*Departamento de Biodiversidad y Biología Evolutiva, Museo Nacional de Ciencias Naturales, CSIC, Madrid, Spain;
Departamento de Biología, Facultad de Ciencias del Mar, Universidad de Cádiz, Puerto Real, Spain
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Abstract |
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Introduction |
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Several recent phylogenetic analyses based on morphological characters support the validity of the Euthyneura clade but tentatively reject the monophyly of both opisthobranchs and pulmonates (e.g., Haszprunar 1985
; Salvini-Plawen and Steiner 1996
; Ponder and Lindberg 1997
) (fig. 1A and B
). Depending on the phylogenetic analysis, different lineages of pulmonates are placed as sister groups of different orders of opisthobranchs (fig. 1
). Additionally, the monophyly of some opisthobranch orders (e.g., Cephalaspidea and Notaspidea) has also been recently questioned (Schmekel 1985
; Mikkelsen 1996
; Wägele and Willan 2000
). Apparently, the high degree of convergence or parallelism exhibited by many morphological characters of opisthobranchs (associated with reduction and loss of the shell and the mantle cavity; Gosliner and Ghiselin 1984
; Gosliner 1985
; Salvini-Plawen and Steiner 1996
) has seriously complicated phylogenetic inferences within the group.
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Several phylogenetic analyses have demonstrated recently that the use of complete mitochondrial genomes in phylogenetic studies significantly increases the confidence of the phylogenetic history inferred compared with phylogenetic hypotheses based on individual or partial mitochondrial genes (Cummings, Otto, and Wakeley 1995
; Russo, Takezaki, and Nei 1996
; Zardoya and Meyer 1996
). So far, the complete mitochondrial DNA sequences of seven mollusks are available: a cephalopod, Loligo bleekeri (Sasuga et al. 1999
); a bivalve, Crassostrea gigas (S. H. Kim, E. Y. Je, and D. W. Park, personal communication; GenBank accession no. NC_001276); a polyplacophoran, Katharina tunicata (Boore and Brown 1994
); and four gastropodsthree pulmonates (Albinaria coerulea [Hatzoglou, Rodakis, and Lecanidou 1995
], Cepaea nemoralis [Terrett, Miles, and Thomas 1996
], and Euhadra herklotsi [Yamazaki et al. 1997
]) and a primitive opisthobranch (Pupa strigosa [Kurabayashi and Ueshima 2000a
]). The incomplete mitochondrial genomes of a bivalve (Mytilus edulis [Hoffmann, Boore, and Brown 1992
]) and two gastropods (a caenogastropodan, Littorina saxatilis [Wilding, Mill, and Grahame 1999
], and a heterostrophan, Omalogyra atomus [Kurabayashi and Ueshima 2000b
]) have also been described.
The mitochondrial DNA of mollusks shows extreme variations in gene organization (Boore and Brown 1994
; Yamazaki et al. 1997
; Boore 1999
; Kurabayashi and Ueshima 2000a;
Rawlings, Collins, and Bieler 2001
). The mitochondrial gene arrangements of pulmonates (Euhadra, Cepaea, and Albinaria), the heterostrophan (insofar as has been determined; Omalogyra), and the opisthobranch (Pupa) are nearly identical (Kurabayashi and Ueshima 2000a,
2000b
). The gene organization of the mitochondrial genomes of Littorina, Katharina, and Loligo shows greater resemblance to the consensus gene arrangement of arthropods (Boore 1999
; Sasuga et al. 1999
; Wilding, Mill, and Grahame 1999
). The lack of atp8 gene in Crassostrea and Mytilus (Hoffmann, Boore, and Brown 1992
; S. H. Kim, E. Y. Je, and D. W. Park, unpublished data; GenBank accession no. NC_001276), the presence of additional tRNA genes in Katharina and Mytilus (Hoffmann, Boore, and Brown 1992
; Boore and Brown 1994
), and an unusual mode of inheritance of Mytilus mitochondrial DNA (Zouros et al. 1994
; Saavedra, Reyero, and Zouros 1997
; Zouros 2000
) are other intriguing features of the mitochondrial genomes of mollusks.
