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J. Moll. Stud. (2000), 66, 565–570 © The Malacological Society of London 2000

RESEARCH NOTES

Evolution within the gastropod molluscs; using the ribosomal RNA gene-cluster as
an indicator of phylogenetic relationships

Christopher M. Wade1* and Peter B. Mordan2

1
Institute of Genetics, University of Nottingham, Queens Medical Centre, Clifton Boulevard,
Nottingham, NG7 2UH, UK.
2
Department of Zoology, The Natural History Museum, Cromwell Road, London, SW7 5BD, UK.

The evolutionary relationships among the pulmonate
land snails have long remained the subject of contro-
versy. While classifications based on morphology have
been relatively consistent in the assignment of taxa to
families, the deeper phylogeny within the group
remains unresolved, with morphological studies giving
confusing and conflicting taxonomies1,2,3. Molecular
techniques have proved valuable in determining the
evolutionary relationships between taxa in cases
where morphological features fail to give clear infor-
mation. Yet, so far, only limited molecular work has
been undertaken on the pulmonates, with studies
restricted to the analysis of relatively short sequence
regions or limited sets of taxa (large subunit rDNA4,5,6;
mitochondrial 16S rDNA7). Consequently, these stu-
dies have been unable to adequately resolve the deep
phylogenetic relationships within the group. Here we
present primer sequences for the amplification of
approximately 1460 nucleotides of the ribosomal (r)
RNA gene-cluster. The region comprises a combina-
tion of conserved, variable and highly variable sections,
and thus will be of utility in addressing evolutionary
questions over a wide taxonomic range. We describe
the genetic variation observed at different taxonomic
levels, and present a preliminary phylogeny confirming
the monophyletic nature of the stylommatophoran
land snails within the Gastropoda. Work is now under
way to generate a phylogeny encompassing all the
major stylommatophoran groups using this rDNA
sequence region.
A procedure for extracting the DNA of plants
(Nucleon Phytopure, Scotlab) has proved effective in
removing excess mucopolysacharides from snail
DNA extracts. Extraction of DNA from snails has
been problematic8, apparently due to mucopolysacha-
rides in the mucus causing the inhibition of DNA
polymerases in downstream reactions9,10. The pro-
cedure does not require the snail to be killed, since it
needs only a small fragment of tissue from the foot
(1–2 mm3, which is ground with glass beads immedi-
ately before extraction). In order to minimise degra-
dation, the extracted DNA was resuspended in TE
*
Corresponding author
Present address: Department of Zoology, The Natural History
Museum, Cromwell Road, London, SW7 5BD, UK.
Email: c.wade@nhm.ac.uk
(10mM Tris-HCL pH8.0, 0.1mM EDTA), heated to
96°C for 20 minutes and stored at –80°C.
PCR primers were designed for conserved rDNA
sequence regions identified from alignments encom-
passing all the major eukaryote groups. The primers
amplify approximately 1460 nucleotides of the rRNA
gene-cluster (positions 2990-5104 in the rat rDNA
sequence), including the 3 end (approx. 80 nucleo-
tides) of the 5.8s gene, the complete ITS-2 region, and
the 5 end (approx. 840 nucleotides) of the large
subunit (LSU; 28s) gene (Fig. 1). PCR was carried out
with two overlapping primer sets (set A, LSU-1 and
LSU-3; set B, LSU-2 and LSU-4, Figure 1) using the
Qiagen Taq DNA polymerase and Q buffer system
(0.2M each primer, 0.5 U Taq and 1.5mM magnes-
ium chloride in a 50l final volume). To maximise
yield, thermal cycling (with a Perkin Elmer cycler)
was performed in two rounds with 1l of the product
from the first round used as the template in the
second round. The cycling parameters were 96°C for
1 min, followed by 30 (first round) or 35 cycles
(second round) of 94°C for 30 sec, 50°C for 30 sec
and 72°C for 1 min. Amplification products were
purified from an agarose gel using a Qiagen gel extrac-
tion kit. Both sense and antisense strands were
sequenced directly on an Applied Biosystems 377
DNA sequencer using the Taq dRhodamine termina-
tor cycle sequencing chemistry.
To examine the potential uses of the amplified
rDNA sequence region we examined variation at a
number of taxonomic levels within the Gastropoda.
The taxa sampled included three species of the genus
Partula, three genera of the family Partulidae, ten
stylommatophoran pulmonate genera (incorporating
the main stylommatophoran infraorders), six non-
stylommatophoran pulmonate genera belonging to the
orders Eupulmonata, Basommatophora and Systellom-
matophora, one representative of the Anaspidea, two
nudibranchs and five prosobranchs (see Table 1).
Of the amplified sequence, the ITS-2 region is the
most variable and shows many substitutional changes
and insertion/deletion events. Unfortunately, this
high degree of variability has produced hardly any
alignable ITS-2 sites in comparisons above the family
level. Nevertheless, the region is suitable for examin-
ing evolutionary relationships at or below the family
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566 RESEARCH NOTES

