RECONSTRUCTION OF THE PHYLOGENY OF THE

OPISTHOBRANCHIA (MOLLUSCA: GASTROPODA) BY MEANS

OF 18S AND 28S rRNA GENE SEQUENCES
VERENA VONNEMANN 1 , MICHAEL SCHRO DL 2 , ANNETTE KLUSSMANN-KOLB 3
AND HEIKE WA GELE 1
1

Lehrstuhl fur Spezielle Zoologie, Ruhr-Universitat Bochum, 44780 Bochum, Germany;

2
Zoologische Staatssammlung Munchen, 81247 Munchen, Germany;
3
Zoologisches Institut der J. W. Goethe Universitat, 60054 Frankfurt am Main, Germany
(Received 20 November 2003; accepted 25 August 2004)

ABSTRACT

INTRODUCTION
The Opisthobranchia are an assemblage of morphologically very
diverse snails and slugs occurring in marine habitats all over the
world. Their appearance ranges from inconspicuous caenogastropod-like animals burrowing in sand to extravagantly coloured
and highly derived swimming organisms. Their smallest representatives (Microhedylidae, Acochlidiacea) measure little more
than 1 mm (Arnaud, Poizat & Salvini-Plawen, 1986), whereas
the largest species (Aplysia vaccaria Winkler, 1955, Anaspidea) can
reach nearly 1m in length (Behrens, 1980). The earliest fossil
opisthobranchs are from the Lower Carboniferous (ca 310 Ma),
resembling members of the extant Scaphandridae and Retusidae
(Cephalaspidea; Gosliner, 1981). The number of species of
Opisthobranchia is of the order of 4500 5000 (Wagele,
unpublished) and is very different among the opisthobranch
subgroups (e.g. Tylodinoidea about 15 species, Nudibranchia
nearly 3000 species).
Despite numerous publications dealing with the Opisthobranchia, their phylogeny is mainly unresolved. This is due to the lack
of comprehensive morphological investigations and the contradictory results produced by molecular studies (Thollesson, 1999;
Dayrat et al., 2001; Wagele, Vonnemann & Wagele, 2003). The
present study is the rst extensive phylogenetic examination of
opisthobranch 28S rRNA gene sequences and also the rst
considering the domains D1, D2 and D3. Dayrat et al. (2001)
investigated the D1 and D2 domains of 31 euthyneuran species
and found that some groups were well supported, but resolution
was poor for other major clades. In order to obtain an increase of
Correspondence:V.Vonnemann;
e-mail: Verena.Vonnemann@ruhr-uni-bochum.de

phylogenetic signal, additional characters (domain D3) have

been included.
The aims of the present paper are to reconstruct opisthobranch
phylogeny and to assess the suitability of the 28S rRNA gene for
phylogenetic questions in comparison with the well-studied 18S
rRNA gene.

MATERIALS AND METHODS

Biological material
Forty-three species of opisthobranchs and six outgroup species
(Pulmonata and Allogastropoda) were collected at various sites
all over the world (Table 1). Within the scope of the present study
the partial 28S rRNA gene of 48 taxa and the complete 18S
rRNA gene of 41 taxa were sequenced, and the remaining
sequences were taken from GenBank.

DNA extraction, amplication and sequencing

Genomic DNA was extracted from alcohol-preserved specimens
by means of the Blood and Tissue Kit (Qiagen), guided by the
enclosed protocol. The amplication of the 18S rRNA gene
region by PCR (Saiki et al., 1988) was performed with primers
developed by Trisha Spears (personal communication): forward
(18A1), 50 -CCT A(CT)C TGG TTG ATC CTG CCA GT-30
and reverse (1800), 50 -GAT CCT TCC GCA GGT TCA CCT
ACG-30 . The PCR was carried out in the thermal cycler Progene
(Techne) under the following conditions: 95 8C for 4 min,
followed by 38 cycles of 30 s at 94 8C, 30 s at 52.5 8C, 2.5 min at
72 8C and a nal extension at 72 8C for 10 min. The domains D1
D3 of the 28S rRNA gene were amplied with forward primer
C10 (Dayrat et al., 2001): 50 -ACC CGC TGA ATT TAA GCA

Journal of Molluscan Studies (2005) 71: 113125

q The Author 2005. Published by Oxford University Studies on behalf of The Malacological Society of London, all rights reserved.

doi: 10.1093/mollus/eyi014

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The complete 18S (SSU) rRNA gene and the partial 28S (LSU) rRNA gene of 49 taxa, including
representatives of the Opisthobranchia, Pulmonata and Allogastropoda, were sequenced to infer the
phylogeny of the Opisthobranchia and their major subordinate taxa Nudibranchia, Pleurobranchoidea,
Tylodinoidea, Sacoglossa, Cephalaspidea s.l. and Anaspidea. For the rst time the enigmatic taxon
Acochlidiacea was included. Several tests were applied to the datasets to investigate relative evolutionary
rates of the taxa, saturation of substitution, base composition and the amount of phylogenetic
information. Maximum parsimony, maximum likelihood and distance methods of tree construction were
used. The opisthobranch subgroups, including the Acochlidiacea, were conrmed as monophyletic in
most analyses, whereas monophyly vs paraphyly of Opisthobranchia could not be resolved. All trees
showed a sister-group relationship between Nudibranchia and Pleurobranchoidea (Nudipleura). In most
trees, sister-group relationships between Cephalaspidea s.s. and Anaspidea, and between Nudipleura and
Acteonoidea (Pupa solidula, Hydatina physis), were supported. The latter sister-group relationship supports
an afnity that has not been mentioned for nearly 40 years (Ghiselin, 1966, Malacologia, 3: 327 378). It
was rediscovered in the molecular analyses by Grande et al. (2004, Molecular Biology and Evolution, 21:
303 313) and represents a valid and interesting opisthobranch clade.

V. VONNEMANN ET AL.V. VONNEMANN ET AL.

Table 1. List of specimens examined in this study, sampling localities, accession numbers and lengths of complete 18S and partial 28Sr DNA genes.