To test the monophyly of opisthobranchs and to clarify their relative phylogenetic position within gastropods, as well as to further investigate variations in the mitochondrial genome organization of mollusks, we have sequenced the complete mitochondrial genome of a nudibranch, Roboastra europaea García-Gómez 1985. We have compared this new mitochondrial genome with the only opisthobranch mitochondrial genome described so far, that of P. strigosa, and with mitochondrial genomes of other gastropods. To further understand opisthobranch systematics, we have also sequenced a mitochondrial DNA fragment of about 2,500 bp (including part of cox1, the complete rrnL and nad6 genes, and a portion of the nad5 gene) in several species that represent different orders of opisthobranchs.
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Materials and Methods |
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The sequences of both fragments were used to design two sets of specific primers (LP-F, LP1-R and LP1-F, LP-R; see table 1 ) that amplified, by long PCR, two fragments of about 7,000 bp each, that covered the remaining mitochondrial genome (fig. 2 ). Long PCRs containing 60 mM Tris-SO4 (pH 9.1), 18 mM (NH4)2SO4, 12 mM MgSO4, 0.2 mM of each dNTP, 0.4 µM of each primer, and elongase enzyme (1 unit; Life Technologies) in a final volume of 50 µl were subjected to 40 cycles of denaturing at 94°C for 30 s, annealing at 52°C for 30 s, and extending at 68°C for 7 min.
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Total cellular DNA was also purified from several species of opisthobranchs that represent five different orders: Chelidonura africana (Cephalaspidea); Ascobulla fragilis (Sacoglossa); Aplysia punctata (Anaspidea); Umbraculum mediterraneum (Notaspidea); and Aeolidia papillosa (Nudibranchia). Two sets of primers, Opis COI-F (5'-ACTTTTTTTCCTCAGCATTTYTT-3')/16Sbr and LP-F/Opis-2R (table 1 ), were used to amplify by standard PCR two overlapping DNA fragments that covered the 3' end of the mitochondrial cox1 gene, the complete mitochondrial rrnL, trnL(cun), trnA, trnP, and nad6 genes, and the 5' end of the mitochondrial nad5 gene. PCR products were cloned into the pGEM-T vector (Promega) and were sequenced using M13 universal primers.
Molecular and Phylogenetic Analyses
Sequence data were analyzed with the GCG program version 10.2 (Devereux, Haeberli, and Smithies 1984
), MacClade version 3.08a (Maddison WP and Maddison DR 1992, pp. 1398
), and PAUP* version 4.0b8 (Swofford 1998
). Nucleotide and amino acid sequences were aligned using CLUSTAL X version 1.62b (Thompson et al. 1997
) followed by refinement by eye. Ambiguous alignments and gaps were excluded from the analysis using GBLOCKS 0.73b (Castresana 2000
). Alignments are available from http://www.molbiolevol.org.
The following five complete mollusk mitochondrial genomes were analyzed in this study: L. bleekeri (Sasuga et al. 1999
); A. coerulea (Hatzoglou, Rodakis, and Lecanidou 1995
); C. nemoralis (Terrett, Miles, and Thomas 1996
); P. strigosa (Kurabayashi and Ueshima 2000a
); and R. europaea (this study). Loligo bleekeri was used as the out-group in all phylogenetic analyses because most authors currently consider cephalopods as the sister group of gastropods (Haszprunar 1988
; Bieler 1992
). The deduced amino acid sequences of all 13 protein-coding genes encoded by the mitochondrial genomes were combined into a single data set that was subjected to maximum parsimony (MP), minimum evolution (ME), maximum likelihood (ML), and Bayesian methods of phylogenetic inference. MP analyses were performed with PAUP* using heuristic searches (TBR branch swapping; MulTrees option in effect) with 10 random additions of taxa. ME analyses (Rzhetsky and Nei 1992
) were carried out with PAUP* using mean character distances. ML analyses were performed with PUZZLE version 4.0.1 (Strimmer and von Haeseler 1996
) using the mtREV model (Adachi and Hasegawa 1996
). The robustness of the resulting MP and ME trees was evaluated by bootstrapping (Felsenstein 1985
) (as implemented in PAUP* with 1,000 pseudoreplicates). The robustness of the resulting ML tree was evaluated by quartet puzzling (as implemented in PUZZLE with 10,000 puzzling steps). A Bayesian inference of gastropod phylogeny was performed with MrBayes 2.01 (Huelsenbeck and Ronquist 2001
) by simulating a Markov chain for 10,000 cycles under the Jones model (Jones, Taylor, and Thornton 1992
). The same phylogenetic analyses at the amino acid level were performed including only the mitochondrial protein-coding genes that were available for the caenogastropodan L. saxatilis (Wilding, Mill, and Grahame 1999
), i.e., cox1, cox2, atp8, atp6, nad1, nad6, and cob. Sequence data of the six protein-coding genes known for the heterostrophan O. atomus (Kurabayashi and Ueshima 2000b
) were not included in the phylogenetic analyses because they were not available on EMBL-GenBank data libraries.
