Characterization of transcriptomes from sexual and asexual
lineages of a New Zealand snail (Potamopyrgus antipodarum)
PETER R. WILTON,†* DANIEL B. SLOAN,‡# JOHN M. LOGSDON JR,† HARSHAVARDHAN
DODDAPANENI†& and MAURINE NEIMAN†
†Department of Biology, University of Iowa, Iowa City, IA 52242, USA‡Department of Biology, University of Virginia,
Charlottesville, VA 22904, USA
Understanding the evolution and maintenance of sexual reproduction is one of the central challenges of evolutionary
biology, yet we know very little about how sex influences molecular evolution. The New Zealand freshwater snail
Potamopyrgus antipodarum is ideally suited to address this knowledge gap because obligately sexual individuals
often coexist with multiple independently derived obligately asexual lineages. This unusual situation allows direct
comparisons both between sexual and asexual P. antipodarum and across populations that differ in the relative fre-
quency of sexual individuals. As such, P. antipodarum has received a great deal of attention as a model system for
the maintenance of sex in nature and is also used as a model for environmental toxicology and biological invasions.
Molecular genetic resources for P. antipodarum will thus be useful to investigators in a variety of biological fields.
We used 454 sequencing of cDNA libraries to generate transcriptomes from two sexual and two asexual P. antipoda-
rum lineages. A de novo assembly of 116.7 Mb of sequence reads produced 41 396 contigs, and sequence similar-
ity-based Gene Ontology annotations were obtained for 3740 contigs. We detected 408 315 SNP loci and 7315
microsatellite loci, which together represent the first genome-scale resource available for P. antipodarum. Raw 454
read sequences, contig sequences, annotation data and polymorphism data are publicly available in a searchable
online database and for download at http://www.biology.uiowa.edu/neiman/transcriptome.php.
Keywords: ESTs, transcriptome, asexual reproduction, sexual reproduction, polyploidy, snail
Received 28 August 2012; revision received 12 November 2012; accepted 19 November 2012
Potamopyrgus antipodarum is a New Zealand freshwater
snail characterized by coexistence between obligately
sexual diploid and obligately asexual polyploid individ-
uals (Wallace 1992; Neiman et al. 2011). The P. antipoda-
rum system has been the focus of a large body of
research into the ecology and evolution of sexual vs.
asexual reproduction (e.g. Fox et al. 1996; Jokela et al.
1997; Neiman et al. 2005, 2010) and host-parasite coevo-
lution (e.g. Lively 1987, 1989; Dybdahl & Lively 1996;
Jokela et al. 2009; King et al. 2011). More recently,
researchers have used P. antipodarum to study invasion
biology (Dybdahl & Drown 2011; Miranda et al. 2011)
and ecotoxicology (Alonso & Camargo 2003; Duft et al.
2003; Wagner & Oehlmann 2009). Sexual and asexual P.
antipodarum have similar phenotypes (Jokela et al. 1997),
and there have been multiple independent and sponta-
neous (non-hybrid) transitions to asexual reproduction
(Dybdahl & Lively 1995; Neiman & Lively 2004) and
higher ploidy levels (Neiman et al. 2011). These attri-
butes make P. antipodarum one of the best available sys-
tems to study the costs and benefits of sexual and
asexual reproduction, the mechanisms underlying the
transition to asexuality, and the causes and consequences
Despite the broad interest in P. antipodarum as a
model for sex, genome evolution, invasion biology,
and ecotoxicology, the lack of genetic or genomic
resources for the P. antipodarum system constitutes a
formidable barrier towards developing the system into
a fully actualized model system. The advent of next-
generation sequencing now permits genomic analyses
of non-model organisms
(Wheat 2008; Ekblom & Galindo 2010) and allowed us
to perform the first foray into P. antipodarum genomics.