Species

Family

Collecting locality

Bathydorididae

Antarctica

Length of

Length of partial

Accession numbers*

complete 18S

28S (D1 D3)

AY165754

2007

1383

1826

1064

1832

1071

1842

1062

1881

1027

1870

1045

1947

1063

2047

1136

2105

1156

2024

1123

2028

1090

2022

1120

2001

1166

1786

1069

1796

1067

1790

1053

1793

1053

1786

1053

1789

1052

1790

1063

1787

1044

1786

1047

1789

1047

1791

1049

1785

1042

NUDIBRANCHIA
Bathydoris clavigera
Thiele, 1912
Chromodoris krohni

AY427444
Chromodorididae

Spain, Atlantic Ocean

(Verany, 1846)
Hypselodoris villafranca

Chromodorididae

Spain, Atlantic Ocean

(Risso, 1818)
Dendrodoris fumata

Dendrodorididae

Great Barrier Reef, Australia

Tergipedidae

Helgoland, Germany

AY165760
AY427448

Flabellinidae

Spain, Mediterranean Sea

AY165768
AY427449

Dendronotidae

USA, Atlantic Ocean

Bergh, 1879

AY165757
AY427450

PLEUROBRANCHOIDEA
Euselenops luniceps

Pleurobranchidae

Great Barrier Reef, Australia

(Cuvier, 1817)
Tomthompsonia antarctica

Pleurobranchidae

Antarctica

AY427492

(Thiele, 1912)
Bathyberthella antarctica

AY427452
Pleurobranchidae

Weddel Sea, Antarctica

Willan & Bertsch, 1987

Berthellina citrina

Pleurobranchidae

Elba, Italy

AY427493
AY427454

Pleurobranchidae

Wollongong, NSW, Australia,

Cuvier, 1804
Berthella stellata

AF249219
AY427453

(Ruppell & Leuckart, 1828)

Pleurobranchus peroni

AF249218
AY427451

AY427494
AY427455

Pleurobranchidae

Elba, Italy

AY427495

(Risso, 1826)

AY427456

TYLODINOIDEA
Umbraculum mediterraneum

Umbraculidae

Meteor Bank, Atlantic Ocean

(Lamarck, 1829)
Tylodina perversa

AY165753
AY427457

Tylodinidae

Spain, Mediterranean Sea

(Gmelin, 1791)

AY427496
AY427458

SACOGLOSSA
Placobranchus ocellatus

Plakobranchidae

Magnetic Island, QLD, Australia

van Hasselt, 1824

Bosellia mimetica

Boselliidae

Elba, Italy

AY427498

Trinchese, 1891
Thuridilla bayeri

AY427460
Elysiidae

Great Barrier Reef, Australia

Marcus, 1965
Elysia viridis

Elysiidae

Mediterranean Sea

Caliphyllidae

Eagle Island, QLD, Australia

AY427500
AY427463

Limapontiidae

Elba, Italy

AY427501

dOrbigny, 1837
Limapontia capitata

AY427499
AY427462

(Pease, 1866)
Calliopaea bellula

AF249220
AY427461

(Montagu, 1804)
Cyerce nigricans

AY427497
AY427459

AY427464
Limapontiidae

North Sea

AJ224920

(O.F. Muller, 1773)

AY427465

ANASPIDEA
Akera bullata

Akeridae

Kattegat, Denmark

Muller, 1776
Aplysia californica

Aplysiidae

Genbank

AY039804

Cooper, 1863
Dolabella auricularia

AY427502
AY427466
AY026366

Aplysiidae

Okinawa, Japan

AY427503

(Lightfoot, 1786)

AY427467

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(Schmekel, 1972)
Dendronotus dalli

AF249216
AY427447

(Alder & Hancock, 1842)

Flabellina babai

AJ224780
AY427446

(Ruppell & Leuckart, 1830)

Cuthona nana

AJ224774
AY427445

PHYLOGENY OF OPISTHOBRANCHIAPHYLOGENY OF OPISTHOBRANCHIA

Table 1. (continued)

Species

Family

Collecting locality

Haminoeidae

Elba, Italy

Length of

Length of partial

Accession numbers*

complete 18S

28S (D1 D3)

AY427504

1790

1037

1787

1036

1787

1034

1789

1039

1790

1034

1796

1039

1788

1034

1784

1041

1788

1035

1787

1036

1792

1042

1783

1044

1804

1087

1842

1065

1787

1045

1787

1045

1788

1043

1786

1035

1790

1049

1791

1050

1786

1046

1788

1058

1800

1042

1791

1044

CEPHALASPIDEA s.s.
Haminoea hydatis
(Linne, 1758)
Phanerophthalmus smaragdinus

AY427468
Haminoeidae

Lizard Island, QLD, Australia

(Ruppell & Leuckart, 1831)

Atys semistriatus

Haminoeidae

Dingo Beach, QLD, Australia

Pease, 1860
Odontoglaja guamensis

Aglajidae

Orpheus Island, QLD, Australia

Aglajidae

La Paz, Gulf of California, Mexico

Aglajidae

Lizard Island, QLD, Australia

Magnetic Island, QLD, Australia

AY427509
AY427474

Philinoglossidae

Rovinj, Croatia

AY427510
AY427475

Retusidae

Elba, Italy

AY427511
AY427476

Bulla cf. striata

Bullidae

Elba, Italy

AY427512

Bruguie`re, 1792
Sagaminopteron psychedelicum

AY427477
Gastropteridae

Magnetic Island, QLD, Australia

Carlson & Hoff, 1974

Runcina adriatica

AY427513
AY427478

Runcinidae

Elba, Italy

AY427514

Thompson, 1980

AY427479

ACTEONOIDEA
Hydatina physis

Hydatinidae

Hastings Point, NSW, Australia

(Linne, 1758)
Pupa solidula

AY427515
AY427480

Acteonidae

Dingo Beach, QLD, Australia

(Linne, 1758)

AY427516
AY427481

ACOCHLIDIACEA
Unela glandulifera

Microhedylidae

Rovinj, Croatia

AY427517

(Kowalewsky, 1901)
Parahedyle cryptophthalma

AY427482
Microhedylidae

Elba, Italy

AY427518

(Westheide & Wawra, 1974)

