Aquatic Invasions (2022) Volume 17, Issue 3: 415–430
CORRECTED PROOF

Research Article

Population structure and demography of non-indigenous Japanese mystery snails
in freshwater habitats of Virginia and Washington, D.C., USA
Amy E. Fowler1,*, Grace A. Loonam1 and April M.H. Blakeslee2
1 Department of Environmental Science and Policy, George Mason University, Fairfax, VA, 22030, USA
2 Biology Department, East Carolina University, Greenville, NC, 27858, USA*Corresponding author
E-mail: afowler6@gmu.edu

Citation: Fowler AE, Loonam GA,
Blakeslee AMH (2022) Population
structure and demography of nonindigenous Japanese mystery snails infreshwater habitats of Virginia andWashington, D.C., USA. Aquatic Invasions
17(3): 415–430, https://doi.org/10.3391/ai.
2022.17.3.06

Received: 12 September 2021
Accepted: 13 April 2022
Published: 23 May 2022
Thematic editor: Elena Tricarico
Copyright: © Fowler et al.
This is an open access article distributed under termsof the Creative Commons Attribution License(Attribution 4.0 International - CC BY 4.0).

OPEN ACCESS.

Abstract
Non-indigenous species may drive declines in global freshwater biodiversity. Japanese
mystery snails, Heterogen japonica (previously Cipangopaludina / Bellamya / Viviparusjaponica), have invaded numerous freshwater systems in North America. To resolve
questions about its population demography and genetics, we surveyed ponds andrivers from six Mid-Atlantic USA locations (Richmond, Virginia to Washington, D.C.)
in 2018 and 2019 for mystery snails and co-occurring indigenous snails. A random
subset of each snail species (max N = 80) per location was assessed for population
demographics (size, sex), and brooding embryos were counted in mystery snails.
Because morphological identification can be difficult to discern from its congener,
the Chinese mystery snail (Cipangopaludina chinensis), we used a mitochondrial
barcoding gene (COI) to confirm identity of H. japonica and also serve as a population
genetics marker. Our barcoding confirmed that the mystery snails detected in our
surveys were H. japonica, and that, compared to the indigenous range, its populations
have low genetic diversity and limited genetic structure. Even so, H. japonica had
the highest overall catch-per-unit-effort among all snails and sites. In demographic
analyses, H. japonica populations skewed towards females, and females brooding
live young were the largest across all sites. The number of live young ranged from
14 to 101/female (average: 52 live young/female). Further, a linear relationship existed
between brooding female shell length and the number of live young for all sites,
except one. Possible explanations could include site-level differences in abiotic or biotic
parameters, but this requires further research. Altogether, the snail’s reproductive
capacity documented here suggests that H. japonica has the potential to undergo
additional population growth, especially if large females spread to new locations.
Moreover, it highlights another example of an invasive species with high population
abundance, demographic performance, and distributional range even with depauperate
genetic diversity.
Key words: Heterogen/Cipangopaludina japonica, gastropod, genetic paradox,
invasion, North America

Introduction
Globally, freshwater biodiversity is at risk due to a number of leading
anthropogenic concerns, including climate change, water pollution, flow
modification, degradation of habitats, and invasion by exotic species(Dudgeon et al. 2006; Reid et al. 2019). Invasive species, in particular, rank
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as the second leading cause of freshwater biodiversity declines around theworld because they may compete with, predate on, or displace indigenous
flora and fauna (Parker et al. 1999; Dudgeon et al. 2006; Reid et al. 2019).
Exotic freshwater gastropods, specifically, have invaded numerous indigenous
ecosystems either through deliberate introductions or accidental anthropogenictransfer (Padilla and Williams 2004).
Two invasive freshwater snail species, the Japanese mystery snail (Heterogen /Cipangopaludina / Bellamya / Viviparus japonica) and the Chinese mysterysnail (C. chinensis), were intentionally transported from Japan to North
America in ~ 1911 and ~ 1892, respectively, to be cultivated for humanconsumption (Prashad 1928; Clench and Fuller 1965). Both species now
occur in tributaries throughout North America (Jokinen 1982; UnitedStates Geological Survey 2021). The exact species identities of the mysterysnails detected in many North American locations remain unclear because
identification via traditional visual morphology is challenging due to theirmorphological similarities (Smith 2000; Van Bocxlaer and Strong 2016).
The presence of two morphs of C. chinensis in North America further
hinders correct identification (Jokinen 1982, 1992; Van Bocxlaer andStrong 2016). In addition, their taxonomies remain in flux as they haveeach been assigned multiple scientific names, and debate still surrounds
the phylogenetic structure of these species and related congeners (Perez etal. 2016). Indeed, recent evidence suggests that Japanese mystery snails
belong to the genus Heterogen, and thus in this paper, we refer to them as
Heterogen japonica (Saito and Kagawa 2020).
Of the two species, snails morphologically identified as C. chinensis havebeen studied more extensively in introduced populations. As non-selective
detritivores, Cipangopaludina chinensis consume benthic and epiphytic
algae. In their introduced range, they compete with indigenous molluscs,
as well as alter nutrient cycling, algal biomass, and microbial communities(Jokinen 1982; Bury et al. 2007; Clark 2009; Johnson et al. 2009; Olden etal. 2013). They have ovoviviparous reproduction (i.e., eggs hatch within themother and develop inside before being expelled as juveniles), and females
can live five years (Jokinen 1982, 1992; Stephen et al. 2013). Similarly, theJapanese mystery snail, H. japonica, acts as a filter-feeder and detritivore
and can improve overall water quality and measures of lake productivity(Solomon et al. 2010; Van Bocxlaer and Strong 2016). However, thisspecies can also negatively impact indigenous snail populations, likely due
in part to exponential population growth stemming from their
ovoviviparous reproductive strategy (Wolfert and Hiltunen 1968). Overall,little information exists about the life history of H. japonica in its invasive
ranges, but this may be partially because of the difficulty in distinguishing
between the two species, which could limit the association of characteristics
to H. japonica. Moreover, few genetic studies have been used to identify
the two mystery snail species in North America (but see Perez et al. 2016
and David and Cote 2019).
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Here, we collected mystery snails and co-occurring indigenous snails
from six freshwater populations in Virginia and Washington, D.C. over
two years. Our study provides a detailed understanding of the demographic
features of indigenous and non-indigenous snails in these communities
and provides further insight into the demographic success and geographic
spread of mystery snails in these systems.