Correspondence: Maurine Neiman, Fax: 319-335-1069;
*Current address: Department of Organismic and Evolutionary
Biology, Harvard University, Cambridge, MA 02138, USA
#Current address: Department of Ecology and Evolutionary Biol-
ogy, Yale University, New Haven, CT 06516, USA
&Current address: Baylor College of Medicine, Houston, TX
© 2012 Blackwell Publishing Ltd
Molecular Ecology Resources (2012)doi: 10.1111/1755-0998.12051
Here, we present the findings of an analysis of 454
transcriptome sequencing of sexual and asexual P. anti-
podarum lineages. The results of this analysis, as well as
both raw and processed read data, are available online in
the NCBI Sequence Read Archive (SRA Accession ID:
SRA051247) or at http://www.biology.uiowa.edu/nei-
Materials and methods
We extracted RNA from four independently isolated
P. antipodarum lineages descended from wild-caught
females originally sampled from three New Zealand
South Island lakes: Lake Alexandrina (one sexual diploid
lineage and one asexual triploid lineage), Lady Lake (sex-
ual diploid) and Lake Mapourika (asexual triploid). We
selected these lineages in part based on prior knowledge
that they contained divergent (between 0.6% and 3.3%
uncorrected pairwise divergence) mitochondrial haplo-
types (Neiman & Lively 2004; Neiman et al. 2010) and
were thus more likely to reveal polymorphisms that could
be used for, e.g., SNP assays. Each lineage was repre-
sented by four arbitrarily selected adult females. Individ-
uals from asexual lineages are the products of apomictic
parthenogenesis (Phillips & Lambert 1989), and are thus
expected to be genetically identical or nearly identical to
one another within their lineage. Snails selected for
sequencing were sacrificed in water, removed from their
shells,and frozenat ?80 °Cuntil RNAextraction.
RNA extraction, preparation, and normalization
Total RNA was extracted from whole snail bodies
(pooled within lineages) using TRIzol reagent as per the
manufacturer’s instructions. RNA was quantified on a
ND–1000 Spectrophotometer (Thermo Fisher Scientific,
Inc.; Wilmington, DE). cDNA synthesis and normaliza-
tion were carried out using the Evrogen Trimmer-Direct
cDNA normalization kit as per the manufacturer’s
instructions. We used 5 lg of the normalized cDNA
obtained from each lineage extraction for 454 sequenc-
ing. Sample fragmentation, single-stranded library prep-
aration, and emulsion PCR were carried out according to
the manufacturer’s protocol.
attached to sequencing beads was then loaded on a four-
chambered PicoTiterPlate and run on a Roche 454
GS-FLX platform using the titanium sequencing chemis-
try. Sequencing was carried out at the University of Iowa
Carver Center for Genomics.
Sequences from all four libraries were initially combined
and assembled with two different methods: Roche’s GS
SearchESTSNPs from the MIRA v3.0.2 software package
(Chevreux et al. 2004). Assembly with Newbler pro-
duced 28 599 contigs with a mean contig length of
411.7 ? 278.8
SearchESTSNPs produced 41 396 contigs with a mean con-
tig length of 614.8 ? 359.1 (SD) bp. When searched against
the ‘nr’ database with BLASTX, the Newbler assembly
produced 3 752 contigs with e-value less than 10?3(13.1%
of all contigs), while the miraSearchESTSNPs assembly
produced 10 866 contigs with e-value less than 10?3
(26.2% of all contigs). Because the miraSearchESTSNPs
assembly produced longer contigs with a greater number
of predicted homologues, we used this assembly in all
To prepare read data for the miraSearchESTSNPs
pipeline, a FASTA file (with associated quality and clip-
ping files) was generated from 454 outputs using the sff_
extract tool distributed with MIRA. Primers sequences
used in cDNA library construction were masked using
cross_match v1.080721 and a custom Perl script. The mir-
aSearchESTSNPs pipeline was run with the quality grade
set to normal, masked base clipping turned on, and
quality clipping disabled. The four different snail lin-
eages were each specified as a different ‘strain’ for this
The miraSearchESTSNPs pipeline is unique in that it
assembles reads into contigs in three distinct stages
(Chevreux et al. 2004). Contigs from the third stage of
assembly are assemblies of second-stage contigs, which
are themselves assemblies of read sequences. Second-
stage contigs contain reads from one lineage only, but
third-stage contigs may be assemblies of second-stage
contigs from multiple lineages. Not all second-stage con-
tigs are assembled into third-stage contigs because not
all transcripts are found in multiple lineages. Our col-
lected 41 396 contigs consist of all contig sequences from
the third stage of assembly and those from the second
stage that were not assembled into third-stage contigs.