Pontohedyle milatchevitchi

AY427483
Microhedylidae

Elba, Italy

AY427519

(Kowalewsky, 1901)
Hedylopsis spiculifera

AY427484
Hedylopsidae

Rovinj, Croatia

AY427520

(Kowalewsky, 1901)

AY427485

PULMONATA
Onchidella floridana

Onchidiidae

Flatts Village, Bermuda

(Dall, 1885)
Onchidium cf. verruculatum

Onchidiidae

Cockle Bay, QLD, Australia

(Cuvier, 1830)
Siphonaria alternata

Siphonariidae

Flatts Village, Bermuda

AY427523
AY427488

Helicidae

Grietherbusch, Rhine, Germany

(OF Muller 1774)

Lymnaea stagnalis

AY427522
AY427487

(Say, 1826)
Cepaea hortensis

AY427521
AY427486

AY427524
AY427489

Lymnaeidae

Grietherbusch, Rhine, Germany

(Linnaeus, 1758)

AY427525
AY427490

ALLOGASTROPODA
Odostomia sp.

Pyramidellidae

Banyuls sur Mer, France,

Mediterranean Sea

AY427526
AY427491

*Accession number for complete 18S rRNA gene sequence above, accession number for partial 28S rRNA gene below.
Specimens have been deposited at either the Zoologische Staatssammlung Munchen (ZSM) or at the Lehrstuhl Spezielle Zoologie, Ruhr-Universitat Bochum.

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Aglajidae

Salvini-Plawen, 1973
cf. Retusa sp.

AY165752
AY427473

(Eliot, 1900)
Philinoglossa praelongata

AY427508
AY427472

Baba, 1949
Philinopsis pilsbryi

AY427507
AY427471

(Cooper, 1863)
Chelidonura inornata

AY427506
AY427470

Rudman, 1978
Navanax inermis

AY427505
AY427469

V. VONNEMANN ET AL.V. VONNEMANN ET AL.

T-30 and reverse primer D3: 50 -GAC GAT CGA TTT GCA CGT
CA-30 under the same conditions as the 18S rRNA gene. Each
PCR reaction mix (50 ml) contained 5 ml of 10 PCR buffer
(Qiagen), 10 ml of Q-Solution (Qiagen), 5 ml of dNTP-mix
(2 mM per dNTP), 0.5 ml of each primer (18A1 and 1800 or C1
and D3), 0.3 ml of Taq polymerase (Qiagen), 0.25 to 3.0 ml of
genomic DNA and 21.55 25.7 ml H2O.
After purication of the PCR products by means of the
QIAquick PCR purication kit (Qiagen), the 18S and 28S rRNA
genes were sequenced directly by the chain-termination method
(Sanger, Miklen & Coulson, 1977) using the Thermo-sequenase
uorescent-labelled primer cycle sequencing kit (Amersham) on
the automated sequencers 4000 and 4000IR2 (Licor). Both
strands of the 18S and 28S rRNA genes were sequenced using the
primers from the PCR and several internal primers (Table 2).

opening 10, gap extension 0.2), but subsequent manual correction was necessary. This was based on different and partly
subjective criteria: elimination of obvious errors like displaced
sequence parts, optimization of the homologies among groups
that are well supported morphologically and maximization of
correspondences by insertion of gaps. This manual treatment was
executed by means of the program GENEDOC (Nicholas &
Nicholas, 1997).

Phylogenetic signal

Sequence alignment
Sequences were aligned with CLUSTAL X (Thompson et al.,
1997) using the default parameters (pairwise parameters: gap
opening 10, gap extension 0.1; alignment parameters: gap
Table 2. Primers used for sequencing the complete 18S and partial 28S
rRNA genes.
Name

Sequence (50 30 )

Reference

18A1seq

CTG GTT GAT CCT GCC

According to PCR primer

AGT CAT ATG C

400F

ACG GGT AAC GGG GAA

Wollscheid, Dreyer & Englisch

TCA GGG
470F

CAG CAG GCA CGC AAA

700F

GTC TGG TGC CAG CAG

Wollscheid, Dreyer & Englisch

TTA CCC
Wollscheid, Dreyer & Englisch

Statistical tests and phylogenetic reconstruction

CCG CG
1155F

CTG AAA CTT AAA GGA

Wollscheid, Dreyer & Englisch

Base composition and saturation of substitution were investigated

by means of the software PAUP 4.0 beta 3a (Swofford, 1999) and
rates of evolution by means of the software k2WuLi (Wu & Li,
1985).
All tree-building methods were conducted using PAUP 4.0 beta
3a (Swofford, 1999). Parameters of maximum-parsimony analyses
were: ACCTRAN, gaps counted as fth base; heuristic search
options: stepwise addition random, branch-swapping option
TBR. The option gaps are missing data was not used, since
elongations in sequences are separate characters, and should be
coded as such. The parameters for distance and maximumlikelihood methods were determined with likelihood ratio tests
using Modeltest Version 3.06 (Posada & Crandall, 1998).
For testing incongruence in combined analyses, the incongruence length difference (ILD) test (Farris et al., 1994) was
applied. With this test, it can be inferred whether combining the
data improves phylogenetic accuracy (P . 0.01) or whether
accuracy of the combined data suffers relative to the individual
partitions (P , 0.001, Cunningham, 1997).

ATT GAC GG
1600F

CGT CCC TGC CCT TTG

Wollscheid, Dreyer & Englisch

TAC ACA CC
1800seq

GAT CCT TCC GCA GGT

According to PCR primer

TCA CCT ACG

1500R

CAT CTA GGG CAT CAC

Wollscheid, Dreyer & Englisch

AGA CC
1155R

CCG TCA ATT CCT TTA

700R

CGC GGC TGC TGG CAC

Wollscheid, Dreyer & Englisch

AGT TTC AG
Wollscheid, Dreyer & Englisch

CAG AC
400R

CCC TGA TTC CCC GTT

28SC1

ACC CGC TGA ATT TAA

Wollscheid, Dreyer & Englisch

ACC CGT
According to PCR primer

GCA T
28SC2F (C20 )*

GAA AAG AAC TTT GAA

Dayrat et al. 2001, modified

GAG AGA GT
28SD2F

CCC GTC TTG AAA CAC

Vonnemann et al., present study

RESULTS

GGA CCA AGG

28SD3

GAC GAT CGA TTT GCA

According to PCR primer

Sequence alignment

CGT CA
28SD2R

CCT TGG TCC GTG TTT

Vonnemann et al., present study

CAA GAC GGG

28SC2R (C2)*

ACT CTC TCT TCA AAG

Dayrat et al., 2001, modified

TTC TTT TC
* Names of unmodified primers in brackets (Dayrat et al., 2001).