Materials and methods
Study design
Field collections and demographic assessments
We surveyed locations from Richmond to Washington D.C. for freshwatersnails in July, August, and September 2018 and June and July 2019. We
chose sites based on previous reports of collected “mystery snails” (i.e.,
species either named as Chinese mystery snails or Japanese mystery snails)
to the United States Geological Surveys’ Invasive Species Program(https://www.usgs.gov/ecosystems/invasive-species-program), and ultimately
we sampled six locations to encompass different Virginia/D.C. rivers or lakes(Table 1). Apple snails (Pomacea maculata) are also sometimes referred toas “mystery snails”; however, we specifically chose sites known to host
Chinese / Japanese mystery snails, and apple snails are easily distinguishedfrom C. chinensis and H. japonica. We used hand and visual surveys tocollect indigenous and non-indigenous snails using a timed collection
method (1 hour) along a ~ 30 m transect at a maximum water depth of 1 m.
This provided a catch-per-unit effort (CPUE) method of assessing relative
species abundance across all encountered snail species.
We brought a random subset of putatively adult individuals of each snail
species (max N = 80) to the laboratory for demographic analyses (sex, size).
We used published keys and expert identification (R. Dillon, pers. comm.)
to visually identify indigenous snails. We used the World Registry of Marine
Species (WORMS, https://www.marinespecies.org/) as our primary taxonomic
source. In mystery snails, the size at maturity for these populations is
unknown, so we assessed all encountered snails > 15 mm shell length. We
measured all snails for shell length (mm) and dissected them to determinesex (i.e., male, female, hermaphroditic). We collectively enumerated all
brooding embryos (e.g., freshly encapsulated, angulated eggs, developing
eggs, and juveniles sensu Van Bocxlaer and Strong 2016) in mystery snails.
We also froze a sample of foot tissue from each mystery snail for species
confirmation and population genetics analyses.
DNA isolation, amplification, and sequencing of mystery snails
We used foot tissue from mystery snails from each location to determine
species identity (N = 10, Anacostia; N = 14, Lake Barcroft; N = 7, Gunston;
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N = 12, Bryan Park; N = 12, Leesylvania; N = 27, Rappahannock). We
extracted DNA using a standard cetyl trimethyl ammonium bromide(CTAB)-ethanol precipitation protocol (France et al. 1996). We used theGeller et al. (2013) Universal COI invertebrate primers and newly designed
Heterogen/Cipangopaludina COI primers (COI-F: CCACCTGCAGGATCAAAGAA; COI-R: TCCGGATTAGTTGGAACTGG) based on a H. japonica
sequence (accession #: MK308324.1) to identify mystery snail species andperform population genetics analyses. We used the following PCR profile:
95 °C for 2 min; 30 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 60 s;
and 72 °C for 5 min (Steinberg et al. 2008). PCR products were purified
using EXOSAP-IT (Applied Biosystems) and submitted for sequencing at
Psomagen, USA (Rockville, MD). These sequences were deposited to
GenBank (https://www.ncbi.nlm.nih.gov/genbank/) as accession numbers
ON323055–ON323149. We compiled new Virginia/D.C. sequences withJapanese and Chinese mystery snail sequences from Asian and North
American populations that were harvested from GenBank and BOLD
(http://www.boldsystems.org/) (Supplementary material Table S1).

Data analyses
Genetic sequence alignment, analysis, and barcoding
We aligned and manually inspected genetic sequences for ambiguities
using Geneious 10.1.2 (Biomatters Ltd). Sequences were aligned without
gaps using the ClustalW algorithm (Larkin et al. 2007). Following alignmentand curation, our COI sequence fragment was 545 base-pairs (bp). We
aligned these sequences with those available in Genbank/BOLD (per above),
and the overlapping sequence fragment among all sequences was 404 bp(i.e., we had to further truncate the overall sequence length to match oursequences with these published sequences). We collapsed sequences into
haplotypes using TCS v.1.21 (Clement et al. 2000). The optimal nucleotide
substitution model was selected based on the Akaike Information Criterion
in jModelTest2 (Darriba et al. 2012) and used in Bayesian phylogeny
reconstructions with MrBayes (Huelsenbeck and Ronquist 2001). The
substitution model was set to GTR with four gamma categories; the ratevariation set to gamma; and MCMC settings were default.
Along with all available GenBank or BOLD mystery snail sequences
overlapping with our H. japonica and C. chinensis sequences, we also
included nine additional sequences of Elimia virginica and four sequencesof Campeloma decisum from Virginia/D.C. for comparative purposes(Table S1). We used a Nodilittorina pyramidalis (GenBank: MN389024)
sequence as our outgroup and to root the tree. We calculated pairwise FSTs
using Arlequin311, constructed a resemblance matrix of populations from
Table S1 using Primer7 and used an nMDS plot and boxplots to compare
population differences in haplotype identities and frequencies.
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While our phylogenetic tree included all published sequences from theindigenous and non-indigenous ranges of H. japonica and C. chinensis(Table S1), our nMDS analysis only included populations that had population
sizes of ≥ 3 individuals per population. For these populations, we also
calculated haplotype (h) and nucleotide (π) diversity values using Arlequin311,
and also compared the total h and π for all indigenous versus nonindigenous sequences. Because there were too few samples from C. chinensis’
non-indigenous range, this latter analysis only examined comparative
population level indices in H. japonica. In Arlequin, we also calculated
Tajima’s D and Fu’s Fs for H. japonica in its indigenous and non-indigenous
ranges (all Japanese and all Virginia/DC populations, respectively) to lookfor evidence of population bottlenecks or recent population expansions in
the two regions.
Population demography
We calculated a catch-per-unit-effort for each species at each site bydividing the total number of snails found, by species, by the total searchtime. We first tested whether there were differences in H. japonica snail
shell length due to sampling year. Finding no differences, we combined alldata and used a general linear model to examine the influence of site andsnail sex (male, brooding female, non-brooding female) on snail shell length,
followed by pairwise comparisons using Tukey’s tests. A generalized linear
model using a negative binomial distribution and log-link function
compared whether the number of live young found in H. japonica females
differed by site, using shell length as a covariate. Pairwise comparisons
between sites were made using sequential Bonferroni corrections. A generallinear model compared the relationships between shell length of brooding
H. japonica females and the number of live young across sites, followed bypairwise comparisons using Tukey’s tests.