To recover phylogenetic relationships among opisthobranchs, nucleotide sequences of part of the mitochondrial cox1 gene, the complete mitochondrial rrnL and nad6 genes, and a fragment of the mitochondrial nad5 gene of several species that represent the main orders of opisthobranchs were analyzed with common methods of phylogenetic inference. MP analyses were performed with PAUP* without weighting based on the estimated empirical value (Ts/Tv = 0.71). ME and ML analyses were performed with PAUP* using the GTR model (Rodríguez et al. 1990
). The robustness of MP, ME, and ML analyses was tested by bootstrapping with 1,000 pseudoreplicates. Bayesian inference of opisthobranch phylogeny was performed with MrBayes 2.01 (Huelsenbeck and Ronquist 2001
) using the GTR model and 10,000 generations.
Statistical differences between alternative phylogenetic hypotheses were evaluated in PAUP* (Swofford 1998
) using the Templeton (1983)
, Kishino and Hasegawa (1989)
, and Shimodaira and Hasegawa (1999)
tests.
The nucleotide sequences of opisthobranchs reported in this article have been deposited at the EMBL-GenBank data libraries under accession numbers AY083457 and AY098927AY098931.
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Results |
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The genetic code of the Roboastra mitochondrial genome is the same as that used by other mollusks. It differs from the universal genetic code in that ATA codes for methionine, TGA for tryptophan, and AGR for serine. A total of 3,665 amino acids are encoded by the Roboastra mitochondrial genome (table 2 ). The most abundant amino acid residue is leucine, whereas the rarest is cysteine (table 2 ). Thymines are preferentially used in third codon positions. Cytosine is generally the rarest nucleotide in third codon positions (table 2 ).
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ML analysis of the combined amino acid data set arrived at a tree (log likelihood = -22,320.08) that strongly supports the monophyly of opisthobranchs (Pupa + Roboastra) and pulmonates (Albinaria + Cepaea) (fig. 6
). Bayesian inference rendered an identical result (fig. 6A
). When MP (one single tree of 3,441 steps; consistency index [CI] = 0.94) and ME (score = 1.03) analyses were performed, only the opisthobranchs were recovered as a monophyletic group (fig. 6A
). But a Wilcoxon signed-ranks test (Templeton 1983
) showed that the second most parsimonious tree that supported the monophyly of pulmonates was not statistically significantly different (3,444 steps; Z = -0.31; P = 0.75). Cepaea exhibits a rather long branch, and its basal position in the MP and ME analyses might be an artifact attributable to long-branch attraction by the out-group (Felsenstein 1978
).