Figure 1. The rRNA gene-cluster, primer locations and primer sequences for PCR amplification. ETS, external
transcribed spacer; ITS, internal transcribed spacer. Primer sequences are as follows:
LSU-1 5-CTAGCTGCGAGAATTAATGTGA-3 (sense 1; positions 2990-3011 in the rat sequence; Genbank
accession number X00133)
LSU-2 5-GGGTTGTTTGGGAATGCAGC-3 (sense 2; position 4143-4162 in the rat sequence)
LSU-3 5-ACTTTCCCTCACGGTACTTG-3 (antisense 1; position 4221-4240 in the rat sequence)
LSU-4 5-GTTAGACTCCTTGGTCCGTG-3 (antisense 2; position 5085-5104 in the rat sequence)

level. Within Partula, 487 of 528 sites could be aligned
of which 5.9% (29) were variable, and 320 of 528 sites
could be aligned within the Partulidae of which 11.6%
(37) were variable (Table 2). The 5.8s and LSU
regions are however more useful for examining evolu-
tionary relationships within the gastropods as a
whole. The 5.8s rDNA sequence is the most con-
served section comprising 80 sites, all of which could
be aligned in comparisons across all the taxa. 1.2% (1
of 80) of sites were variable at the genus level (within
Partula), 2.5% (2 of 80) were variable at the family
level (within the Partulidae), 7.5% (6 of 80) were vari-
able within the Stylommatophora, 10% (8 of 80)
within the Pulmonata, and 30% (24 of 80) were vari-
able in comparisons of all gastropods (Table 2). The
843 nucleotide region of the large subunit gene shows
more variability than the 5.8s. It includes a combina-
tion of conserved and variable sections. In alignments,
834 of 843 sites could be aligned in comparisons of
Partula , 819 within the Partulidae, 792 in the Stylom-
matophora, 770 in the Pulmonata and 581 across the
Gastropoda as a whole. Of these, 0.6% (5 of 834)
were variable at the genus level (within Partula),
1.3% (11 of 819) were variable at the family level
(within the Partulidae), 20.1% (159 of 792) were
variable within the Stylommatophora, 26.5% (204 of
770) within the Pulmonata, and 27.5% (160 of 581)
were variable in comparisons of all gastropods (Table
2). Over the whole amplified sequence, 1401 sites
could be aligned within Partula (35 (2.5%) variable),
1219 sites could be aligned within the Partulidae (50
(4.1%) variable), 872 sites could be aligned within the
Stylommatophora (165 (18.9%) variable), 850 sites
could be aligned within the Pulmonata (212 (24.9%)
variable), and 661 across the Gastropoda as a whole
(184 (27.8%) variable) (Table 2). Thus the region
shows enough variation to be useful for examining
evolutionary relationships at several taxonomic levels
within the gastropods. Deep phylogenetic relation-
ships can be examined using only the most conserved
sites, and additional, progressively more variable,
sites can be recruited for revealing closer phylo-
genetic relationships.
We have constructed a preliminary phylogeny of
deep-level relationships within the gastropods based
on the amplified rDNA sequence region (Figure 2).
Evolutionary trees were reconstructed using distance
(neighbour-joining11 and Fitch-Margoliash12), maxi-
mum likelihood13 and maximum parsimony14 methods
(full methodological details are provided in the legend
to Figure 2). Phylogenetic analyses were based on an
alignment of 661 nucleotides. These sites include
those regions that were unambiguously aligned in
comparisons between all pulmonate, anaspidean,
nudibranch, and prosobranch taxa. They comprise
184 variable sites of which 115 are parsimony infor-
mative. All methods of constructing trees were highly
consistent in their inferred phylogenies, with only
minor variations in the terminal branches of the tree.
Our data confirms the monophyletic nature of all
the major groups within the study, including the
Stylommatophora, Pulmonata and Euthyneura. The
Stylommatophora are clearly and consistently resolved
with all methods of tree construction, and strongly
supported in bootstrap analyses (93% NJ, 97% FM,
95% ML, 80% MP). This is consistent with current
taxonomy15 and agrees with the findings of Tillier et
al.6 based on a smaller section of the LSU gene. The
non-stylommatophoran pulmonates, from the orders
Eupulmonata, Systellommatophora and Basommato-
phora, fall immediately outside the Stylommatophora
thus confirming the monophyly of the Pulmonata
within this tree. This group is resolved by all tree
building methods, and again is strongly supported in
bootstrap analyses (92% NJ, 94% FM, 71% ML, 92%
MP). This also is expected from the current taxon-
omy15. We note, however, that Tillier et al.6 found that
Amphibola clustered separately from the other pul-
monate taxa, resulting in the polyphyly of the group,
but this taxon was not sampled in our study. The pre-
cise relationships among the non-stylommatophoran
pulmonates remain unclear. Currently-defined ordinal
groupings do not appear to be supported by the tree;
the Eupulmonata are paraphyletic and the Systellom-
matophora are polyphyletic. The only clear phylo-
genetic structure within the non-stylommatophorans
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RESEARCH NOTES 567