Results
Genetic analyses
We included 193 sequences in our dataset (Table S1), including Heterogenjaponica from indigenous (N = 20) and non-indigenous (N = 90) ranges(82 new sequences from Virginia/D.C.), C. chinensis from indigenous (N =85) and non-indigenous (N = 4) ranges and Elimia virginica (N = 9) andCampeloma decisum (N = 4) from Virginia. Sequences were collapsed into
37 haplotypes: 11 haplotypes were H. japonica (8 only in the indigenous
range, 2 only in the non-indigenous range, and 1 shared between ranges),
20 were C. chinensis (18 only in the indigenous range, 1 only in the nonindigenous range, and 1 shared between ranges), 5 were E. virginica from
Virginia, and 1 was C. decisum from Virginia (Figure 1A).

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Virginia/D.C. Japanese mystery snail population genetics and demography

Figure 1. A) Phylogenetic reconstruction of the COI-fragment of indigenous and non-indigenous
snails. Posterior probabilities for each clade are noted on the nodes. Two major clades are represented
for the two mystery snail species: H. japonica (HJ) (haplotypes 21–31) and C. chinensis (CC)(haplotypes 1–20) from indigenous Asia and non-indigenous North America. North American
indigenous snails, Campeloma decisum (CD) and Elimia virginica (EV), are represented by
haplotype 37 and haplotypes 32–26, respectively. The tree is rooted using an outgroup sequence
from Nodilittorina pyramidalis (MN389024). The green coloring on the tree signifies shared
haplotypes between the indigenous and non-indigenous regions for each mystery snail species.
(B) nMDS plot of pairwise FSTs from H. japonica and C. chinensis populations in the indigenous
and non-indigenous ranges, based on previous records and newly sampled Virginia/D.C.
locations in our study (represented by the oval surrounding these sites).

While we detected no C. chinensis in our North American sampling, we
included North American C. chinensis sequences (i.e., Hawaii, New York,and Ontario, Canada) that had been extracted from GenBank (Table S1).
The New York (David and Cote 2019) and Hawaiian (Hayes et al. 2019)sequences shared haplotype h16 and were subsumed within a C. chinensis
clade that contained multiple sequences (h17–h20) from the indigenous
range (Figure 1A). The Ontario sequence (Center for Biodiversity Genomics)
shared a frequent hapolotype (h1) that was part of a C. chinensis clade (h1–Fowler et al. (2022), Aquatic Invasions 17(3): 415–430, https://doi.org/10.3391/ai.2022.17.3.06

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Virginia/D.C. Japanese mystery snail population genetics and demographyh4) that only included individuals from Japan. Given the limited numberof non-indigenous C. chinensis samples available for comparison, we did
not further analyze the C. chinensis data for indigenous/non-indigenous
comparisons, except for the nMDS plot where we included C. chinensis
from the indigenous range (Figure 1B).
Within the H. japonica clade, all non-indigenous samples, including
Virginian/D.C. samples, fell within a clade containing Japanese records(Figure 1A). However, Japan was the only indigenous region where publishedsequences existed. All non-indigenous North American records (N = 90)
were assigned to three haplotypes: haplotype h23 was identified in New York
(David and Cote 2019) and Rappahannock and shared with several populationsin the indigenous range; haplotype h21 was found at all Virginian/D.C.
sites, Texas (Perez et al. 2016) and Florida (Sengupta et al. 2009); haplotype
h22 was only found in two Virginian (Gunston Cove and Rappahannock)sites. Haplotypes h21 and h22 did not match any of the indigenous records
extracted from GenBank, most likely because the indigenous range is vastand undersampled (Figure 1A). This result can also be seen in the nMDS
plot (Figure 1B) where the non-indigenous North American sequenceswere not subsumed within the Japanese indigenous sequences.
Haplotype (h) and nucleotide diversity (π) analyses of indigenous versus
non-indigenous populations found nearly 7x greater h diversity and 48x
greater π diversity in the indigenous range. Tajima’s D was not significant
for either region. Fu’s Fs was significant and negative for the non-indigenous
range (Table 2), suggesting a recent population expansion of the species inthis region following introduction events.

Snail field surveys
Invasive mystery snails were hand-collected at all locations during thetimed survey, with highest counts at the Rappahannock site and lowest at
Leesylvania State Park (Figure 2). Mystery snails were most often foundattached to hard surfaces (e.g., wooden docks, tree limbs and downed tree
trunks, cement walls, and rock boulders) and were very rarely found attached
to living plant material or on the bare sediment surface (except for
Leesylvania, where they were found along the edge of a macrophyte bed).
In addition to the invasive species, the following nine indigenous snail species
were also encountered and collected: Amnicola limosa, Campeloma decisum,
Elimia virginica, Lymnaea humilis, Lyogyrus granum, Physella acuta,
Physella pomilia, and Helisoma trivolvis (Figure 2). Including the invasive
mystery snail, the species richness of snails at our sites ranged from two
species in Gunston Cove to seven species in Lake Barcroft. The invasive
mystery snails made up between 18% (N = 19/104 from Leesylvania) and
56% (N = 98/174 from Gunston Cove) of the total number of handcollected snails. Heterogen japonica had the highest overall catch-per-uniteffort (CPUE) across all sites at 0.92 individuals hand collected/minute
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Table 1. Total number and male to female ratios of Heterogen japonica hand collected during a timed 60 min search from eachlocation July–September 2018 and June–July 2019 (*only sampled in 2018). The abundance and percent of brooding female snails
and the average number of embryos (± standard error) and maximum and minimum number for those that had embryos is also given.
Site
Anacostia*
Gunston Cove
Bryan Park
Lake Barcroft
Leesylvania*
Rappahannock

