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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unpublished) and is very different among the opisthobranch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
doi: 10.1093/mollus/eyi014
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.
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
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
(Verany, 1846)
Hypselodoris villafranca
Chromodorididae
(Risso, 1818)
Dendrodoris fumata
Dendrodorididae
Tergipedidae
Helgoland, Germany
AY165760
AY427448
Flabellinidae
AY165768
AY427449
Dendronotidae
Bergh, 1879
AY165757
AY427450
PLEUROBRANCHOIDEA
Euselenops luniceps
Pleurobranchidae
(Cuvier, 1817)
Tomthompsonia antarctica
Pleurobranchidae
Antarctica
AY427492
(Thiele, 1912)
Bathyberthella antarctica
AY427452
Pleurobranchidae
Pleurobranchidae
Elba, Italy
AY427493
AY427454
Pleurobranchidae
Cuvier, 1804
Berthella stellata
AF249219
AY427453
AF249218
AY427451
AY427494
AY427455
Pleurobranchidae
Elba, Italy
AY427495
(Risso, 1826)
AY427456
TYLODINOIDEA
Umbraculum mediterraneum
Umbraculidae
(Lamarck, 1829)
Tylodina perversa
AY165753
AY427457
Tylodinidae
(Gmelin, 1791)
AY427496
AY427458
SACOGLOSSA
Placobranchus ocellatus
Plakobranchidae
Boselliidae
Elba, Italy
AY427498
Trinchese, 1891
Thuridilla bayeri
AY427460
Elysiidae
Marcus, 1965
Elysia viridis
Elysiidae
Mediterranean Sea
Caliphyllidae
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
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
114
(Schmekel, 1972)
Dendronotus dalli
AF249216
AY427447
AJ224780
AY427446
AJ224774
AY427445
Table 1. (continued)
Species
Family
Collecting locality
Haminoeidae
Elba, Italy
Length of
Length of partial
Accession numbers*
complete 18S
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
Haminoeidae
Pease, 1860
Odontoglaja guamensis
Aglajidae
Aglajidae
Aglajidae
AY427509
AY427474
Philinoglossidae
Rovinj, Croatia
AY427510
AY427475
Retusidae
Elba, Italy
AY427511
AY427476
Bullidae
Elba, Italy
AY427512
Bruguie`re, 1792
Sagaminopteron psychedelicum
AY427477
Gastropteridae
AY427513
AY427478
Runcinidae
Elba, Italy
AY427514
Thompson, 1980
AY427479
ACTEONOIDEA
Hydatina physis
Hydatinidae
(Linne, 1758)
Pupa solidula
AY427515
AY427480
Acteonidae
(Linne, 1758)
AY427516
AY427481
ACOCHLIDIACEA
Unela glandulifera
Microhedylidae
Rovinj, Croatia
AY427517
(Kowalewsky, 1901)
Parahedyle cryptophthalma
AY427482
Microhedylidae
Elba, Italy
AY427518
AY427483
Microhedylidae
Elba, Italy
AY427519
(Kowalewsky, 1901)
Hedylopsis spiculifera
AY427484
Hedylopsidae
Rovinj, Croatia
AY427520
(Kowalewsky, 1901)
AY427485
PULMONATA
Onchidella floridana
Onchidiidae
(Dall, 1885)
Onchidium cf. verruculatum
Onchidiidae
(Cuvier, 1830)
Siphonaria alternata
Siphonariidae
AY427523
AY427488
Helicidae
AY427522
AY427487
(Say, 1826)
Cepaea hortensis
AY427521
AY427486
AY427524
AY427489
Lymnaeidae
(Linnaeus, 1758)
AY427525
AY427490
ALLOGASTROPODA
Odostomia sp.
Pyramidellidae
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.
115
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
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
TCA GGG
470F
700F
TTA CCC
Wollscheid, Dreyer & Englisch
CCG CG
1155F
ATT GAC GG
1600F
TAC ACA CC
1800seq
AGA CC
1155R
700R
AGT TTC AG
Wollscheid, Dreyer & Englisch
CAG AC
400R
28SC1
ACC CGT
According to PCR primer
GCA T
28SC2F (C20 )*
GAG AGA GT
28SD2F
RESULTS
Sequence alignment
CGT CA
28SD2R
TTC TTT TC
* Names of unmodified primers in brackets (Dayrat et al., 2001).
116
Maximum-parsimony analysis
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
117
Phylogenetic signal
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
118
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.
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.
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,
119
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.
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
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.
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.
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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124
the remaining taxa the Acteonoidea show the highest evolutionary rates.
The suitability of 18S and partial 28S rRNA genes for
investigating the phylogeny of the Opisthobranchia must be
assessed critically. Although monophyly of the individual
subgroups is supported, the relationships of several major
clades remain uncertain. The analyses with PHYSID reveal a
relatively low content of phylogenetic information in both
genes. This is probably due to the many highly conserved
positions and the loss of phylogenetic signal in the variable
positions by multiple hits. The content of phylogenetic
information is higher in the 18S than in the (investigated
part) of the 28S rRNA gene, probably attributed to the
greater sequence length of the 18S rRNA gene.
125