All calculations reported here account for the fact that
third-stage contigs are assemblies of other contigs.
Assemblerv2.3 (‘Newbler’) and mira-
(SD)bp, and assembly withmira-
Contigs were searched for sequence similarity with
NCBI’s non-redundant protein database ‘nr’ and Uni-
prot’s protein database ‘Swiss-Prot’. These searches were
carried out at the University of Iowa using the Tera-
BLASTX®implementation of the BLASTX algorithm on a
TimeLogic DeCypher®system (Active Motif Inc., Carls-
bad, CA) using default BLASTX parameters with default
e-value or maximum-hit cutoffs. In addition to the search
against the nr database, we also carried out TBLASTX
searches against recent transcriptomes from the snail
© 2012 Blackwell Publishing Ltd
2 P. R. WILTON ET AL.
Lymnaea stagnalis (Bou? etard et al. 2012; Sadamoto et al.
2012). For searches against these transcriptomes we
chose to carry out TBLASTX searches for ease of compar-
ison with BLASTX results. BLASTX results from the nr
search were then provided to the annotation pipeline
BLAST2GO (G€ otz et al. 2008) for Gene Ontology (GO,
Ashburner et al. 2000) annotation. BLAST2GO annota-
tion was performed with the default settings of pre-
annotation-cut-off set to 10?6, annotation cut-off set to
55, and the GO weight set to 5.
We used MEGAN version 4.40 (Huson et al. 2011) to
assess the taxonomic distribution of BLAST hits across
the NCBI taxonomic classification tree. MEGAN assigns
a sequence to the node of the NCBI taxonomic classifica-
tion tree that is the most recent common ancestor of the
sequence’s BLAST hits meeting certain stringency crite-
ria. For this analysis, we supplied MEGAN with the
BLASTX results from the search of the NCBI ‘nr’ data-
base and the TBLASTX results from searches against the
L. stagnalis transcriptomes. We used default stringency
parameters. Specifically, under the default settings we
used, in order for a BLASTX hit to be included in the cal-
culation of the most recent common ancestor of a contig’s
BLASTX hits, it was required to have a bit score that was
within 10% of the highest bit score of the contig’s other
BLASTX hits, with a minimum bit score of 35.
Identification of sequence variants
The miraSearchESTSNPs pipeline automates the process
of identifying SNPs during the assembly process. We
retained all of the potential SNP loci reported by mira-
SearchESTSNPs; many of these will be artefacts created
by errors in assembly, base calling, or sequencing, and
we caution that the validity of these loci needs to be
checked empirically. We used the software msatcom-
mander v0.8.2 (Faircloth 2008) to identify microsatellite
repeats of 2–6 bp motifs in the assembled contigs. To be
identified as a potential microsatellite locus, dinucleotide
repeats were required to extend at least six repeats, and
repeats of all other motif lengths were required to extend
at least four repeats. msatcommander works in tandem
with primer3 (Rozen & Skaletsky 1999 p. 3) to locate
primers for PCR amplification of the putative microsatel-
lite loci identified by msatcommander. We provide the
PCR primers found by primer3 with the microsatellite
Sequencing and assembly
Sequencing produced 494 054 reads with a total length
of 116.7 Mbp, yielding a mean read length of 236.3 bp.
The miraSearchESTSNPs pipeline assembled 52.5% of all
reads into 41 396 contigs representing a total of
20.52 Mbp. Of these contigs, 3 517 were shared among
all four lineages, 20 900 were shared among at least two
lineages, 2 822 were shared among sexual lineages only,
and 1 276 were shared among asexual lineages only
(Fig. 1). The mean and maximum contig lengths were
614.7 ? 359.1 (SD) bp and 4154 bp, respectively, and
mean contig coverage was 2.08 9 ? 2.38 (SD) (Fig. 2).