Molecular Phylogeny Working group, Department of Spezielle Zoologie,

Ruhr-Universitat Bochum.

116

The alignment of the 18S rRNA gene (28S rRNA gene)

sequences comprises 2211 (1149) positions, of which 1124 (441)
positions are constant, 293 (149) are variable but parsimonyuninformative and 794 (559) are parsimony-informative.
Parts with uncertainties of homology were removed from the
alignments (unpublished data of secondary structure analyses of
senior author). In the 18S rRNA gene the Pleurobranchoidea
possess long non-alignable insertions (positions 865 1004 in

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The software PHYSID (Phylogenetic Signal Detection, Wagele

& Rodding, 1998) allows an a priori estimation of the information
content of aligned sequences without reference to a tree topology
or to a model of sequence evolution. The method consists of a
search for splits in an alignment supported by putative
apomorphies. If the most frequent partitions are compatible a
strong phylogenetic signal is assumed (Wagele, 1996; Wagele &
Rodding, 1998). An a posteriori evaluation can follow by
comparing the splits with the dendrograms obtained by
maximum parsimony, maximum likelihood or distance methods
and examinating the t of the splits on the trees.
Another (a posteriori) approach for evaluating the phylogenetic
signal of the 18S and 28S rRNA gene alignments is the
permutation-tail probability (PTP) test (Archie, 1989). This
test is based on comparison of the length of the most parsimonious
tree with the lengths of trees obtained after randomly permutating the character state assignments within each character. The
permutation-tail probability is then dened as the estimate of the
proportion of times that a tree can be found that is as short
or shorter than the original tree (Faith & Cranston, 1991). The
PTP test was conducted by means of PAUP 4.0 beta 3a
(Swofford, 1999).

PHYLOGENY OF OPISTHOBRANCHIAPHYLOGENY OF OPISTHOBRANCHIA

Pleurobranchoidea the very high rates of Berthella stellata are
striking (18S, 7.3 14.7; 28S, 2.3 9.3). Its Z-scores are much
higher than the values of the other Pleurobranchoidea and
even higher than those of the Anthobranchia. Among the other
groups the substitution rates are comparatively homogeneous
(18S, 0 5.0; 28S, 0 4.7), except for Pupa solidula and Hydatina
physis, which are distinguished by slightly higher Z-scores in the
18S gene (0.1 5.9)

structure E23_3) in region V4 (Wuyts, van de Peer &

De Wachter, 2001). In structure 43 (region V7: positions
1892 1974) the sequence lengths of Nudibranchia, and
especially of Bathydoris clavigera, are much higher than of the
other opisthobranch taxa and show a high divergence. These two
regions were excluded from the analyses. In the 28S rRNA gene,
length variation and extensive sequence divergence occur in the
domains D2 and D3. Therefore, the alignment positions 491 539
(D2), 667 729 (D2) a nd 1113 1398 (D3) were not considered
in the phylogenetic analyses.

Maximum-parsimony analysis

The application of PHYSID (Wagele & Rodding, 1998) reveals

that the signal-to-noise relationship is adverse in both alignments.
Most opisthobranch subgroups are supported by as many or
fewer putative apomorphies as nonsense groups, i.e. the
phylogenetic signals for these clades are not higher than
background noise. The most frequent partition separates Cuthona
and Flabellina (Nudibranchia) from the remaining taxa, supported by 217/102 (18S dataset/28S dataset) putative apomorphies. Concerning the major opisthobranch clades the
Pleurobranchoidea show the best signal (70/42 putative apomorphies), followed by Nudibranchia (50/34), Nudipleura (31/
24), Acteonoidea (31/21), Sacoglossa (10/14), Cephalaspidea s.s.
(12/4), Tylodinoidea (6/10), Anaspidea Cephalaspidea s.s.
(7/4), Anaspidea (2/8) and Acochlidiacea (1/1).
The permutation-tail probability (PTP) test on the other hand
reveals signicant phylogenetic information in the 18S as well as
in the 28S dataset. After generation of 100 random data sets,
computed tree lengths were compared with the MP trees (50%
majority rule consensus; Figs 1, 2) of the original data. In the 18S
rRNA gene dataset, the shortest tree length of the unpermutated
data is 2903 and the shortest random set length is 5996, resulting
in the minimum possible PTP of 0.01. In the 28S the PTP is also
0.01 (actual length 3413, shortest random set length 5678).

Statistical tests
Execution of the chi-squared test shows a signicant heterogeneous base composition in the 18S (P 0) as well as the 28S
(P 0.02) rRNA gene data, caused primarily by the sequences of
the Nudibranchia. After exclusion of this taxon, P values of 0.89
(18S) and 0.92 (28S) are obtained. Additional exclusion of the
Pleurobranchoidea leads to a homogeneous composition of bases
(18S, P 1.0; 28S, P 0.99). Regarding the AT and GCcontent of the 18S rRNA gene sequences a progressive increase of
AT is observed from Cladobranchia (Nudibranchia, 38.4%) to
Anthobranchia (Nudibranchia, 42.4%), Pleurobranchoidea
(43.7%), Acteonoidea (46.8%) and the remaining taxa
(48.1%). This trend is not as distinct in the 28S rRNA gene
(Cladobranchia, 31.7%; Anthobranchia, 32.5%; Pleurobranchoidea, 34.2%; Acteonoidea, 32.9%; others, 36.8%). Investigation of the nucleotide substitutions by plotting the P-distances
against the absolute number of transitions and transversions (not
shown) indicated that the 18S and 28S rRNA gene sequences are
not saturated.
The relative-rate test (Sarich & Wilson, 1973) reveals that
evolutionary rates are signicantly different in both genes. The
Cladobranchia (Nudibranchia) show the highest rates in both
genes, with a maximum Z-score of 15.9 between Dendronotus dalli
and Siphonaria alternata (Pulmonata) in the 18S rRNA gene and
10.9 between Dendronotus dalli and Dolabella auricularia
(Anaspidea) in the 28S rRNA gene. The Anthobranchia
(Nudibranchia) show higher rates than the other taxa except
Cladobranchia (18S, 5.9 10.1; 28S, 2.6 7.4), and the Pleurobranchoidea evolve faster than the remaining taxa except
Nudibranchia (18S, 5.0 9.8; 28S, 1.7 7.0). Within the
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A maximum-parsimony analysis of the complete 18S rRNA gene