Latitude, Longitude
38°52′16.2″N; 76°59′19.4″W38°46′40.7″N; 77°02′55.7″W37°35′48.1″N; 77°28′14.3″W38°50′48.6″N; 77°10′04.3″W38°35′06.2″N; 77°15′47.2″W38°14′39.4″N; 77°13′33.6″WTotal #
collected
59
98
136
113
19
156

M:F ratio% Female

1: 1.68
1: 2.06
1: 1.49
1: 1.51
1: 2.8
1: 1.5

63%
67%
60%
60%
74%
60%

No. (%)
Brooding
32 (86%)52 (79%)63 (83%)49 (72%)14 (100%)
77 (86%)

Average Brood No. ±SE (Min–Max)
63 ± 3.6 (1–97)
101 ± 5.6 (1–188)
47 ± 2.6 (2–95)
55 ± 7.1 (2–197)
56 ± 4.2 (24–78)
14 ± 0.9 (1–42)

Table 2. Regional and population-level diversity indices and neutrality tests for Heterogen japonica using newly contributed
sequences and published sequences (see Methods). Population-level indices include populations with > 3 individuals. Regional
analyses include all sequences from Table S1. Per population or region, N = number of individuals; a = number of alleles; h =haplotype diversity; π = nucleotide diversity. For regional analyses, Tajima’s D and Fu Fs statistics, along with their respective pvalues, are denoted. Bolded values represent significant deviation from neutrality.
Site
INDIGENOUS
Aichi, Japan
Hyogo, Okayama, Japan
Osaka, Nara, Wakayama, Japan
Shiga, JapanAll Japanese Populations
NON-INDIGENOUS
Anacostia, Washington, D.C.
Belle Haven, Virginia
Bryan Park, Virginia
Potomac, Virginia
Lake Barcroft, Virginia
Leesylvania, Virginia
Rappahannock, Virginia
All Virginian/D.C. Populations

Nah ± S.E.

π ± S.E.

Tajima’s D p (Dsim < Dobs)

4
3
4
5
20

2
1
2
4
9

0.6667 ± 0.2041
0.0000 ± 0.0000
0.5000 ± 0.2652
0.8000 ± 0.1721
0.8579 ± 0.0611

0.00179 ± 0.00166
0.00000 ± 0.00000
0.00124 ± 0.00153
0.00941 ± 0.00635
0.00771 ± 0.00466−0.770

10
7
12
3
14
12
17
90

1
2
1
1
2
1
2
3

0.0000 ± 0.0000
0.2857 ± 0.1964
0.0000 ± 0.0000
0.0000 ± 0.0000
0.2637 ± 0.1360
0.0000 ± 0.0000
0.2206 ± 0.1208
0.1281 ± 0.0474

0.00000 ± 0.00000
0.00000 ± 0.00000
0.00000 ± 0.00000
0.00000 ± 0.00000
0.00065 ± 0.00086
0.00000 ± 0.00000
0.00000 ± 0.00000
0.00016 ± 0.00038−0.784

Fu’s Fs

p (Fssim < Fsobs)

0.245−1.619

0.193

0.214

−3.1590.000(Figure 2). However, there were some indigenous snails with higher CPUEs
at particular sites (i.e., C. decisum at Anacostia – 1.33 and E. virginica at
Leesylvania – 1.33 and Rappahannock – 1.36) (Figure 2).

Heterogen japonica population demographics
The largest snails (shell length) across all snail species and sites were brooding
H. japonica females (average shell length 48.93 ± 9.76 mm standard error).
Non-brooding females from Gunston Cove measured significantly smaller(average 34.79 ± 4.69 mm) than all other site and sex combinations(average 44.81 ± 11.16 mm) (Figure 3), resulting in a significant interaction
between site and sex on H. japonica shell length (N = 566, LM, F(10,555) =20.86, p < 0.001).
In the majority of locations, male H. japonica were less abundant thanfemales, with an average male to female sex ratio of 1:1.63 (Table 1). In
addition, of those females collected, the majority (between 72% and 100%
depending on the site) were brooding live young. The average number ofbrooding young in a single female across sites was 52 live young. Afteraccounting for shell length, there was a significant difference in thenumber of embryos between sites (N = 286, GLM, df = 5, Wald Chi-Square
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Figure 2. Catch-per-unit-effort for each indigenous and non-indigenous snail species hand collectedduring a timed 60 min search from each location July–September 2018 and June–July 2019
(except Anacostia and Leesylvania, which were only sampled in 2018). Values represent how manysnails could be hand collected per minute.

Figure 3. Shell lengths (mm) by site of male, brooding female, and non-brooding female Heterogenjaponica hand collected during a timed 60 min search from each location July–September 2018
and June–July 2019 (except Anacostia and Leesylvania, which were only sampled in 2018).
Box plots represent the interquartile (IQ) range (i.e., the middle 50% of the records), the verticalline is the median, and the whiskers are the highest and lowest values which are no greater than
1.5 times the IQ range. Outliers (i.e., black circles) are values between 1.5 and 3 times the IQ
range, beyond the whiskers. The dashed line at 45 mm represents the overall average size ofinvasive mystery snails across all sexes and sites.

= 134.07, p < 0.001), with the highest average number of embryos infemales from Gunston Cove (101 embryos) and the lowest in females fromthe Rappahannock (14 embryos) (Table 1, Figure 4A). With the exceptionof females from the Rappahannock and Gunston Cove, larger females were
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Figure 4. (A) Number of live young found in brooding female Heterogen japonica by site. The boxes represent the interquartile(IQ) range (i.e., the middle 50% of the records), the horizontal line is the median, and the whiskers are the highest and lowest
values which are no greater than 1.5 times the IQ range. Outliers (i.e., black circles) are values between 1.5 and 3 times the IQ range,
beyond the whiskers. Different letters above each box plot indicate significant differences (p < 0.05). (B) Linear relationships andthe strength of those relationships (R2 value) between the shell lengths (mm) of brooding female H. japonica and the number oflive, internal embryos counted during dissection.

found with higher numbers of live young during dissections (Figure 4B).
Therefore, the relationship between shell length of a brooding female andthe number of live embryos that was counted during dissection differed
between sites (N = 286, LM, F(5,280) = 54.05, p < 0.001, Adjusted R2 = 0.482).
Brooding females from the Rappahannock were never observed with morethan 42 live young, while there were clear positive associations between
shell length and numbers of live young at all other sites (Figure 4B).