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The nucleotide sequences of the mitochondrial cox1, rrnL, nad6, and nad5 genes were combined into a single data set that produced an alignment of 2,631 positions. Of these, 942 were excluded because of ambiguity in the homology assignment, 437 were invariant, and 900 were parsimony-informative. The mean pairwise uncorrected p distance among opisthobranchs is 0.32 ± 0.03. The minimum and maximum uncorrected p distances are between Aplysia and Umbraculum (0.25) and between Pupa and both Roboastra and Aplysia (0.36), respectively. The uncorrected p distance between Albinaria and Cepaea is 0.47. The mean pairwise uncorrected p distance between opisthobranchs and pulmonates is 0.43 ± 0.02. ML (log likelihood = -15,722.75) and Bayesian methods of phylogenetic inference arrived at identical topologies (fig. 7
). ME (score = 2.65) only differed from the ML tree in that Aplysia was recovered as sister group of Umbraculum to the exclusion of Chelidonura. MP recovered two trees of 3,779 steps (CI = 0.62), one with the topology shown in figure 7
and the other supporting the monophyly of pulmonates. In all cases, opisthobranchs were monophyletic with strong statistical support (fig. 7
). The Kishino and Hasegawa (1989)
and Shimodaira and Hasegawa (1999)
tests rejected statistical differences between the ML tree and a tree with a monophyletic pulmonate clade (log likelihood = -15,724.52; P = 0.78 and P = 0.38, respectively). Within opisthobranchs, cephalaspideans (Pupa and Chelidonura) were polyphyletic. Pupa was consistently the sister group of Ascobulla (order Sacoglossa), and both species were placed basal to the rest of opisthobranchs (fig. 7
). Chelidonura was placed in a derived position either as sister group of Aplysia (order Anaspidea) (MP, ML, and Bayesian analyses) or as sister group of Aplysia + Umbraculum (order Notaspidea) (ME analyses). Because of the low bootstrap support, the relationships between Chelidonura, Aplysia, and Umbraculum remain unresolved. The nudibranchs (Roboastra and Aeolidia) were monophyletic (fig. 7 ).
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Discussion |
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The gene arrangement of Roboastra is similar to that of Pupa (Kurabayashi and Ueshima 2000a
) but differs in the transposition of the trnC gene. Moreover, the relative position of the trnC gene in pulmonates is different from that of both opisthobranchs (fig. 9
). The rearrangement of tRNA genes is very frequent in invertebrate mitochondrial genomes and may mobilize adjacent protein-coding and rRNA genes (Boore 1999
). The Heterostropha, Pulmonata, and Opisthobranchia mitochondrial genomes described so far share a rather conserved gene arrangement (Hatzoglou, Rodakis, and Lecanidou 1995
; Terrett, Miles, and Thomas 1996
; Yamazaki et al. 1997
; Kurabayashi and Ueshima 2000a,
2000b
) (fig. 9
). In contrast, a group traditionally considered within Prosobranchia, Caenogastropoda, shows a highly divergent gene arrangement that could be related to the gene arrangement of nongastropod mollusks (Boore 1999
) (fig. 9
). Therefore, the conserved gene arrangement of heterostrophans, pulmonates, and opisthobranchs (all together Heterobranchia) may present a derived state with respect to the ancestral state represented by the caenogastropodan Littorina (Boore 1999
).
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The amino acid sequence divergence between Pupa and Roboastra is almost half that between Albinaria and Cepaea. Furthermore, the amino acid sequence divergence within pulmonates is as much as that found between pulmonates and opisthobranchs. All phylogenetic analyses based on the combined protein-coding gene data set strongly support the monophyly of opisthobranchs (Roboastra and Pupa) (fig. 6A
). The monophyly of opisthobranchs is also highly supported when Littorina is included in the phylogenetic analyses (fig. 6B
). The genus Pupa is a representative of the most ancestral order (Cephalaspidea) of opisthobranchs. In fact, members of its family (Acteonidae) have been proposed as a model of the archetypal opisthobranch because their external morphology is similar to that of prosobranchs (Rudman 1972
). In contrast, the genus Roboastra represents the most derived order (Nudibranchia) of the group with numerous morphological innovations (Wägele and Willan 2000
).
Our results are in full agreement with the traditional view that opisthobranchs are a natural group of gastropods (Thiele 19291935
) and contradict recent molecular studies (e.g., Thollesson 1999b;
Wollscheid and Wägele 1999
; Dayrat et al. 2001
) (fig. 1C
E). Our larger sequence data set and the lack of phylogenetically informative sites of previous molecular data sets (either because of their shorter size or their higher among-site rate variation) likely explain the discrepancy in resolution and statistical support between our results and those of previous studies. To test the validity of morphological hypotheses (Salvini-Plawen and Steiner 1996
; Ponder and Lindberg 1997
; fig. 1A
and B) that challenge the monophyly of opisthobranchs, more opisthobranch as well as heterostrophan and pulmonate taxa need to be included in future phylogenetic analyses based on molecular data.