Table 1. The material, collectors and sampling localities.

Family Species Collection/Location Collector

Subclass Pulmonata, Order Eupulmonata, Suborder Stylommatophora

Infraorder Orthurethra
Partulidae Partula suturalis Pfeiffer, 1855 Moorea B. Clarke
Partula varia Broderip, 1832 Huahine B. Clarke
Partula turneri Pfeiffer, 1860 New Hebrides W. Sutherland
Samoana conica (Gould, 1848) Samoa R. Cowie
Eua zebrina (Gould, 1848) Samoa R. Cowie
Buliminidae Buliminus labrosus (Olivier, 1804) Saladin’s Castle, Syria J. Payne and
P. Mordan
Infraorder Mesurethra
Clausiliidae Cochlodina laminata South Downs, East Sussex, B. Clarke
(Montagu, 1803) UK
Infraorder Elasmognatha
Succineidae Succinea putris (L. 1758) Southampton, UK C. MacDonald
Infraorder Sigmurethra
Achatinidae Achatina fulica Bowdich, 1882 Unknown (Zoological Society P. Pearce-Kelly
of London Collection)
Milacidae Milax budapestensis (Hazay, 1881) Kirkdale, Derbyshire, UK C. Wade
Helicidae Cepaea nemoralis (L., 1758) Marlborough Downs, A. Davison
Wiltshire, UK.
Rhytidae Rhytida stephenensis (Powell, 1930) Manaaki Whenua, New D. Gleeson
Zealand
Subclass Pulmonata, Order Eupulmonata, Suborder Actophila
Ellobiidae Melampus luteus (Quoy & Gaimard, Souilla, Mauritius O. Griffiths
1832)
Laemodonta sp. Suralaya, W. Java B. Dharma
Carychiidae Carychium tridentatum (Risso, 1826) Abelheira, San Miguel, P. Mordan
Azores
Subclass Pulmonata, Order Basommatophora
Siphonariidae Siphonaria pectinata (L., 1758) Zamara Los Atunes, Spain S. Hawkins
Subclass Pulmonata, Order Systellommatophora
Veronicellidae Laevicaulis alte (Férussac, 1823) Dubai, United Arab Emirates A. Green
Rathouisiidae Atopos australis (Heynemann, 1876) Malanda, Queensland, J. Stanisic
Australia

Subclass Opisthobranchia, Order Anaspidea

Aplysiidae Aplysia punctata Cuvier, 1803 Tröndelag, Norway J. Evertsen and
T. Bakken
Subclass Opisthobranchia, Order Nudibranchia
Onchidoridae Acanthodoris pilosa (Müller, 1798) Kinkell Braes, St Andrews, UK C. Todd
Goniodoridae Goniodoris nodosa (Montagu, 1808) Kinkell Braes, St Andrews, UK C. Todd
Subclass Prosobranchia
Hydrobiidae Potamopyrgus antipodarum Morpeth, Northumberland, A. Davison
(Gray, 1843) UK
Thiaridae Melanoides turberculata Tropical Fish Tank, Kettering C. Wade
(Müller, 1774)
Pomatiasidae Pomatias elegans (Müller, 1774) France E. Platts
Cyclophoridae Cyclophorus herklotsi Martens, 1861 Mino, Osaka City, Japan P. Callomon
Ampullaridae Pomacea sp. London Zoo Collection Unknown
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568 RESEARCH NOTES

