h ps://doi.
o g/10.1111/jzs.12174
Akseno a OV, Vina ski MV, I agaki T, Oha i Y, Oshida T, Kim SK, Lee JH, Kondako AV, Kh eb o-
a IS, Sobole a AA, T a ina OV, Sokolo a SE, Pala o DM, Bespalaya YV, Vikh e IV, Go-
a o MY, Bolo o IN (2024) Taxonomy and ans-Be ingian biogeog aphy o he pond
snails (Gas opoda: Lymnaeidae) o Eas Asia: an in eg a i e iew. Zoological Jou nal o
he Linnean Socie y 201(4): zlae083. h ps://doi.o g/10.1093/zoolinnean/zlae083
Be n M, Dona h A, Jühling F, Ex e nb ink F, Flo en z C, F i zsch G, Pü z J, Middendo
M, S adle PF (2013) MITOS: Imp o ed de no o me azoan mi ochond ial genome
anno a ion. Molecula Phylogene ics and E olu ion 69(2): 313–319. h ps://doi.
o g/10.1016/j.ympe .2012.08.023
Bolo o I, Bespalaya Y, Akseno a O, Akseno A, Bolo o N, Go a o M, Kondako A, Pal s-
e I, Vikh e I (2014) A axonomic e ision o wo local endemic Radix spp. (Gas op-
oda: Lymnaeidae) om Khodu ka geo he mal a ea, Kamcha ka, Russian Fa Eas .
Zoo axa 3869(5): 585–593. h ps://doi.o g/10.11646/zoo axa.3869.5.9
Bo ay JC (1964a) S udies on he ecology o Lymnaea omen osa, he in e media e hos
o Fasciola hepa ica. 1. His o y, geog aphical dis ibu ion, and en i onmen . Aus a-
lian Jou nal o Zoology 12(2): 217–230. h ps://doi.o g/10.1071/ZO9640217
Bo ay JC (1964b) S udies on he ecology o Lymnaea omen osa, he in e media e hos
o Fasciola hepa ica. 2. The sexual beha iou o Lymnaea omen osa. Aus alian Jou -
nal o Zoology 12(2): 231–238. h ps://doi.o g/10.1071/ZO9640231
Bo ay JC (1969) Expe imen al Fascioliasis in Aus alia. In: Dawes B (Ed.) Ad ances in Pa -
asi ology. Academic P ess, 95–210. h ps://doi.o g/10.1016/S0065-308X(08)60435-2
Bo ay JC (1978) The po en ial impac o exo ic Lymnaea spp. on ascioliasis in Aus-
alasia. Ve e ina y Pa asi ology 4(2): 127–141. h ps://doi.o g/10.1016/0304-
4017(78)90004-3
Bo ay JC, McMichael DF (1961) The iden i y o he Aus alian lymnaeid snail hos o Fas-
ciola hepa ica L. and i s esponse o en i onmen . Ma ine and F eshwa e Resea ch
12(2): 150–163. h ps://doi.o g/10.1071/MF9610150
Co on BC (1942) Some Aus alian eshwa e Gas opoda. T ansac ions o he Royal
Socie y o Sou h Aus alia 66: 75–82.
Co on BC, God ey FK (1938) A sys ema ic lis o he Gas opoda, he ma ine eshwa-
e and land uni al e Mollusca o Sou h and Cen al Aus alia. Malacological Socie y
o Sou h Aus alia Publica ion No. 1, 44 pp.
60
ZooKeys 1255: 41–62 (2025), DOI: 10.3897/zookeys.1255.164109
Zhe-Yu Chen e al.: Molecula and mo phological compa ison o wo Aus opeplea spp.