The monophyly of opisthobranchs is further supported by all phylogenetic analyses based on mitochondrial cox1, rrnL, nad6, and nad5 genes at the nucleotide level (fig. 7
). The relative position of the trnP gene between the trnA and nad6 genes seems to be a shared derived character of opisthobranchs. Within opisthobranchs, the monophyly of cephalaspideans is rejected because Pupa is closely related to Ascobulla (order Sacoglossa), and Chelidonura appears as sister group to either Aplysia (order Anaspidea) or Aplysia+Umbraculum (fig. 7
). This result is consistent with recent phylogenetic analyses based on morphological (Mikkelsen 1996
) and molecular (Thollesson 1999b
) data. According to morphological and molecular evidence, cephalaspideans can be separated into at least two groups: one basal to other opisthobranchs and the other related to anaspideans (Mikkelsen 1996
; Thollesson 1999b
). Our results only differ from these hypotheses in suggesting a close relationship of sacoglossans to basal cephalaspideans. This relationship is not that surprising because Ascobulla, which exhibits typical sacoglossan-type radular teeth, shares many external morphological characters with cephalaspideans (Mikkelsen 1998
). The order Notaspidea is represented in our molecular phylogeny by the genus Umbraculum which is placed close to Aplysia and Chelidonura (fig. 7
). This result supports previous morphological evidence that related some lineages of notaspideans to anaspideans (Schmekel 1985
). The order Notaspidea includes two distinct groups, Umbraculomorpha and Pleurobranchomorpha (Rudman and Willan 1998
). Several morphological characters such as an open seminal groove, a nonretractile penis, an albumen gland, plates in the gizzard, and the absence of a blood gland suggest that the Umbraculomorpha are closer to the Anaspidea than to the Pleurobranchomorpha (Schmekel 1985
). Furthermore, the latter group seems to be more closely related to nudibranchs (Schmekel 1985
; Wägele and Willan 2000
). In this regard, a recent molecular phylogeny based on mitochondrial rrnL gene sequence data suggests that Pleurobranchomorpha may even be placed deep within the nudibranchs (Thollesson 1999b
). The monophyly of nudibranchs (without considering Pleurobranchomorpha) is well supported by several morphological (Schmekel 1985
; Haszprunar 1988
; Salvini-Plawen and Steiner 1996
; Wägele and Willan 2000
) and molecular (Wollscheid and Wägele 1999
; Wollscheid et al. 2001
) studies. The two nudibranchs (Roboastra and Aeolidia) included in our study are grouped together in the recovered phylogeny.
In conclusion, our analyses show that the newly determined mitochondrial genome of the nudibranch R. europaea is similar in size and gene arrangement to the mitochondrial genome of the cephalaspidean P. strigosa. Both species represent the most derived and basal lineages of opisthobranchs, respectively. These two mitochondrial genomes show the greatest sequence similarity when compared with other gastropod mitochondrial genomes. All phylogenetic analyses performed in this study both at the amino acid (including two orders) and at the nucleotide (including five orders) level, as well as the relative position of the trnP gene, support the monophyly of opisthobranchs with respect to pulmonates. The monophyly of this latter group is supported by ML and Bayesian analyses and is not rejected by MP analyses. Nevertheless, more representatives of heterostrophans, opisthobranchs, and pulmonates need to be included in future analyses to confirm further the monophyly of these gastropod groups.
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Acknowledgements |
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Footnotes |
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Address for correspondence and reprints: Rafael Zardoya, Departamento de Biodiversidad y Biología Evolutiva, Museo Nacional de Ciencias Naturales, CSIC, José Gutiérrez Abascal, 2, 28006 Madrid, Spain. rafaz{at}mncn.csic.es
.