with Odostomia sp. (Pyramidellidae) as outgroup produced two
shortest topologies (length 2903 steps, CI 0.63, RI 0.78,
RC 0.49). The strict consensus tree is shown in Figure 1. All
opisthobranch subgroups appear monophyletic, with the Nudipleura (Nudibranchia and Pleurobranchoidea), Acteonoidea
(Pupa solidula and Hydatina physis) and Sacoglossa forming one
major clade and Cephalaspidea s.s., Anaspidea, Tylodinoidea
and Acochlidiacea forming the other. The Acteonoidea are the
sister group of the Nudipleura and the members of the Pulmonata
are basal in the tree. The bootstrap analysis (1000 replicates)
reveals low support for the position of the major opisthobranch
groups. Monophyletic groups with high bootstrap support are,
for example, Nudipleura (100%), Pleurobranchoidea (100%),
Nudibranchia (90%), Acteonoidea (100%), Nudipleura plus
Acteonoidea (98%), Tylodinoidea (97%), Sacoglossa (100%)
and Cephalaspidea s.s. except Runcina adriatica (100%). The
sister-group relationship of Cephalaspidea s.s. and Anaspidea is
supported by a bootstrap value of only 56%; the sister-group
relationship between these two groups and the Tylodinoidea by
only 59%. The positions of the Sacoglossa, Acochlidiacea and
Pulmonata are not resolved at all and the acochlidiacean species
Hedylopsis spiculifera does not form a monophyletic group with the
other Acochlidiacea.
The result of the maximum-parsimony analysis of the 28S
rRNA gene (Fig. 2; 16 shortest trees, length 3413 steps,
CI 0.45, RI 0.63, RC 0.28) shows several correspondences with the results based on the 18S rRNA gene alignment.
Subordinate taxa of the Opisthobranchia are monophyletic
except the Acochlidiacea (as above). The sister-group relationships between Nudipleura and Acteonoidea as well as between
Cephalaspidea s.s. and Anaspidea are conrmed, and a sistergroup relationship between Sacoglossa and Tylodinoidea is
proposed. The bootstrap tree does not differ signicantly from
the maximum-parsimony tree, but resolution is lower. Monophyletic groups with high bootstrap support are, for example,
Nudibranchia (97%), Pleurobranchoidea (99%), Nudipleura
(100%), Acteonoidea (100%), Cephalaspidea s.s. (88%),
Anaspidea (100%), Sacoglossa (100%), Acochlidiacea without
Hedylopsis spiculifera (99%) and Tylodinoidea (91%). The sistergroup relationship between Nudipleura and Acteonoidea has a
bootstrap value of only 54%, and that between Cephalaspidea
s.s. and Anaspidea has a bootstrap value of 76%.
The incongruence length-difference test (Farris et al., 1994)
shows a P value of 0.003 (1000 replicates), indicating a signicant
degree of incongruence. Cunningham (1997) showed that
combining genes generally improved phylogenetic accuracy,
even in cases where incongruence was detected. Only when
P values were lower than 0.001 did the combined data suffer
relative to the individual partitions. Therefore, the 18S and 28S
rRNA gene data sets have also been analysed combined.
The combined analysis of the two genes (Fig. 3) shows a
topology (8 shortest trees, length 6352 steps, CI 0.53,
RI 0.7, RC 0.37) that is better resolved than the 28S tree,
but not as good as the 18S tree. The Pulmonata are presented as
the sister group of the Opisthobranchia, the remaining taxa form
three major clades, of which the relationships are unresolved.

Phylogenetic signal

V. VONNEMANN ET AL.V. VONNEMANN ET AL.

One clade consists of Nudipleura Acteonoidea Sacoglossa,

the second of Cephalaspidea s.s. Anaspidea Tylodinoidea
and the third of the Acochlidiacea. The bootstrap analysis shows
higher resolution compared with the bootstrap trees of the single
genes. All opisthobranch subgroups are supported by high
bootstrap values, as well as Nudibranchia plus Pleurobranchoidea (100%), Nudipleura plus Acteonoidea (98%) and Cephalaspidea s.s. plus Anaspidea (91%).

gamma distribution shape parameters of a 0.31 (18S) and

a 0.52 (28S). The proportions of invariable sites are 0.27
(18S) and 0.23 (28S). The phylogram of the 18S dataset is
shown in Figure 4, revealing that branch lengths are
extremely short in all groups except Nudibranchia, Pleurobranchoidea and Acteonoidea. The results of the distance trees
and bootstrap values (18S and 28S gene) are presented in
Figure 5A, B. For better clarity and easier comparison of
opisthobranch intrarelationships we prefer to present the
distance and maximum-likelihood trees as cladograms and
not as phylograms, and tree presentation is limited to the
relationships of the major opisthobranch groups. If they are
not monophyletic, the excluded taxa are specied separately.
In Figure 5A, B it can be seen that several taxa have varying
positions within the cladograms (Sacoglossa, Anaspidea,
Acochlidiacea, Pulmonata), and even the monophyly of

Distance methods
For choosing the most appropriate model of sequence
evolution, hierarchical likelihood-ratio tests (Goldman, 1993)
are conducted by means of the program Modeltest Version
3.06 (Posada & Crandall, 1998). For both datasets the best-t
model is the Tamura-Nei model (Tamura & Nei, 1993) with
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Figure 1. Strict consensus tree of two shortest maximum-parsimony trees based on complete 18S rRNA gene (length 2903 steps, CI 0.63, RI 0.78,
RC 0.49). Bootstrap indices (50% majority rule) are given.

