Molecular Biology and Evolution 19:1672-1685 (2002)
© 2002 Society for Molecular Biology and Evolution
The Complete Mitochondrial Genome of the Nudibranch Roboastra europaea (Mollusca: Gastropoda) Supports the Monophyly of Opisthobranchs
*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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The complete nucleotide sequence (14,472 bp) of the mitochondrial genome of the nudibranch Roboastra europaea (Gastropoda: Opisthobranchia) was determined. This highly compact mitochondrial genome is nearly identical in gene organization to that found in opisthobranchs and pulmonates (Euthyneura) but not to that in prosobranchs (a paraphyletic group including the most basal lineages of gastropods). The newly determined mitochondrial genome differs only in the relative position of the trnC gene when compared with the mitochondrial genome of Pupa strigosa, the only opisthobranch mitochondrial genome sequenced so far. Pupa and Roboastra represent the most basal and derived lineages of opisthobranchs, respectively, and their mitochondrial genomes are more similar in sequence when compared with those of pulmonates. All phylogenetic analyses (maximum parsimony, minimum evolution, maximum likelihood, and Bayesian) based on the deduced amino acid sequences of all mitochondrial protein-coding genes supported the monophyly of opisthobranchs. These results are in agreement with the classical view that recognizes Opisthobranchia as a natural group and contradict recent phylogenetic studies of the group based on shorter sequence data sets. The monophyly of opisthobranchs was further confirmed when a fragment of 2,500 nucleotides including the mitochondrial cox1, rrnL, nad6, and nad5 genes was analyzed in several species representing five different orders of opisthobranchs with all common methods of phylogenetic inference. Within opisthobranchs, the polyphyly of cephalaspideans and the monophyly of nudibranchs were recovered. The evolution of mitochondrial tRNA rearrangements was analyzed using the cox1+rrnL+nad6+nad5 gene phylogeny. The relative position of the trnP gene between the trnA and nad6 genes was found to be a synapomorphy of opisthobranchs that supports their monophyly.
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Introduction |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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Sea slugs and their relatives (Gastropoda: Opisthobranchia) are one of the most diverse and successful groups of mollusks. The 5,000 or so opisthobranch species currently recognized are classified into nine orders (Cephalaspidea, Acochlidea, Rhodopemorpha, Sacoglossa, Anaspidea, Notaspidea, Thecostomata, Gymnostomata, and Nudibranchia) (Rudman and Willan 1998
). They are distributed throughout the world and occur in almost every marine environment. Regression of the shell, development of aposematic colorations, and acquisition of toxic defenses are general evolutionary trends within the group (Poulicek, Voss-Foucart, and Jeuniaux 1991
). Traditionally, opisthobranchs have been recognized as a natural group and have been included within the gastropods together with prosobranchs and pulmonates (Thiele 1929–1935, pp. 1–1134
). Prosobranchs (which used to include caenogastropodans, heterostrophans, retigastropodans and other related groups) are currently considered to be a paraphyletic group (Haszprunar 1988
; Ponder and Lindberg 1997
; Winnepenninckx et al. 1998
; Yoon and Kim 2000
) and represent the most basal lineages of gastropods, with pulmonates as the closest relatives of opisthobranchs (Spengel 1881
; Fretter and Graham 1962, pp. 1–755
; Morton 1979, pp. 1–264
; Rudman and Willan 1998
). The latter two groups have several synapomorphies and are commonly referred to as Euthyneura (Spengel 1881
).
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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Molecular data can help in these cases by providing an independent set of characters. So far, most molecular studies on the phylogeny of opisthobranchs have been based on nuclear 18S and 28S rRNA sequence data (Tillier et al. 1994
; Winnepenninckx et al. 1998
; Wollscheid and Wägele 1999
; Yoon and Kim 2000
; Dayrat et al. 2001
). These studies recovered poorly resolved phylogenetic trees in which the relationships of opisthobranch orders were unclear and the monophyly of opisthobranchs was rejected (fig. 1C and D
). The lack of resolution of nuclear rRNA gene sequence data is likely because of an extensive rate heterogeneity among sites that significantly reduces the number of positions that are phylogenetically informative at any level of divergence (Olsen and Woese 1993
). A recent phylogeny of Euthyneura based on partial sequences of the mitochondrial rrnL gene rejected the monophyly of opisthobranchs as well as those of the orders Cephalaspidea and Nudibranchia (Thollesson 1999b
) (fig. 1E
). But these results were based on rather short sequence data. The phylogenies of some orders of opisthobranchs (Anaspidea and Nudibranchia) based on partial sequences of nuclear 18S rRNA and mitochondrial rrnS, rrnL, and cox1 genes were also published recently (Thollesson 1999a;
Medina and Walsh 2000
; Wollscheid et al. 2001
).