Discussion
Here, we genetically confirmed that mystery snails present in Virginia andWashington, D.C., USA were the Japanese mystery snail, H. japonica, andthat these non-indigenous populations were genetically depauperate. In
demographic analyses, we determined that mystery snails were the mostabundant snails present and that their populations were skewed towards
large brooding females, suggesting stable and successfully reproducing
populations.

Genetic confirmation and population genetics of H. japonica
While Van Bocxlaer and Strong (2016) previously identified H. japonica in
the Potomac River in Alexandria, Lake Barcroft, and Gunston Cove,
Virginia via morphological distinctions, here we confirm identification of
H. japonica via genetic methods. Moreover, we detected little to no genetic
structure in the populations of H. japonica from the United States. All
sequences, regardless of collection site, were comprised of just three
haplotypes, and diversity indices (h and π) were 7 and 48 times lower in the
non-indigenous regions, suggesting very limited genetic diversity for theinvasive snail in its non-indigenous range.
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Many non-indigenous populations exhibit significantly lower genetic
diversity, which may occur as a result of founder effects (Dlugosch andParker 2008), where just a subset of a species’ source genetic diversity istransported (naturally or anthropogenically) to a new region, resulting insubstantially lower genetic diversity compared to the source (Barton andCharlesworth 1984; Grosberg and Cunningham 2001). Frequent additions
to the founding population could introduce new genotypes and increase
standing genetic variation in the non-indigenous range that could help
alleviate deleterious effects of low genetic diversity (Roman and Darling2007; Dlugosch and Parker 2008; Lejeusne et al. 2014). However, eventhough we observed lower haplotype and nucleotide diversity in its nonindigenous populations, H. japonica was the most abundant snail at the
majority of our sample sites and also demonstrated a very high reproductive
capacity (discussed further below). This species is another example of ademographically successful invasive species demonstrating limited genetic
diversity—and thus represents a “genetic paradox”.
The genetic paradox refers to species that exhibit significantly reduced
genetic diversity in founding populations, yet are demographically successful
by adapting to novel environments (Estoup et al. 2016). While seemingly
paradoxical, these species may have biological traits that allow them tocircumvent possible deleterious effects predicted in founding populations(e.g., accumulation of deleterious alleles due to low founding sizes andsubsequent genetic drift) (Schrieber and Lachmuth 2017). However, neutral
markers commonly used in single-gene approaches may not reflect genetic
diversity across the genome or genes associated with ecologically relevent
traits (Estoup et al. 2016). Thus, further examination of genetic diversity
across multiple markers could reveal genetic mechanisms for the demographic
success of mystery snails under a scenario of depauperate genetic diversity,
as in the invaded North American range.
In addition, it was not possible for us to pinpoint source locations fromthe indigenous range for the species’ introduction using genetic data
presently available. This is because the genetic diversity in that region isvast in comparison to what we sampled in the non-indigenous range (e.g.,
Blakeslee et al. 2008). Greater spatiotemporal sampling of indigenous andnon-indigenous ranges could provide better understanding of the impactof genetic diversity on the species’ distribution and its spread in the nonindigenous range, as well as likely sources of introduction.

Population demography of H. japonica
While the invasive Japanese mystery snail H. japonica was the mostfrequently encountered snail across all sites, two indigenous snail species
had higher catch-per-unit-efforts at particular sites (i.e., Campeloma decisumat Anacostia and Elimia virginica at Leesylvania and Rappahannock). Oneof these, the pointed campeloma – C. decisum, was the largest indigenous
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Virginia/D.C. Japanese mystery snail population genetics and demographysnail we encountered and can reach sizes of up to 40 mm shell length(Brown et al. 1989). Brown et al. (1989) observed that individuals canaggregate at high densisties of up to 800/m2, but we did not observe large
aggregations at our sites. Similar to H. japonica, C. decisum is also a deposit
and filter feeder and has ovoviviparous (broods live young) reproduction(Brown et al. 1989). The Piedmont elimia, E. virginica, can reach sizes of
up to 33 mm shell length, is dioecious, and has been observed at densities
over 400 individuals/m2 (Smith 1980). This species grazes on epilithic
periphyton (Harman 2000), in contrast to H. japonica and C. decisum.
Previous work has also noted that invasive populations of H. japonica
can attain high densities and likely has significant negative effects on thelocal ecosystem (Wolfert and Hiltunen 1968). Wolfert and Hiltunen (1968)
reported that seine fishermen in Lake Erie, Pennsylvania, USA could catch
two tons of the species in a single haul. Similar to our study, Wolfert and
Hiltunen (1968) found that the second most frequently encountered snails
were either C. decisum or a pleurocerid snail similar to E. virginica. It is
likely that these species overlap in diet and may even compete for food inareas where there are high densities; yet studies thus far examining
competition among indigenous freshwater snails and non-indigenous
mystery snails have concentrated on C. chinesis (e.g., Jokinen 1982; Bury et
al. 2007; Clark 2009; Johnson et al. 2009; Olden et al. 2013). While it islikely that the two mystery snails have similar ecological impacts because
they share many of the same demographic and morphological traits, moreexperimental work describing the impacts of H. japonica on indigenous
species and systems is needed to compare their ecosystem influences.
The broad size range of H. japonica collected for both sexes and the highproportion of brooding females in the populations suggests that these
multigenerational populations successfully reproduce in these locations.
The reproductive mode (i.e., ovoviviparous; eggs hatch within the motherbefore being expelled as juveniles) and reproductive capacity of this species
both contribute to its success as an invader (Van Bocxlaer and Strong2016). Organisms that brood live young are often less constrained bypossible Allee effects (i.e., reduced or negative population growth at low
population densities) that could emerge in founding populations(Johannesson 1988). Because ovoviviparous species can release numerous,
crawl-away young that do not disperse far from the parent settlement, thisreproductive strategy can enhance their colonization success and population
growth in newly founded regions; indeed, some of the most globallywidespread and invasive aquatic gastropods include brooders (e.g.,
Potamopyrgus antipodarum; Alonso and Castro-Diez 2008). Ovoviviparity
has likely enhanced both H. japonica’s and C. chinensis’ colonization
success, spread, and population growth in freshwater systems throughout
North America. For example, the estimated annual production of an invasive
population of C. chinensis from a Nebraska reservoir was ~ 2.2–3.7 million
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Virginia/D.C. Japanese mystery snail population genetics and demography

young (Stephen et al. 2013). This reproductive strategy may have also beena contributing factor in its demographic success and geographic spread inNorth America despite low genetic diversity in the region as detected inour study.