Euthyneura

[Systellommatophora]
[Systellommatophora]
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RESEARCH NOTES 569

Figure 2. rDNA phylogeny for the gastropods. Evolutionary trees were constructed using the neighbour join-
ing (NJ; shown), Fitch-Margoliash (FM), maximum likelihood (ML) and maximum parsimony (MP) methods
in PAUP* (version 4.0d6516). For NJ, FM and ML methods, correction for multiple hits was performed using
the general time reversible (GTR) model. The rate matrix, base frequencies, proportion of invariant sites (pinv
= 0.225) and gamma shape parameter ( = 0.333; based on 16 rate categories) were estimated using a likeli-
hood-based iterative procedure. FM, ML and MP tree searching was carried out using a heuristic search pro-
cedure with tree-bisection-reconnection branch swapping. Branches highlighted in bold indicate key branches
defining major divisions within the phylogeny. Bootstrap values (NJ/FM/ML/MP) indicate the percentage
support for these branches based on 1000 (NJ, FM and MP) or 100 (ML) bootstrap replicates. The scale bar
corresponds to 1 substitutional change per 100 nucleotide positions. The phylogeny is rooted on the Proso-
branchia.
* This branch is not supported in the parsimony bootstrap consensus tree.

Table 2. Genetic variation in the amplified rDNA sequence.

Within Within Within Within Within

Partula Partulidae Stylommatophora Pulmonata Gastropoda
Aligned Variable Aligned Variable Aligned Variable Aligned Variable Aligned Variable
Sites Sites Sites Sites Sites Sites Sites Sites Sites Sites

5.8s 80 1 80 2 80 6 80 8 80 24
ITS-2 487 29 320 37 0 – 0 – 0 –
LSU 834 5 819 11 792 159 770 204 581 160
Complete 1401 35 1219 50 872 165 850 212 661 184
rDNA region

is produced by the basommatophoran Siphonaria
which consistently falls out before all other pul-
monate taxa, with all tree methods and with strong
support from the bootstrap analyses (95% NJ, 97%
FM, 92% ML, 82% MP). This position was suggested
by Tillier et al.6, but was only weakly supported in
their analyses.
Outside the Pulmonata, the anaspidean Aplysia
emerges as the sister group of the Nudibranchia in
all the trees, with strong bootstrap support with all
methods except maximum parsimony, where the
branch is unresolved in the bootstrap consensus tree
(88% NJ, 84% FM, 87% ML). Finally, the Euthy-
neura as a whole also form a clear monophyletic
group, strongly supported in bootstrap analyses with
all methods (100% NJ, 100% FM, 99% ML, 100%
MP).
Our preliminary analyses of the genetic variation in
the amplified region of the rRNA gene-cluster indi-
cate its general suitability for examining land snail
evolution. The combination of conserved and vari-
able regions suggests that the region will be suitable
for addressing evolutionary questions over a wide
range of taxonomic levels. We intend to use this
sequence to conduct a broad study of the evolution-
ary relationships within the Stylommatophora. The
phylogenetic analyses presented here confirm both
the monophyly of the Stylommatophora and the suit-
ability of the non-stylommatophoran pulmonates as
potential outgroups for this study.
We are especially indebted to Professor Bryan
Clarke for his assistance and comments on the manu-
script. We also wish to thank all those who provided
biological material. This work was carried out with
the support of NERC grant GST/02/1338 to B. Clarke
and P. Mordan.
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Figure 1. ITS-1 situated between 18S and 5.8S rDNA. Black boxes I-VI indicate the conserved regions, their
relative position and size in stylommatophoran and basommatophoran snails (see Fig. 2). To amplify the ITS-1
of Cochlicopa and Azeca, the following primers were used (for further information, see 16): within the conserved
18S  5‘-TAACAAGGTTTCCGTATGTGAA-3‘, and within the conserved 5.8S  5‘-GCGTTCTTCATC-
GATGC-3‘. For Arianta the rDNA region consisting ITS-1, 5.8S, and ITS-2 was amplified with primers 18SB 
5‘-TTCCGTAGTGAACCTGCGG, at the 3‘-end of 18S, and ITS4  5‘-TCCTCCGCTTATTGATATGC at
the 5‘-end of 28S (for further information see 17).