A BLASTX search of the contigs against the NCBI ‘nr’
database produced 992 287 hits for 22 434 contigs. Of
these, 608 431 hits for 9 468 contigs had e-values less
than 10?5. TBLASTX searches against the L. stagnalis
transcriptomes produced 646 134 hits for 10 604 contigs,
with 493 394 hits for 9 282 contigs having e-values less
than 10?5. Annotation by BLAST2GO yielded a total of
24 159 annotations for 3 740 contigs (Ashburner et al.
Fig. 1 Venn diagram of the contigs between
and within lineages. The number in each
area represents the number of contigs
shared by all lineages whose ellipses
enclose that area. For ease of reading,
numbers are underlined with the colours
associated with the lineages that enclose
that area. The counts of contigs in this dia-
gram correspond to the miraSearchEST
© 2012 Blackwell Publishing Ltd
TRANSCRIPTOMES FROM A SNAIL MODEL FOR SEX 3
2000). Of the 3 106 contigs shared only among the two
sexual lineages, 316 received 1 254 GO annotations and
98 of the 1 854 contigs shared only among the two asex-
ual lineages received 362 GO annotations.
Analysis with MEGAN placed 9 194 contigs on the
NCBI taxonomic tree. Most of these contigs were placed
with Lymnaea stagnalis (Fig. 3), reflecting both the com-
paratively recent split between P. antipodarum and L.
stagnalis and the large number of L. stagnalis sequences
against which the contigs were searched. A total of 7 438
contigs (80.9% of the contigs placed on the tree) were
placed within molluscs. Other relatively well-repre-
sented groups featured in the taxonomic distribution of
contigs include chordates (286 contigs assigned), bacteria
(83), arthropods (49), cnidarians (49) and echinoderms
(45). Of the contigs placed within molluscs, 7 397 were
placed within the gastropods and 24 were placed with
Littorina littorea, another littorinimorph snail. Because of
the additional sequence resources we searched, Lymnaea
stagnalis was by far the most represented taxonomic
group on the taxonomic tree, with 7 283 contigs assigned
to the species. Other well-represented gastropod species
include Haliotus discus (20 contigs assigned) and Biom-
phalaria glabrata (8). The contigs placed within bacteria
suggest that the transcriptome was partially contami-
nated with bacteria, but these bacterial sequences com-
prise only a small fraction (0.9%) of all assignments on
the NCBI taxonomic tree and should not interfere with
careful use of this resource.
Identification of sequence variants
Assembly by miraSearchESTSNPs produced 408 315
putative SNPs for 24 367 contigs. The mean (? SD)
number of SNPs per contig was 9.9 (? 15.2) and was 30.2
(? 36.8) per kb. The program msatcommander identified
7 315 potential microsatellite loci for 5 471 contigs, and
primer3 found suitable PCR primers for 2 289 of these
loci. These loci are available online in tabular form
php). The utility of each putative microsatellite locus will
need to be confirmed experimentally, but this resource
nevertheless represents a potentially large increase in the
availability of microsatellite markers in P. antipodarum,
which until now has been limited to seven loci (Weetman
et al. 2001).
Here we have presented an analysis of transcriptomes
sampled from two sexual and two asexual P. antipodarum
lineages. We assembled 116.7 Mb of sequence reads into
20.52 Mb of contig sequence and annotated 3 740 of
these contigs based on sequence similarity. We identified
408 315 putative SNPs and 7 315 putative microsatellite
loci. We make all of these data available online for explo-
ration and for download. These data represent the first
genomic resource available for P. antipodarum.
These transcriptomes add to a growing literature fea-
turing genome-scale data from both sexual and asexual
organisms (e.g., Gallot et al. 2012; Ollivier et al. 2012) and
open the door to large-scale genomic analyses of the
many transitions from sexual to asexual reproduction
and from lower to higher ploidy level in P. antipodarum.
For example, studies of patterns of molecular evolution
across the transcriptomes as a whole can be applied to
largely unanswered questions regarding the genomic
consequences of sex, asexuality, and polyploidy. In par-
allel, particular categories of loci identified within the
transcriptomes can be used to evaluate how the absence
of sex influences evolution in transcripts associated with,
for example, spermatogenesis (GO:0007283, 18 annotated
contigs) and recombination (GO:0006310, three anno-
The work presented here also adds to a body of recent
studiesof snail transcriptomes.Usinganexperimentalset-
up very similar to the one used here, Galindo et al. (2010)
analysed pooled transcriptomes to compare two ecotypes
number of individuals into their samples, Galindo et al.
were able to estimate SNP frequencies within the two
ecotypes and identify potential drivers of phenotypic dif-
ferentiation by scanning for SNPs that showed excep-
tional frequency differences between the two ecotypes.