Site-level differences in H. japonica
In general, brooding female H. japonica were significantly larger thanmales, due mainly to the presence of the large brood pouch located in thesame location as the much smaller testes in males (Van Bocxlaer andStrong 2016). However, at the Rappahannock site, we found a smaller size
range of brooding females in comparison to other locations. Female
H. japonica in this survey contained developing embryos of several sizes
and stages of development, a trait found in other species of Viviparidae snails(Jakubik 2007). However, this characteristic also varied across sites. In
general, female shell length and embryo number were positively related to
one another except for two locations – Gunston Cove and Rappahannock.
Female H. japonica in their indigenous range in Japan release live young
between April and October, and the number of live brooding young was
between 10 and 120 (Taki 1981). Stephen et al. (2013) found that C. chinensis
females (N = 29) (e.g., species identification was not genetically confirmed)
collected in southeast Nebraska contained an average of 25 embryos per
female, while the average across all sites in this survey was 2.5x higher (N =132, average 62, range 16–109). An earlier survey of only seven female
H. japonica from northern Virginia collected in September 2012 found an
average of 108 live young (range 46–220) (Van Bocxlaer and Strong 2016).
It is possible that there were various abiotic and biotic factors that could
explain these site-level differences detected between our study and others;
for example, the Stephen et al. (2013) survey occurred in September, possiblyat the end of the breeding season, while the survey presented here occurredearlier during the summer months. The warmer water temperatures in
Virginia/D.C. may have also contributed to the higher abundances ofembryos in females as compared to other northern populations. Moreover,snail density, water quality, nutrients, day length, parasites and foodavailability all likely vary by location and could influence snail growth andreproductive output in these different locations (Ter Maat 2007; Sandland
and Josephson 2017; Song et al. 2017; Dickens et al. 2018; Muñoz et al.
2018). Conversely, a genetic explanation for these demographic differences
across sites in Virginia/D.C. is unlikely given the little genetic differentiation
we observed among populations and low genetic diversity in all nonindigenous populations. Even so, the reproductive mode and the numberof developing embryos documented in H. japonica in Virginia/D.C.
populations suggest that this species has the potential to undergo additional
population growth, especially if large females spread to new locations(Keller et al. 2007).
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Virginia/D.C. Japanese mystery snail population genetics and demography

Although the populations studied here are genetically depauperate, thesnails are successfully reproducing, and thus appear to represent anotherinvasive species demonstrating a genetic paradox. The species’ reproductive
strategy as well as the presence of ecologically relevant genes may havehelped it avoid deleterious effects often associated with low genetic
diversity. Finally, while much of the literature from laboratory and fieldstudies have identified North American mystery snails as C. chinensis, ourstudy suggests that Japanese mystery snails are the dominant mystery snail
in Virginia. Given the morphological similarities between the two species,
genetic confirmation is needed going forward to ensure any new reports
and research correctly identifies these two invasive species.

Acknowledgements
We thank the Fowler lab for dissecting snails (Darby Pochtar, Alex Mott, Allison Assur, andJulia Czarnecki) and Dr. Robert Dillon for identifications. We also thank the anonymous
reviewers for their comments, which greatly improved the manuscript.

Funding declaration
George Mason University start up funds to AEF, East Carolina University start-up funds to
AMHB, a Washington Field Biologists’ Club grant to AEF, and GMU’s undergraduate research
program (OSCAR) grant to GAL supported this work. The funders had no role in study design,
data collection and analysis, decision to publish, or preparation of the manuscript.

Author’s contribution
AEF conceptualized the research, designed the sampling and methods, collected the data,
completed the data analysis, interpreted the data, wrote the original draft, and reviewed andedited all other drafts. GAL collected the data and reviewed and edited all other drafts. AMHB
designed the sampling and methods, collected the data, completed the data analysis, interpreted
the data, wrote the original draft, and reviewed and edited all other drafts.

Ethics and permits
With submission of this article, the authors have complied with the institutional and/or national
policies governing the humane and ethical treatment of the experimental subjects. Maryland
Department of Natural Resources Scientific Collection Permit Number SCP2018861. Virginia
Department of Game and Inland Fisheries Scientific Collection Permit Number 062276.

References
Alonso A, Castro-Diez P (2008) What explains the invading success of the aquatic mud snail
Potamopyrgus antipodarum (Hydrobiidae, Mollusca)? Hydrobiologia 614: 107–116,
https://doi.org/10.1007/s10750-008-9529-3

Barton NH, Charlesworth B (1984) Genetic revolutions, founder effects, and speciation. Annual
Review of Ecology and Systematics 15: 133–164, https://doi.org/10.1146/annurev.es.15.110184.001025
Blakeslee AMH, Byers JE, Lesser ME (2008) Resolving cryptogenic histories using host andparasite genetics. Molecular Ecology 17: 3684–3696, https://doi.org/10.1111/j.1365-294X.2008.
03865.x

Brown K, Varza D, Richardson T (1989) Life histories and population dynamics of two
subtropical snails (Prosobranchia: Viviparidae). Journal of the North American
Benthological Society 8: 222–228, https://doi.org/10.2307/1467325
Bury JA, Sietman BE, Karns BN (2007) Distribution of the non-native Viviparid snails,
Bellamya chinensis and Viviparus georgianus, in Minnesota and the first record of
Bellamya japonica from Wisconsin. Journal of Freshwater Ecology 22: 697–703,
https://doi.org/10.1080/02705060.2007.9664830