Da is JI, Nixon KC (1992) Popula ions, gene ic a ia ion, and he delimi a ion o phylogene -
ic species. Sys ema ic Biology 41(4): 421–435. h ps://doi.o g/10.1093/sysbio/41.4.421
Doyle JJ (1992) Gene ees and species ees: Molecula sys ema ics as one-cha ac e
axonomy. Sys ema ic Bo any 17(1): 144–163. h ps://doi.o g/10.2307/2419070
Falniowski A, Jaszczyńska A, Ho man S (2023) Radix u escens (J. E. G ay, 1822) (Gas-
opoda: Lymnaeidae), a new species o Oman and A abian Peninsula. Ac a Zoolog-
ica Academiae Scien ia um Hunga icae 69(3): 303–312. h ps://doi.o g/10.17109/
AZH.69.3.303.2023
Fe ei a APPN, Cos a ALO, Beca ini RM, Fe ei a MAND, da Paixão HPR, Cosca elli D, Vi-
digal THDA, Lima W dos S, Pe ei a CA de J (2021) In eg a i e axonomy: Combining
molecula and mo phological cha ac e is ics o iden i y Lymnaea (Galba) cubensis, in-
e media e hos o Fasciola hepa ica. Re is a B asilei a de Pa asi ologia Ve e iná ia =
B azilian Jou nal o Ve e ina y Pa asi ology : Ó gão O icial do Colégio B asilei o de Pa -
asi ologia Ve e iná ia 30(2): e026320. h ps://doi.o g/10.1590/s1984-29612021052
Fou d ilis S, de F ias Ma ins AM, Backeljau T (2018) Rela ion be ween mi ochond i-
al DNA hype di e si y, mu a ion a e and mi ochond ial genome e olu ion in Mela -
haphe ne i oides (Gas opoda: Li o inidae) and o he Caenogas opoda. Scien i ic
Repo s 8(1): 17964. h ps://doi.o g/10.1038/s41598-018-36428-7
Ghiselli F, Gomes-dos-San os A, Adema CM, Lopes-Lima M, Sha b ough J, Boo e JL
(2021) Molluscan mi ochond ial genomes b eak he ules. Philosophical T ans-
ac ions o he Royal Socie y o London. Se ies B, Biological Sciences 376(1825):
20200159. h ps://doi.o g/10.1098/ s b.2020.0159
G an JR, Enns E, Ma inie E, Mandal A, He man EK, Chen C, G aham M, Van Domselaa G, S o-
ha d P (2023) P oksee: In-dep h cha ac e iza ion and isualiza ion o bac e ial genomes.
Nucleic Acids Resea ch 51(W1): W484–W492. h ps://doi.o g/10.1093/na /gkad326
Ha ison RG, La son EL (2014) Hyb idiza ion, in og ession, and he na u e o species bound-
a ies. The Jou nal o He edi y 105: 795–809. h ps://doi.o g/10.1093/jhe ed/esu033
Hubendick B (1951) Recen Lymnaeidae: Thei a ia ion, mo phology, axonomy, no-
mencla u e and dis ibu ion. Kungliga S enska Ve enskapsakademiens Handlinga .
Fjä de Se ien 3: 1–223.
I edale T (1943) A basic lis o he eshwa e Mollusca o Aus alia. Aus alian Zoologis
10(2): 188–230.
I edale T (1944) Guide o he eshwa e shells o New Sou h Wales. P II. Aus alian
Na u alis 11: 113–127.
Kalyaanamoo hy S, Minh BQ, Wong TKF, on Haesele A, Je miin LS (2017) ModelFind-
e : Fas model selec ion o accu a e phylogene ic es ima es. Na u e Me hods 14(6):
587–589. h ps://doi.o g/10.1038/nme h.4285
Liu G-H, Wang S-Y, Huang W-Y, Zhao G-H, Wei S-J, Song H-Q, Xu M-J, Lin R-Q, Zhou D-H,
Zhu X-Q (2012) The comple e mi ochond ial genome o Galba pe ia (Gas opo-
da: Mollusca), an in e media e hos snail o Fasciola spp. PLoS ONE 7(7): e42172.
h ps://doi.o g/10.1371/jou nal.pone.0042172
Lounnas M, Co ea AC, Alda P, Da id P, Dubois M-P, Cal opiña M, Ca on Y, Celi-E azo M,
Dung BT, Ja ne P, Loke ES, Noya O, Rod íguez-Hidalgo R, To y C, U ibe N, Poin ie
J-P, Hu ez-Boussès S (2018) Popula ion s uc u e and gene ic di e si y in he in a-
si e eshwa e snail Galba schi azensis (Lymnaeidae). Canadian Jou nal o Zoology
96(5): 425–435. h ps://doi.o g/10.1139/cjz-2016-0319
Luch el DL, Dey up-Olsen I (2001) Body wall: o m and unc ion. In: Ba ke GM (Ed.)
The biology o e es ial molluscs. CABI Publishing, Oxon, 147–178. h ps://doi.
o g/10.1079/9780851993188.0147
61
ZooKeys 1255: 41–62 (2025), DOI: 10.3897/zookeys.1255.164109
Zhe-Yu Chen e al.: Molecula and mo phological compa ison o wo Aus opeplea spp.