Although in principle we could have used a similar
approach to compare sexual and asexual P. antipodarum,
the low number of individuals pooled in each sequencing
sample (four) and the likelihood that polymorphism will
Fig. 2 Distribution of lengths of contigs assembled by Mira-
SearchESTSNPs. The mean contig length is represented by a ver-
tical dashed line.
© 2012 Blackwell Publishing Ltd
4 P. R. WILTON ET AL.
be extremely low within each of the two asexual P. anti-
podarum lineages that weused precluded thistype of anal-
ysis. We were also able to use BLAST searches of our
contigs against the recent Lymnaea stagnalis transcripto-
mes produced by Bou? etard et al. (2012) and Sadamoto
et al. (2012); interestingly, we detected only marginally
better rates of significant BLAST hit production compared
to searches against the NCBI ‘nr’ database. This is not sur-
prising given the large number of molluscs already repre-
sented in the ‘nr’ database and the fact that both the
L. stagnalis transcriptomes and the transcriptomes pre-
sented here are incomplete and do not overlap completely
with respect tothe transcriptspresent.
We generated transcriptomes for only two sexual
and two asexual lineages of P. antipodarum, yet there
are many additional independent transitions between
sexual and asexual reproduction in P. antipodarum
(Dybdahl & Lively 1995; Neiman & Lively 2004) as well
as to higher ploidy levels (Neiman et al. 2011). Addi-
tional sequencing of other sexual and asexual lineages
and including more ploidy-level diversity will likely
enable the identification of loci that are represented
and/or evolving differentially between sexual and
asexual P. antipodarum or across ploidy levels. This line
of enquiry represents important early steps towards a
genomic understanding of how sex and ploidy influ-
ence genomic evolution.
We thank Austin Baldwin for snail care and help with RNA
extractions, Gery Hehman of the Carver Center for Genomics
for his assistance with transcriptome sequencing, and Mark Ben-
nett and Matthew Brockman for assistance with setting up
online databases. The Carver Trust, the University of Iowa, and
NSF grant MCB 1122176 provided funding for this project.
Alonso A, Camargo J (2003) Short-term toxicity of ammonia, nitrite, and
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Fig. 3 Taxonomic assignment of contigs by MEGAN on the NCBI taxonomic tree. The size of the circles located at the nodes of the tree
represents the relative number of contigs assigned to that node. Non-terminal nodes with assignments represent contigs with multiple
well-matched BLASTX hits to sequences from different species. When multiple taxa are matched, MEGAN assigns the contig to the
node representing the most recent common ancestor of the hits (see text). Nodes are constrained to be drawn no larger than a maximum
size; the nodes associated with L. stagnalis, ‘No hits’, and ‘Not assigned’ are drawn smaller than they would otherwise be. For these
nodes, the number of contigs assigned to the node is shown below the label and can be compared to the Bacteria node, which also dis-
plays the number of contigs assigned.
© 2012 Blackwell Publishing Ltd
TRANSCRIPTOMES FROM A SNAIL MODEL FOR SEX 5
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P.W: Analysed data, designed research, wrote the paper;
D.S: Analysed data, contributed to writing; J.L: Designed
and funded research; H.D: Prepared libraries, carried out
sequencing; M.N: Designed research, collected speci-
mens, contributed to writing, funded research.
Read sequences: NCBI SRA SRA051247; http://www.
biology.uiowa.edu/neiman/download.php; Dryad entry:
Contig sequences: http://www.biology.uiowa.edu/
GO annotations: http://www.biology.uiowa.edu/neiman/
Polymorphic loci data: http://www.biology.uiowa.
edu/neiman/download.php; Dryad entry: doi:10.5061/
© 2012 Blackwell Publishing Ltd
6 P. R. WILTON ET AL.