Clark GT (2009) Distribution, growth, and competitive impacts of the exotic Chinese mysterysnail (Bellamya chinesis) in the James River, southwest Missouri. MSc Thesis, Missouri
State University
Clench WJ, Fuller SLH (1965) The genus Viviparus (Viviparidae) in North America.
Occasional Papers on Mollusks 2(32): 385–412
Fowler et al. (2022), Aquatic Invasions 17(3): 415–430, https://doi.org/10.3391/ai.2022.17.3.06

428

Virginia/D.C. Japanese mystery snail population genetics and demography

Clement M, Posada DCKA, Crandall KA (2000) TCS: a computer program to estimate genegenealogies. Molecular Ecology 9: 1657–1659, https://doi.org/10.1046/j.1365-294x.2000.01020.x
Darriba D, Taboada GL, Doallo R, Posada D (2012) jModelTest 2: more models, new heuristics
and parallel computing. Nature Methods 9: 772–772, https://doi.org/10.1038/nmeth.2109
David AA, Cote SC (2019) Genetic evidence confirms the presence of the Japanese mysterysnail, Cipangopaludina japonica (von Martens, 1861) (Caenogastropoda: Viviparidae) innorthern New York. BioInvasions Records 8: 793–803, https://doi.org/10.3391/bir.2019.8.4.07
Dickens KL, Capinera JL, Smith TR (2018) Effects of density and food deprivation on growth,
reproduction, and survival of Lissachatina fulica. American Malacological Bulletin 36: 57–61,
https://doi.org/10.4003/006.036.0115

Dlugosch KM, Parker IM (2008) Founding events in species invasions: genetic variation,
adaptive evolution, and the role of multiple introductions. Molecular Ecology 17: 431–449,
https://doi.org/10.1111/j.1365-294X.2007.03538.x

Dudgeon D, Arthington AH, Gessner MO, Kawabata ZI, Knowler DJ, Lévêque C, Naiman RJ,
Prieur-Richard A-H, Soto D, Stianssny MLJ, Sullivan CA (2006) Freshwater biodiversity:
importance, threats, status and conservation challenges. Biological Reviews 81: 163–182,
https://doi.org/10.1017/S1464793105006950

Estoup A, Ravigné V, Hufbauer R, Vitalis R, Gautier M, Facon B (2016) Is there a genetic
paradox of biological invasion? Annual Review of Ecology, Evolution, and Systematics 47:
51–72, https://doi.org/10.1146/annurev-ecolsys-121415-032116
France SC, Rosel PE, Ewann J (1996) DNA sequence variation of mitochondrial large-subunit
rRNA. Molecular Marine Biology and Biotechnology 5(1): 15–28
Geller J, Meyer C, Parker M, Hawk H (2013) Redesign of PCR primers for mitochondrial
cytochrome c oxidase subunit I for marine invertebrates and application in all‐taxa biotic
surveys. Molecular Ecology Resources 13: 851–861, https://doi.org/10.1111/1755-0998.12138
Grosberg R, Cunningham CW (2001) Genetic structure in the sea. In: Bertness MD, Gaines S,
Hay ME (eds), Marine Community Ecology, Sinauer Associates, Sunderland, Massachusetts,
pp 61–84
Harman WN (2000) Diminishing species richness of mollusks in Oneida Lake, New York State,USA. Nautilus 114: 120–126
Hayes KA, Cowie RH, Thiengo SC (2009) A global phylogeny of apple snails: Gondwanan
origin, generic relationships, and the influence of outgroup choice (Caenogastropoda:
Ampullariidae). Biological Journal of the Linnean Society 98: 61–76, https://doi.org/10.1111/j.
1095-8312.2009.01246.x

Huelsenbeck JP, Ronquist F (2001) MRBAYES: Bayesian inference of phylogenetic trees.
Bioinformatics 17: 754–755, https://doi.org/10.1093/bioinformatics/17.8.754
Jakubik B (2007) Egg number-female body weight relationship in freshwater snail (Viviparus
viviparus L.) population in a reservoir. Polish Journal of Ecology 55(2): 325–336
Johannesson K (1988) The paradox of Rockall: why is a brooding gastropod (Littorina saxatilis)
more widespread than one having a planktonic larval dispersal stage (L. littorea)? Marine
Biology 99: 507–513, https://doi.org/10.1007/BF00392558
Johnson PT, Olden JD, Solomon CT, Vander Zanden MJ (2009) Interactions among invaders:
community and ecosystem effects of multiple invasive species in an experimental aquatic
system. Oecologia 159: 161–170, https://doi.org/10.1007/s00442-008-1176-x
Jokinen EH (1982) Cipangopaludina chinensis (Gastropoda: Viviparidae) in North America,
review and update. Nautilus 96: 89–95
Jokinen EH (1992) The Freshwater Snails (Mollusca: Gastropoda) of New York State. New
York State Museum, Albany, NY, 112 pp
Keller RP, Drake JM, Lodge DM (2007) Fecundity as a basis for risk assessment of nonindigenous
freshwater molluscs. Conservation Biology 21: 191–200, https://doi.org/10.1111/j.1523-1739.
2006.00563.x

Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam, H, Valentin F,
Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins, DG (2007) Clustal W
and Clustal X version 2.0. Bioinformatics 23: 2947–2948, https://doi.org/10.1093/
bioinformatics/btm404

Lejeusne C, Saunier A, Petit N, Béguer M, Otani M, Carlton JT, Rico C, Green AJ (2014) Highgenetic diversity and absence of founder effects in a worldwide aquatic invader. Scientific
Reports 4: 1–9, https://doi.org/10.1038/srep05808
Muñoz JCV, Hilt S, Horák P, Knopf K (2018) Impact of trematode infections on periphyton
grazing rates of freshwater snails. Parasitology Research 117: 3547–3555, https://doi.org/10.
1007/s00436-018-6052-y

Olden JD, Ray L, Mims MC, Horner-Devine MC (2013) Filtration rates of the non-native
Chinese mystery snail (Bellamya chinensis) and potential impacts on microbialcommunities. Limnetica 32: 107–120, https://doi.org/10.23818/limn.32.11
Padilla DK, Williams SL (2004) Beyond ballast water: aquarium and ornamental trades assources of invasive species in aquatic ecosystems. Frontiers in Ecology and theEnvironment 2: 131–138, https://doi.org/10.1890/1540-9295(2004)002[0131:BBWAAO]2.0.CO;2Parker IM, Simberloff D, Lonsdale WM, Goodell K, Wonham M, Kareiva PM, Williamson
MH, von Holle B, Moyle PB, Byers JE, Goldwasser L (1999) Impact: toward a framework
Fowler et al. (2022), Aquatic Invasions 17(3): 415–430, https://doi.org/10.3391/ai.2022.17.3.06