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References |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Adachi J., M. Hasegawa, 1996 Model of amino acid substitution in proteins encoded by mitochondrial DNA J. Mol. Evol 42:459-468[ISI][Medline]
Arnason U., X. Xu, A. Gullberg, 1996 Comparison between the complete mitochondrial DNA sequences of Homo and the common chimpanzee based on nonchimeric sequences J. Mol. Evol 42:145-152[ISI][Medline]
Bieler R., 1992 Gastropod phylogeny and systematics Annu. Rev. Ecol. Syst 23:311-338.[ISI]
Boore J. L., 1999 Animal mitochondrial genomes Nucleic Acids Res 27:1767-1780
Boore J. L., W. M. Brown, 1994 Complete DNA sequence of the mitochondrial genome of the black chiton, Katharina tunicata Genetics 138:423-443
Castresana J., 2000 Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis Mol. Biol. Evol 17:540-552
Clary D. O., D. R. Wolstenholme, 1985 The mitochondrial DNA molecule of Drosophila yakuba: nucleotide sequence, gene organization and genetic code J. Mol. Evol 22:252-271[ISI][Medline]
Cummings M. P., S. P. Otto, J. Wakeley, 1995 Sampling properties of DNA sequence data in phylogenetic analysis Mol. Biol. Evol 12:814-822[Abstract]
Dayrat B., A. Tillier, G. Lecointre, S. Tillier, 2001 New clades of Euthyneuran Gastropods (Mollusca) from 28S rRNA sequences Mol. Phylogenet. Evol 19:225-235[ISI][Medline]
Devereux J., P. Haeberli, O. Smithies, 1984 A comprehensive set of sequence analysis programs for the VAX Nucleic Acids Res 12:387-395[Abstract]
Felsenstein J., 1978 Cases in which parsimony and compatibility methods will be positively misleading Syst. Zool 27:401-410[ISI]
. 1985 Confidence limits on phylogenies: an approach using the bootstrap Evolution 39:783-791[ISI]
Fretter V. G., A. Graham, 1962 British prosobranch molluscs Ray Society, London
Gosliner T. M., 1985 Parallelism, parsimony and testing of phylogenetics hypotheses: the case of opisthobranch gastropods Pp. 105107 in E. S. Vrba, ed. Species and speciation. Transvaal Museum, Pretoria
Gosliner T. M., M. T. Ghiselin, 1984 Parallel evolution in opisthobranch gastropods and its implications for phylogenetic methodology Syst. Zool 33:255-274[ISI]
Haszprunar G., 1985 The Heterobranchiaa new concept of the phylogeny of the higher Gastropoda Z. Zool. Syst. Evolutionsforsch 23:15-37[ISI]
. 1988 On the origin and evolution of major gastropods group, with special reference to the streptoneura J. Molluscan Stud 54:367-441[ISI]
Hatzoglou E., G. C. Rodakis, R. Lecanidou, 1995 Complete sequence and gene organization of the mitochondrial genome of the land snail Albinaria coerulea Genetics 140:1353-1366
Hoffmann R. J., J. L. Boore, W. M. Brown, 1992 A novel mitochondrial genome organization for the blue mussel, Mytilus edulis Genetics 131:397-412
Huelsenbeck J. P., F. R. Ronquist, 2001 MrBayes: Bayesian inference of phylogeny Bioinformatics 17:754-755
Jones D. T., W. R. Taylor, J. M. Thornton, 1992 The rapid generation of mutation data matrices from protein sequences Comp. Appl. Biosci 8:275-282[Abstract]
Kishino H., M. Hasegawa, 1989 Evaluation of the maximum likelihood estimate of the evolutionary tree topologies from DNA sequence data, and the branching order in Hominoidea J. Mol. Evol 29:170-179[ISI][Medline]
Kocher T. D., W. K. Thomas, A. Meyer, S. V. Edwards, S. Paabo, F. X. Villablanca, A. C. Wilson, 1989 Dynamics of mitochondrial DNA evolution in animals: amplification and sequencing with conserved primers Proc. Natl. Acad. Sci. USA 86:6196-6200[Abstract]