PHYLOGENY OF OPISTHOBRANCHIAPHYLOGENY OF OPISTHOBRANCHIA

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Figure 2. Strict consensus tree of 16 shortest maximum-parsimony trees based on partial 28S rRNA gene (length 3413 steps, CI 0.45, RI 0.63,
RC 0.28). Bootstrap indices (50% majority rule) are given.

some major taxa is not supported (Pulmonata by exclusion of

Cepaea hortensis and/or Lymnaea stagnalis, Cephalaspidea s.s. by
exclusion of Runcina adriatica). Bootstrap values in the 18S tree
are high for e.g. Cladobranchia (100%), Nudipleura (96%),
Acteonoidea (100%), the latter plus Nudipleura (86%),
Sacoglossa (92%), Cephalaspidea s.s. except Runcina adriatica
(99%), Anaspidea (84%) and Tylodinoidea (99%). For 28S,
they are high, for example, Anthobranchia (98%), Cladobranchia (100%), Nudibranchia (98%), Pleurobranchoidea
(99%), Nudipleura (99%), Acteonoidea (100%), Anaspidea
(99%), Cephalaspidea s.s. except Runcina adriatica (90%),
Sacoglossa (100%) and Tylodinoidea (97%).
A distance analysis of the combined datasets is not carried out
because the ILD test is presently only implemented with
parsimony.

Maximum-likelihood analysis
The phylogenetic reconstructions by means of the maximumlikelihood method were also conducted with the settings obtained
from the likelihood-ratio tests (see above).
The trees based on 18S and 28S rRNA genes (Fig 5C, D) again
show differences. Monophyly is shown for the major opisthobranch groups except for the Acochlidiacea (because of Hedylopsis
spiculifera). Moreover Runcina adriatica is excluded again from the
Cephalaspidea s.s. in the 18S tree. Bootstrap analyses could not
be carried out because of the computation time required. The
two datasets were not analysed simultaneously under the
maximum-likelihood criterion (see above).
Comparing all four analyses (Fig. 5A D), the sister-taxa
relationship of Nudipleura and Acteonoidea occurs three times,
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V. VONNEMANN ET AL.V. VONNEMANN ET AL.

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Figure 3. Strict consensus tree of 8 shortest maximum-parsimony trees based on the combined dataset of 18S and 28S rRNA genes (length 6352 steps,
CI 0.53, RI 0.7, RC 0.37). Bootstrap indices (50% majority rule) are given.

Guerdoux & Tillier, 1994; Tillier, Masselot & Tillier, 1996;

Ponder & Lindberg, 1997; Thollesson, 1999; Dayrat et al., 2001).
A third phylogenetic hypothesis for the Euthyneura has
recently been presented by Grande et al. (2003), based on the
analysis of eight mitochondrial genes. They recovered the
pulmonates as a polyphyletic group with the Stylommatophora
as an early split and the basommatophoran pulmonate Siphonaria
placed within the opisthobranchs.
Because of the well-known rampant parallelism in opisthobranch gastropods, there are very few uniquely derived
attributes in the Opisthobranchia (Gosliner & Ghiselin, 1984).
Mikkelsen (2002) mentioned the following synapomorphies:
loss of the adult operculum, presence of parapodia (with
three reversals) and loss of the posterior stomach chamber.
Many phylogenetic investigations concerning the various

and the sister-taxa relationship of Cephalaspidea s.s. and

Anaspidea twice.

DISCUSSION
To date, the phylogeny of the Opisthobranchia is unresolved,
both in respect of the origin of the whole group as well as of the
interrelationships of its major clades. Two possible evolutionary
scenarios have been discussed: Opisthobranchia and Pulmonata
evolved from a common ancestor and are therefore sister-groups
(e.g. Gosliner, 1981, 1994; Gosliner & Ghiselin, 1984; Wade &
Mordan, 2000; Mikkelsen, 2002; Wagele et al., 2003); Pulmonata
originated within the Opisthobranchia, rendering the
latter paraphyletic (e.g. Haszprunar, 1985; Tillier, Masselot,
120

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Figure 4. Distance phylogram based on the complete 18S rRNA gene. Model of sequence evolution: Tamura-Nei model (Tamura & Nei, 1993), gamma
distribution shape parameter 0.31, proportions of invariable sites 0.27.

opisthobranch subgroups have been carried out and have

produced some good results. The Cephalaspidea s.l. are
commonly accepted as a paraphyletic group consisting of the
monophyletic group Cephalaspidea s.s. and a cluster of primitive
forms, the Acteonoidea (Acteonidae, Hydatinidae and Ringiculidae). Apomorphies of Cephalaspidea s.s. are the ciliated
strips exed at the mantle margin and the presence of three
gizzard plates (Mikkelsen, 1996). The Acteonoidea are either
a basal and probably paraphyletic opisthobranch group
(e.g. Gosliner, 1994) or have even been excluded from the
Opisthobranchia, belonging to the lower heterobranchs

(e.g. Mikkelsen, 1996). The lower heterobranchs comprise

several families, for example Rissoellidae, Architectonicidae and
Pyramidellidae, that in the past have been variously aligned with
prosobranchs or opisthobranchs.
The Anaspidea (including Akera) are well supported as a
monophyletic group by several synapomorphies from the
reproductive system, defensive glands, radula, gizzard and
nervous system (Morton & Holme, 1955; Ghiselin, 1966;
Mikkelsen, 1996; Medina & Walsh, 2000). The Sacoglossa,
with reallocation of the genus Cylindrobulla, are conrmed as
monophyletic by, for example, the uniserial radula and the ascus
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V. VONNEMANN ET AL.V. VONNEMANN ET AL.