429

Virginia/D.C. Japanese mystery snail population genetics and demography

for understanding the ecological effects of invaders. Biological Invasions 1: 3–19,
https://doi.org/10.1023/A:1010034312781

Perez B, Segrest A, Minton R, Campos S, Burks R (2016) First record of Japanese mysterysnail Cipangopaludina japonica (von Martens, 1861) in Texas. Check List 12: 1–17,
https://doi.org/10.15560/12.5.1973

Prashad B (1928) Recent and fossil Viviparidae. A study in distribution, evolution andpalaeogeography. Memoirs of the Indian Museum 8: 153–252
Reid AJ, Carlson AK, Creed IF, Eliason EJ, Gell PA, Johnson PT, Kidd KA, MacCormack TJ,
Olden JD, Ormerod SJ, Smol JP, Taylor WW, Tockner K, Vermaire JC, Dudgeon D, Cooke
SJ (2019) Emerging threats and persistent conservation challenges for freshwater biodiversity.
Biological Reviews 94: 849–873, https://doi.org/10.1111/brv.12480
Roman J, Darling JA (2007) Paradox lost: genetic diversity and the success of aquatic invasions.
Trends in Ecology and Evolution 22: 454–464, https://doi.org/10.1016/j.tree.2007.07.002
Saito T, Kagawa O (2020) New insights from museum specimens: a case of Viviparidae(Caenogastropoda: Mollusca) in Iwakawa’'s collection preserved in the National Museum of
Nature and Science, Tokyo. Biodiversity Data Journal 8: e52233, https://doi.org/10.3897/BDJ.8.e52233
Sandland GJ, Josephson KA (2017) Nutrient availability modulates both host and parasite life
histories in a snail-trematode interaction. Diseases of Aquatic Organisms 126: 135–142,
https://doi.org/10.3354/dao03169

Schrieber K, Lachmuth S (2017) The genetic paradox of invasions revisited: the potential roleof inbreeding × environment interactions in invasion success. Biological Reviews 92): 939–
952, https://doi.org/10.1111/brv.12263
Sengupta ME, Kristensen TK, Madsen H, Jørgensen A (2009) Molecular phylogenetic investigationsof the Viviparidae (Gastropoda: Caenogastropoda) in the lakes of the Rift Valley area ofAfrica. Molecular Phylogenetics and Evolution 52: 797–805, https://doi.org/10.1016/j.
ympev.2009.05.007

Smith DG (1980) Goniobasis virginica (Gastropoda: Pleuroceridae) in the Connecticut River.
The Nautilus 94(2): 50–54
Smith DG (2000) Notes on the taxonomy of introduced Bellamya (Gastropoda: Viviparidae)
species in northeastern North America. The Nautilus 114: 31–37
Solomon CT, Olden JD, Johnson PT, Dillon RT, Vander Zanden MJ (2010) Distribution andcommunity-level effects of the Chinese mystery snail (Bellamya chinensis) in northern
Wisconsin lakes. Biological Invasions 12: 1591–1605, https://doi.org/10.1007/s10530-009-9572-7
Song Z, Zhang J, Jiang X, Xie Z (2017) Linking environmental factors, life history andpopulation density in the endangered freshwater snail Margarya melanioides (Viviparidae)
in Lake Dianchi, China. Journal of Molluscan Studies 83: 261–269, https://doi.org/10.1093/
mollus/eyx024

Steinberg MK, Krimsky LS, Epifanio CE (2008) Induction of metamorphosis in the Asian shore
crab Hemigrapsus sanguineus: effects of biofilms and substratum texture. Estuaries andCoasts 31: 738–744, https://doi.org/10.1007/s12237-008-9063-6
Stephen BJ, Allen CR, Chaine NM, Fricke KA, Haak DM, Hellman ML, Kill RA, Nemec KT,
Pope KL, Smeenk NA, Uden DR, Unstad KM, VanderHam AE, Wong A (2013) Fecundityof the Chinese mystery snail in a Nebraska reservoir. Journal of Freshwater Ecology 28:
439–444, https://doi.org/10.1080/02705060.2013.769127
Taki A (1981) The fecundity of a mud snail, Cipangopaludina japonica. Internationale
Vereinigung für theoretische und angewandte Limnologie: Verhandlungen 21: 1637–1639,
https://doi.org/10.1080/03680770.1980.11897251

Ter Maat A, Zonneveld C, de Visser JAG, Jansen RF, Montagne-Wajer K, Koene JM (2007)
Food intake, growth, and reproduction as affected by day length and food availability in thepond snail Lymnaea stagnalis. American Malacological Bulletin 23: 113–120, https://doi.org/
10.4003/0740-2783-23.1.113

United States Geological Survey (2021) NAS - Nonindigenous Aquatic Species. https://nas.er.
usgs.gov/default.aspx (accessed 14 April 2021)
Van Bocxlaer B, Strong EE (2016) Anatomy, functional morphology, evolutionary ecology andsystematics of the invasive gastropod Cipangopaludina japonica (Viviparidae: Bellamyinae).
Contributions to Zoology 85: 235–263, https://doi.org/10.1163/18759866-08502005
Wolfert DR, Hiltunen JK (1968) Distribution and abundance of the Japanese snail, Viviparusjaponicus, and associated macrobenthos in Sandusky Bay, Ohio. The Ohio Journal of
Science 68(1): 32–40
Supplementary material
The following supplementary material is available for this article:
Table S1. Site names, country, and description of genetic sequences obtained for indigenous and non-indigenous populations of
Cipangopaludina chinensis and Heterogen japonica used to construct phylogenetic trees.
This material is available as part of online article from:http://www.reabic.net/aquaticinvasions/2022/Supplements/AI_2022_Fowler_etal_SupplementaryMaterial.xlsx

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