Kurabayashi A., R. Ueshima, 2000a. Complete sequence of the mitochondrial DNA of the primitive opisthobranch gastropod Pupa strigosa: systematics implication of the genome organization Mol. Biol. Evol 17:266-277
. 2000b. Partial mitochondrial genome organization of the heterostrophan gastropod Omalogyra atomus and its systematic significance Venus 59:7-18
Maddison W. P., D. R. Maddison, 1992 MacClade: analysis of phylogeny and character evolution Sinauer Associates, Sunderland, Mass
Medina M., P. J. Walsh, 2000 Molecular systematics of the order Anaspidea based on mitochondrial DNA sequence (12S, 16S, and COI) Mol. Phylogenet. Evol 15:41-58[ISI][Medline]
Mikkelsen P. M., 1996 The evolutionary relationships of Cephalaspidea s. l. (Gastropoda: Opisthobranchia): a phylogenetic analysis Malacologia 37:375-442[ISI]
. 1998 Cylindrobulla and Ascobulla in the western Atlantic (Gastropoda, Opisthobranchia, Sacoglossa): systematic review, description of a new species, and phylogenetic reanalysis Zool. Scrip 27:49-71
Morton J. E., 1979 Molluscs. 5th edition Hutchinson & Co. Ltd., London
Olsen G. J., C. R. Woese, 1993 Ribosomal RNA: a key to phylogeny FASEB J 7:113-123
Palumbi S., A. Martin, S. Romano, W. O. McMillan, L. Stice, G. Grabowwski, 1991 The simple fool's guide to PCR Department of Zoology, University of Hawaii, Honolulu
Ponder W. F., D. R. Lindberg, 1997 Towards a phylogeny of gastropod molluscs: an analysis using morphological characters Zool. J. Linn. Soc 119:83-265[ISI]
Poulicek M., M.-F. Voss-Foucart, C. Jeuniaux, 1991 Regressive shell evolution among opisthobranch gastropods Malacologia 32:223-232[ISI]
Rawlings T. A., T. M. Collins, R. Bieler, 2001 A major mitochondrial gene rearrangement among closely related species Mol. Biol. Evol 18:1604-1609
Rodríguez F., J. F. Oliver, A. Marín, J. R. Medina, 1990 The general stochastic model of nucleotide substitution J. Theor. Biol 142:485-501[ISI][Medline]
Rudman W. B., 1972 A study of the anatomy of Pupa and Maxacteon (Acteonidae, Opisthobranchia) with an account of the breeding cycle of Pupa kirki J. Nat. Hist 6:547-560[ISI]
Rudman W. B., R. C. Willan, 1998 Opistobranchia Pp 9151035 in P. L. Beesley, G. J. B. Ross, and A. Wells, eds. Mollusca: the southern synthesis. Fauna of Australia, Vol. 5. CSIRO publishing, Melbourne
Russo C. A. M., N. Takezaki, M. Nei, 1996 Efficiencies of different genes and different tree-building methods in recovering a known vertebrate phylogeny Mol. Biol. Evol 13:525-536[Abstract]
Rzhetsky A., M. Nei, 1992 A simple method for estimating and testing minimum-evolution trees Mol. Biol. Evol 9:945-967
Saavedra C., M. I. Reyero, E. Zouros, 1997 Male-dependent doubly uniparental inheritance of mitochondrial DNA and female-dependent sex-ratio in the mussel Mytilus galloprovincialis Genetics 145:1073-1082
Salvini-Plawen L., G. Steiner, 1996 Synapomorphies and plesiomorphies in higher classification of Mollusca Pp. 2951 in J. Taylor, ed. Origin and evolutionary radiation of the Mollusca. The Malacological Society of London, London
Sasuga J., S. Yokobori, M. Kaifu, T. Ueda, K. Nishikawa, K. Watanave, 1999 Gene content and organization of a mitochondrial DNA segment of the squid Loligo bleekeri J. Mol. Evol 48:692-702[ISI][Medline]
Schmekel L., 1985 Aspects of the evolution within the opisthobranchs Pp. 221267 in E. R. Trueman and M. R. Clarke, eds. The Mollusca. Academic Press, London