McQui k KA, DeCo e JM, Cas illo MG, Adema CM (2025) Rewilding shows di e en ial i -
ness o sympa ic Physella acu a (D apa naud, 1805) snail lineages. Aqua ic Ecology
59(2): 435–454. h ps://doi.o g/10.1007/s10452-025-10172-3
Minh BQ, Nguyen MAT, on Haesele A (2013) Ul a as app oxima ion o phyloge-
ne ic boo s ap. Molecula Biology and E olu ion 30(5): 1188–1195. h ps://doi.
o g/10.1093/molbe /ms 024
Mülle OF (1774) Ve mium e es ium e lu ia ilium, seu animalium in uso ium, Hel-
min hico um, e es aceo um, non ma ino um, succinc a his o ia. ol 2. Ha niae e
Lipsiae, apud Heineck e Fabe , ex o icina Mölle iana. I-XXXVI, 1–214 pp. [10 unnum-
be ed pages] h ps://doi.o g/10.5962/bhl. i le.46299
P ei e B, Wi elsbü ge U, Ramos-Onsins SE, Le che MJ (2014) PopGenome: An e i-
cien Swiss A my kni e o popula ion genomic analyses in R. Molecula Biology and
E olu ion 31(7): 1929–1936. h ps://doi.o g/10.1093/molbe /msu136
P ei e L (1855) Desc ip ions o i y-se en new species o Helicea, om M Cun-
ning’s collec ion. P oceedings o he Zoological Socie y o London 1854: 286–298.
h ps://doi.o g/10.1111/j.1469-7998.1854. b07277.x
Ponde WF, Wa e house JH (1997) A new genus and species o Lymnaeidae om he
lowe F anklin Ri e , sou h wes e n Tasmania, Aus alia. The Jou nal o Molluscan
S udies 63(3): 441–468. h ps://doi.o g/10.1093/mollus/63.3.441
Ponde WF, Hallan A, Shea ME, Cla k SA, Richa ds K, Klunzinge MW, Kessne V (2024)
Aus alian F eshwa e Molluscs. Re ision 2A. h ps://keys.lucidcen al.o g/keys/ 3/
eshwa e _molluscs/
Puslednik L, Ponde WF, Dow on M, Da is AR (2009) Examining he phylogeny o he
Aus alasian Lymnaeidae (He e ob anchia: Pulmona a: Gas opoda) using mi ochon-
d ial, nuclea and mo phological ma ke s. Molecula Phylogene ics and E olu ion
52(3): 643–659. h ps://doi.o g/10.1016/j.ympe .2009.03.033
Ra inesque CS (1815) Analyse de la na u e ou Tableau de l’uni e s e des co ps o ganisés.
Published by he au ho , Pale mo, 224 pp. h ps://doi.o g/10.5962/bhl. i le.106607
Rambau A, D ummond AJ, Xie D, Baele G, Sucha d MA (2018) Pos e io Summa iza-
ion in Bayesian phylogene ics using T ace 1.7. Sys ema ic Biology 67(5): 901–904.
h ps://doi.o g/10.1093/sysbio/syy032
Ronquis F, Teslenko M, an de Ma k P, Ay es DL, Da ling A, Höhna S, La ge B, Liu L,
Sucha d MA, Huelsenbeck JP (2012) M Bayes 3.2: E icien Bayesian phylogene ic
in e ence and model choice ac oss a la ge model space. Sys ema ic Biology 61(3):
539–542. h ps://doi.o g/10.1093/sysbio/sys029
Russell-Hun e WD (1978) Ecology o eshwa e pulmona es. In: F e e V, Peake J (Eds)
Pulmona es, ol 2A. Academic P ess, London, 335–384.
Smi h EA (1882) On he eshwa e shells o Aus alia. Jou nal o he Linnean Socie y
o London, Zoology 16(92): 255–316. h ps://doi.o g/10.1111/j.1096-3642.1882.
b02283.x
S adnichenko AP (2004) Pond snails and limpe snails (Lymnaeidae and Ac oloxidae) o
Uk aine. Tsen uchebnoi li e a u y, Kie , 327 pp. [in Russian]
Sukee T, Koehle AV, Webs e BL, Gauci CG, Foga y CE, Ponde WF, Gasse RB, Young
ND (2024) Mi ochond ial genome o he luke pond snail, Aus opeplea c . b azie i
(Gas opoda: Lymnaeidae). Pa asi es & Vec o s 17(1): 283. h ps://doi.o g/10.1186/
s13071-024-06358-7
Suwancha oen C, Phuangs i C, Si iwech i iya P, Bunsong T, Japa O (2023) Di e si y o em-
a ode ce ca iae among na u ally in ec ed lymnaeid snails om Phayao, Thailand. Pa -
asi ology Resea ch 122(11): 2691–2708. h ps://doi.o g/10.1007/s00436-023-07971-8
62
ZooKeys 1255: 41–62 (2025), DOI: 10.3897/zookeys.1255.164109
Zhe-Yu Chen e al.: Molecula and mo phological compa ison o wo Aus opeplea spp.