The pelagic Gymnosomata and Thecosomata have not yet

been studied phylogenetically and are not considered in the
present paper.
Our knowledge about the phylogeny of the subgroups within
the opisthobranchs is fragmentary because extensive morphological analyses based on cladistic principles are lacking and
molecular analyses have produced contradictory or poorly
resolved trees (e.g. compare the molecular studies by Thollesson,
1999, and Dayrat et al., 2001 with the morphological analysis by
Wagele & Willan, 2000). Mikkelsen (1996), in her thorough
morphological analyses of Cephalaspidea s.l., found a sistergroup relationship of Cephalaspidea s.s. and Anaspidea, together
representing the sister-group of the Sacoglossa. In 2002 she
conducted a combined analysis of characters from four published
phylogenies based on morphological characters (Willan, 1987;
Mikkelsen, 1996; Jensen, 1996, 1997; Wagele & Willan, 2000),
although the resulting cladogram was not well resolved. The
most derived clade consisted of Tylodinoidea and Nudipleura,
forming a monophyletic group with Anaspidea and Cephalaspidea s.s. All groups together were the sister taxon of the
Sacoglossa. A sister-group relationship of Anaspidea and
Cephalaspidea s.s. was also evident in molecular analyses of the
16S rRNA gene in the study by Thollesson (1999) and of the 18S
rRNA gene, 16S rRNA gene and COI gene by Wagele et al.
(2003).
Practically nothing can be said about the position of
Acochlidiacea, Thecosomata and Gymnosomata, because only
very few data are available. Gosliner & Ghiselin (1984) presented
the Acochlidiacea as the sister-group of the Sacoglossa based
on morphological characters. Dayrat et al. (2001) found a
monophyletic group consisting of Thecosomata, Gymnosomata

(Mikkelsen, 1998, 2002). Monophyly of the Notaspidea

(Pleurobranchoidea plus Tylodinoidea) has been rejected by
several workers (Minichev & Starobogatov, 1978; Schmekel,
1985; Salvini-Plawen, 1991; Salvini-Plawen & Steiner, 1996;
Wagele & Willan, 2000). Characters presented as apomorphies
by Willan (1987), for example a bipinnate gill on the right side of
the body and longitudinally slit rhinophores, have since been
shown to be plesiomorphies. Monophyly of the Tylodinoidea is
supported by a cuticularized thickened ring just inside the mouth
(Willan, 1987) and the concentration of the nervous system with
paired cerebral, pleural-intestinal and pedal ganglia (Schmekel,
1985). The Pleurobranchoidea are united by the internal,
rectangular shell and the presence of a pedal gland (Willan,
1987). Schmekel (1985) was the rst to propose a sister-group
relationship of Pleurobranchoidea and Nudibranchia, conrmed
by Salvini-Plawen (1990, 1991) and Wagele & Willan (2000).
The latter authors named the new taxon Nudipleura for
Pleurobranchoidea plus Nudibranchia, characterized by the
possession of a blood gland, an androdiaulic reproductive system
and the loss of the osphradium.
A polyphyletic origin has often been assumed for the
Nudibranchia (e.g. Minichev, 1970) but recent analyses
support their monophyly (Wagele & Willan, 2000). Apomorphies are, for example, the presence of a vacuolated
epithelium and a longitudinally orientated pericardial complex
(Wagele & Willan, 2000).
The monophyly of the interstitial Acochlidiacea is supported
by their unique external organization. They possess a distinct
visceral hump separated from the rest of the body, in which the
head-foot complex can be partially or completely retracted
(Schrodl, personal communication; Rankin, 1979).
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Figure 5. Distance (branch lengths not considered) and maximum-likelihood trees (branch lengths not considered) based on complete 18S rRNA and
partial 28S rRNA genes. For better understanding and easier comparison, the terminal taxa are condensed into monophyletic major groups. Bootstrap
indices (50% majority rule) are given. Models of sequence evolution were determined by means of Modeltest Version 3.06 (Posada & Crandall, 1998).
A. Distance tree based on the complete 18S rRNA gene. B. Distance tree based on the partial 28S rRNA gene. C. Maximum-likelihood tree based on the
complete 18S rRNA gene. D. Maximum-likelihood tree based on the partial 28S rRNA gene.

PHYLOGENY OF OPISTHOBRANCHIAPHYLOGENY OF OPISTHOBRANCHIA

and Anaspidea, based on analyses of the 28S rRNA gene, whereas
Gymnosomata was not part of the Euthyneura but grouped with
the caenogastropods in the study of Thollesson (1999).
An important contribution to the solution of opisthobranch
phylogeny was made by Grande et al. (2004) by means of their
molecular study of the Euthyneura, based on eight mitochondrial
genes. They recovered the Sacoglossa as the most basal group and
a well-supported clade consisting of Anaspidea, Cephalaspidea
and Tylodinoidea. The Nudibranchia formed a paraphyletic
group, because the Pleurobranchoidea were located within them.
Certainly the most striking afnity was the sister-group
relationship between Acteonoidea and Nudibranchia plus
Pleurobranchoidea (discussed below).
The results of the present study, based on the 18S and 28S
rRNA genes and reconstructed by means of maximum parsimony, maximum likelihood and distance methods, can be
summarized as follows.