Shimodaira H., M. Hasegawa, 1999 Multiple comparisons of log-likelihoods with applications to phylogenetic inference Mol. Biol. Evol 16:1114-1116
Spengel J., 1881 Die Geruchsorgane und das Nervensystem der Mollusken Z. Wiss. Zool. Leipzig 35:333-383
Strimmer K., A. von Haeseler, 1996 Quartet puzzling: a quartet maximum-likelihood method for reconstructing tree topologies Mol. Biol. Evol 13:964-969
Swofford D. L., 1998 PAUP*: phylogenetic analysis using parsimony (* and other methods). Version 4.0 Sinauer Associates, Inc., Sunderland, Mass
Templeton A. R., 1983 Phylogenetic inference from restriction endonuclease cleavage site maps with particular reference to the evolution of human and the apes Evolution 37:221-244[ISI]
Terrett J. A., S. Miles, R. H. Thomas, 1996 Complete DNA sequence of the mitochondrial genome of Cepaea nemoralis (Gastropoda: Pulmonata) J. Mol. Evol 42:160-168[ISI][Medline]
Thiele J., 19291935 Handbuch der Systematischen Weichtierkunde 4 volumes. Jena, Germany
Thollesson M., 1999a. Phylogenetic analysis of dorid nudibranchs (Gastropoda: Doridacea) using the mitochondrial 16S rRNA gene J. Molluscan Stud 65:335-353[Abstract]
. 1999b. Phylogenetic analysis of Euthyneura (Gastropoda) by means of the 16s rRNA gene: use of a fast gene for higher-level phylogenies Proc. R. Soc. Lond. B 266:75-83[ISI]
Thompson J. D., T. J. Gibson, F. Plewniak, F. Jeanmougin, D. G. Higgins, 1997 The CLUSTAL X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools Nucleic Acids Res 25:4876-4882
Tillier S., M. Masselot, J. Guerdoux, A. Tillier, 1994 Monophyly of major gastropod taxa tested from partial 28S rRNA sequences, with emphasis on Euthyneura and hot-vent limpets peltospiroidea Nautilus 2:122-140
Wägele H., R. C. Willan, 2000 Phylogeny of nudibranchia Zool. J. Linn. Soc 130:83-181[ISI]
Wilding C. S., P. J. Mill, J. Grahame, 1999 Partial sequence of the mitochondrial genome of Littorina saxatilis: relevance to gastropod phylogenetics J. Mol. Evol 48:348-359[ISI][Medline]
Winnepenninckx B., G. Steiner, T. Backeljau, R. de Wachter, 1998 Details of gastropod phylogeny inferred from 18S rRNA sequences Mol. Phylogenet. Evol 9:55-63[ISI][Medline]
Wollscheid E., J. L. Boore, W. M. Brown, H. Wägele, 2001 The phylogeny of Nudibranchia (Opisthobranchia, Gastropoda, Mollusca) reconstructed by three molecular markers Org. Diver. Evol 1:241-256
Wollscheid E., H. Wägele, 1999 Initial results on the molecular phylogeny of the Nudibranchia (Gastropoda, Opisthobranchia) based on 18s rRNA Mol. Phylogenet. Evol 13:215-226[ISI][Medline]
Yamazaki N., R. Ueshima, J. A. Terret, et al. (12 co-authors) 1997 Evolution of pulmonate gastropod mitochondrial genomes: comparisons of gene organizations of Euhadra, Cepaea and Albinaria and implications of unusual tRNA secondary structures Genetics 145:749-758
Yokobori S. I., S. Pääbo, 1995 Transfer RNA editing in land snail mitochondria Proc. Natl. Acad. Sci. USA 92:10432-10435[Abstract]
Yoon S. H., W. Kim, 2000 Phylogeny of some gastropod mollusks derived from 18s rRNA sequences with emphasis on the Euthyneura Nautilus 114:84-92
Zardoya R., A. Meyer, 1996 Phylogenetic performance of mitochondrial protein-coding genes in resolving relationships among vertebrates Mol. Biol. Evol 13:933-942
Zouros E., 2000 The exceptional mitochondrial DNA system of the mussel family Mytilidae Genes Genet. Syst 75:313-318[ISI][Medline]
Zouros E., A. O. Ball, C. Saavedra, K. R. Freeman, 1994 Mitochondrial DNA inheritance Nature 368:818.[ISI][Medline]