Ta e R (1880) Desc ip ions o some new species o sou h Aus alian Pulmoni e a. T ans-
ac ions o he Royal Socie y o Sou h Aus alia 3: 102–104.
Tenison Woods JE (1876) On he eshwa e shells o Tasmania. Pape s and P oceedings
o he Royal Socie y o Tasmania 1875: 66–82. h ps://doi.o g/10.5962/bhl.pa .9781
Vázquez AA, Alba A, Alda P, Vi ecoq M, Chapuis E, Faugè e D, Poin ie J-P, Hu -
ez-Boussès S (2023) Lymnaeid snails and he ansmission o Fasciolosis: Unde -
s anding he di e en ial isks om local o global scale. In: Vina ski MV, Vázquez
AA (Eds) The Lymnaeidae: A Handbook on Thei Na u al His o y and Pa asi olog-
ical Signi icance. Sp inge In e na ional Publishing, Cham, 359–394. h ps://doi.
o g/10.1007/978-3-031-30292-3_13
Vina ski MV, Poin ie J-P (2023) Conchological and ana omical iden i ica ion o he lym-
naeid snails. In: Vina ski MV, Vázquez AA (Eds) The Lymnaeidae: A Handbook on
Thei Na u al His o y and Pa asi ological Signi icance. Sp inge In e na ional Publish-
ing, Cham, 103–120. h ps://doi.o g/10.1007/978-3-031-30292-3_4
Vina ski M, Akseno a O, Bespalaya Y, Bolo o I, Go a o M, Kondako A (2016) La-
disla ella um okensis: The i s molecula e idence o a Nea c ic clade o lymnaeid
snails inhabi ing Eu asia. Sys ema ics and Biodi e si y 14(3): 276–287. h ps://doi.o
g/10.1080/14772000.2016.1140244
Vina ski MV, Clewing C, Alb ech C (2019) Lymnaeidae Ra inesque, 1815. In: Lydea d
C, Cummings S (Eds) F eshwa e Mollusks o he Wo ld. Johns Hopkins Uni e si y
P ess, Bal imo e, 158–162.
Vina ski MV, Von Oheimb PV, Akseno a OV, Go a o MY, Kondako AV, Nekhae IO, Bolo o
IN (2022) T apped on he oo o he wo ld: axonomic di e si y and e olu iona y pa -
e ns o Tibe an Pla eau endemic eshwa e snails (Gas opoda: Lymnaeidae: Tibe o-
adix). In eg a i e Zoology 17(5): 825–848. h ps://doi.o g/10.1111/1749-4877.12600
Vina ski MV, Akseno a OV, Bolo o IN, Vázquez AA, Alda P, Poin ie J-P, Hu ez-Boussès S
(2023) Biogeog aphy o he li ing Lymnaeidae. In: Vina ski MV, Vázquez AA (Eds) The Lym-
naeidae: A Handbook on Thei Na u al His o y and Pa asi ological Signi icance. Sp inge
In e na ional Publishing, Cham, 183–206. h ps://doi.o g/10.1007/978-3-031-30292-3_7
Wang M, Kong L (2019) pbla : A mul i h ead bla algo i hm speeding up aligning se-
quences o genomes. BMC Bioin o ma ics 20(1): 28. h ps://doi.o g/10.1186/
s12859-019-2597-8
Wickham H (2016) ggplo 2. Elegan G aphics o Da a Analysis. Second Edi ion. Sp inge
In e na ional Publishing, Cham, 260 pp. h ps://doi.o g/10.1007/978-3-319-24277-4
Supplemen a y ma e ial 1
Loca ion, code, and NCBI GenBank accession numbe s o he lymnaeid
snail nucleo ide sequences used in his s udy
Au ho s: Zhe-Yu Chen, Tanapan Sukee, Anson V. Koehle , Bonnie L. Webs e , Robin B.
Gasse , Wins on F. Ponde , Neil D. Young
Da a ype: pd
Copy igh no ice: This da ase is made a ailable unde he Open Da abase License
(h p://openda acommons.o g/licenses/odbl/1.0/). The Open Da abase License
(ODbL) is a license ag eemen in ended o allow use s o eely sha e, modi y, and
use his Da ase while main aining his same eedom o o he s, p o ided ha he
o iginal sou ce and au ho (s) a e c edi ed.
Link: h ps://doi.o g/10.3897/zookeys.1255.164109.suppl1