Nucleotide composition bias and rate heterogeneity, found in

both genes, are estimated to have no affect on the accuracy of the
reconstructed phylogenies. The detected differences in the ATcontent reect phylogenetic relationships already established on
the basis of morphological investigations, i.e. Cladobranchia,
Anthobranchia and Pleurobranchoidea (Wagele & Willan,
2000). The relative-rate test supports the same groups by
different classes of substitution rates (Cladobranchia .
Anthobranchia . Pleurobranchoidea . remaining taxa). The
only case where higher substitution rates can potentially mislead
phylogeny estimation is that of the Acteonoidea, because within
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Opisthobranchia is mainly presented as a monophyletic group

if Pupa solidula and Hydatina physis are considered opisthobranchs. Opisthobranchia can be either para- or monophyletic, because Pulmonata is arranged among Opisthobranchia
in the maximum-likelihood and distance trees, or as a basal
paraphyletic group in the maximum-parsimony analyses.
Monophyly of the major opisthobranch clades is supported in
most analyses, usually with high bootstrap values. However,
Acochlidiacea forms a monophyletic group only in the
maximum-parsimony (18S and combined tree) and distance
analyses, and a high bootstrap value is found only in the
maximum-parsimony tree of the combined dataset. Furthermore Runcina adriatica is excluded from the Cephalaspidea s.s.
in the maximum-likelihood and distance tree of the 18S rRNA
gene. In addition to the topological support, the monophyly of
the pleurobranchoidean taxa is corroborated by the corresponding insertion (structure E23_3) in region V4 of the 18S
rRNA gene.
The arrangement of the major opisthobranch clades shows
variability caused by the lack of phylogenetic signal in both
alignments, with exceptions of the following three points.
The sister-group relationship of Nudibranchia and Pleurobranchoidea (Nudipleura) is conrmed in all analyses with
high bootstrap values (96 100%).
The monophyly of Cephalaspidea s.s. plus Anaspidea is also
conrmed (except in distance trees), although bootstrap
support is lower than for Nudipleura (56 91%).
A sister-group relationship of Nudipleura and Acteonoidea
(28/21 putative apomorphies detected by PHYSID) is
shown in all trees (except the maximum-likelihood tree of
the 18S rRNA gene). This is remarkable because the
Acteonoidea are usually considered the most basal opisthobranchs or are even excluded from this taxon (Mikkelsen
1996, 2002), whereas the Nudipleura are usually regarded
as highly derived (e.g. Wagele et al., 2003). At rst sight this
relationship therefore seems to be due to a long-branch
problem, because Pupa, Hydatina and especially the Nudipleura shows higher evolutionary rates than the other taxa.
On the other hand, it is possible that there are real sister
taxa, because there are some similar morphological characters. Ghiselin, who united Acoela (Nudipleura and
Tylodinoidea) with Hydatinidae and Acteonidae on the
basis of their reproductive system, mentioned that Eliot
(1910) notes a number of [] similarities which imply that
there is an afnity between the Acoela and the Hydatinidae
and Acteonidae (Ghiselin, 1966: 371). In contrast to the
phylogeny proposed by Ghiselin (1966), Nudipleura and
Acteonoidea represent a very derived clade in the maximum-parsimony and distance trees of the present study. In
the maximum-likelihood analyses on the other hand they

partly appear as basal taxa. Although this is the second

study to show this sister-group relationship, the introduction
of a name for the clade Nudipleura Acteonoidea is
postponed until further support is available.
In the present study Tylodinoidea does not form a clade
with Nudipleura but shows different positions in the
different analyses due to the lack of phylogenetic information in both genes. A relationship with Cephalaspidea
and Anaspidea, as suggested by Grande et al. (2003), is
shown in the maximum-parsimony trees of the18S rRNA
(Fig. 1), the combined dataset (Fig. 3) and the maximumlikelihood tree of the 18S rRNA gene (Fig. 5C).
The position of Sacoglossa is also variable. In the
phylogenetic reconstructions of the 18S rRNA gene and
the combined datasets, this taxon shows an afnity with
Nudipleura and Acteonoidea, but in the trees based on the
28S rRNA gene no such relationship can be recognized. A
basal position, as lin Grande et al. (2004), is not supported
at all.
Acochlidiacea is not recognized as a monophyletic group in
several analyses, because Hedylopsis spiculifera, a member of
the family Hedylopsidae, does not group with the three
members of the Microhedylidae (Unela, Pontohedyle, Parahedyle). Except for the 28S maximum-likelihood tree,
Acochlidiacea shows a more or less basal position, mostly
in close proximity to the Pulmonata.
The amount of phylogenetic information in the 18S and
28S rRNA gene sequences at the family and genus levels is
rather different concerning the several opisthobranch
subgroups. In all analyses the phylogeny of Nudibranchia
is in accordance with Wagele & Willan (2000). The
position of the pleurobranchoidean genera, on the other
hand, is extremely variable, dependent on which gene and
method is used. The arrangement of Cephalaspidea s.s. is
comparatively consistent. The Aglajidae (Chelidonura, Odontoglaja, Navanax, Philinopsis) is recovered as a monophyletic
group in each analysis, and the Haminoeidae (Phanerophthalmus, Atys, Haminoea) in each except in the 18S
distance tree. Runcina, if included, occupies the most basal
position within the Cephalaspidea s.s. and Retusa represents
the sister-group of the Haminoeidae except in the 18S
distance tree where it groups within the Haminoeidae. The
positions of Philinoglossa, Sagaminopteron and Bulla are
variable. The basal position of Runcina adriatica is compatible with morphological observations, because Runcina
possesses four gizzard plates, whereas all other members
of the Cephalaspidea s.s. have three (Mikkelsen, 1996),
implying that four plates represent the plesiomorphic
character state. Concerning Sacoglossa, Limapontiidae
(Limapontia, Calliopaea) is recovered in each tree and a
close afnity between Thuridilla, Elysia, Placobranchus and
Bosellia (Placobranchoidea) is always shown except in the
28S distance tree. The position of Cyerce is variable.
Microhedylidae, containing Unela, Parahedyle and Pontohedyle, is always monophyletic.

V. VONNEMANN ET AL.V. VONNEMANN ET AL.

ACKNOWLEDGEMENTS
This project was supported by several grants from the German
Science Foundation to the authors: Wa 618/5 (HW), Wa
618/7 (HW, VV, MS) and KL 1303/1 (AKK). We wish to
express our sincere thanks to some colleagues who provided
material for this study: J. Brodie (Townsville), B. Bolland
(Okinawa), J. Bohn (Munich), P. Duran (Santiago de Chile),
A. Fahrner (Munich), G. Haszprunar (Munich), N. Michiels
(Munster), M. Raupach (Bochum) and G. Steiner (Vienna).
Thanks also to two unknown reviewers and M. Thollesson for
valuable criticism.

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the remaining taxa the Acteonoidea show the highest evolutionary rates.
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