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 gastropods—three 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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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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DNA Extraction, Polymerase Chain Reaction Amplification, Cloning, and Sequencing
A single fresh specimen from Cabo de Trafalgar (Cádiz, Spain) was used to determine the sequence of the complete mitochondrial genome of R. europaea. Total cellular DNA was purified following a standard phenol-chloroform extraction. Universal primers were used to amplify fragments of the mitochondrial rrnS (H1478 and L1091; Kocher et al. 1989
) and rrnL (16Sar-L and 16Sbr-H; Palumbi et al. 1991, pp. 1–45
) genes by polymerase chain reaction (PCR). Standard PCR reactions containing 67 mM Tris-HCl, pH 8.3, 1.5 mM MgCl2, 0.4 mM of each dNTP, 2.5 µM of each primer, template DNA (10–100 ng), and Taq DNA polymerase (1 unit, Biotools) in a final volume of 25 µl were subjected to 30 cycles of denaturing at 94°C for 60 s, annealing at 42°C for 60 s, and extending at 72°C for 90 s. The amplified fragments were sequenced with the BigDye Deoxy Terminator cycle sequencing kit (Perkin-Elmer Biosystems) in an automated DNA sequencer (ABI PRISM 3100) using the PCR primers and following the manufacturer's instructions. 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, 1–2 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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Twenty-two more primers were designed on the basis of gastropod mitochondrial genome DNA sequences to amplify by standard PCR (see conditions described previously) overlapping fragments using the long PCR products as DNA templates (table 1 and fig. 2 ). The PCR products were cloned into the pGEM-T vector (Promega) and were sequenced using M13 universal primers (see conditions described previously). PCR amplification of a mitochondrial genome might introduce up to 0.25% mutations (Arnason, Xu, and Gullberg 1996
). Divergences of 0.25% correspond to variation at the intraspecific level. 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. 1–398
), 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 AY098927–AY098931.
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Results |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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Genome Organization and Structural Features
The gene order and main features of the mitochondrial genome of R. europaea are summarized in figure 3 . The total length of the mitochondrial DNA is 14,472 bp. The overall base composition of the major strand is A, 27.6%; T, 38.5%; C, 14.5%; and G, 19.4%. The mitochondrial genome of R. europaea encodes 2 rRNAs, 22 tRNAs, and 13 protein-coding genes (figs. 2 and 3 ). The major strand encodes 13 out of the 37 genes (trnQ; trnL(uur); atp8; trnN; atp6; trnR; trnE, rrnS; trnM; nad3; trnS(ucn), trnT; cox3). All protein-coding genes are separated by tRNA genes except two sets of genes (nad6/nad5/nad1 and nad4L/cob). A potential stem-loop secondary structure which could putatively serve as a signal for RNA-processing enzymes (Boore and Brown 1994
) was found between nad4L and cob genes, as well as between the nad6 and nad5 genes, but not between the nad5 and nad1 genes (fig. 4A
). The largest noncoding regions are located between trnC and trnQ genes (93 bp) and between cox3 and trnI genes (54 bp) (fig. 3
). The latter region has the potential to fold into a hairpin secondary structure (fig. 4B
).
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The mitochondrial rrnL (1,109 bp) and rrnS (740 bp) genes are located between trnV and trnL(cun) genes and between trnE and trnM genes, respectively (figs. 2 and 3 ). The mitochondrial genome of R. europaea contains 22 tRNA genes that range in size from 54 to 69 nucleotides (fig. 5 ). All the deduced tRNAs can be folded into a cloverleaf secondary structure with the exception of tRNA(S-UCN) and tRNA(S-AGN) that lack the DHU arm. Mismatches in the acceptor stems of tRNA(L-CUN) and tRNA(Y) (fig. 5 ) might be corrected by the tRNA editing described by Yokobori and Pääbo (1995)
. Interestingly, the trnC gene is located between trnH and trnQ genes and not between trnN and atp6 genes as in the mitochondrial genome of Pupa (Kurabayashi and Ueshima 2000a
). The anticodons of all Roboastra mitochondrial tRNAs are preceded by a thymine and followed by a purine.
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Seven out of the 13 protein-coding genes use ATG as the start codon (cox1, cob, cox2, atp8, atp6, cox3, and nad2). Other genes begin with TTG (nad6, nad5, and nad4L), ATA (nad3), ATT (nad1), and GTG (nad4) (fig. 3 ). Most Roboastra open reading frames end with TAA (nad6, cob, atp8, nad4, and nad2) or TAG (cox1, nad5, cox2, and atp6). The remaining have incomplete stop codons, either T (nad4L, nad3, and cox3) or TA (nad1) (fig. 3 ). In many mitochondrial genomes, nad4L has a complete stop codon and overlaps with a contiguous protein-coding gene. An alternative complete stop codon for nad4L can be postulated if it overlaps cob by eight nucleotides.
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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Sequence Variation and Phylogenetic Analyses
The deduced amino acid sequences of all 13 mitochondrial protein-coding genes were combined into a single data set that produced an alignment of 3,869 positions. Of these, 1,227 were excluded from the analyses because of ambiguity in the homology assignment, 980 were invariant, and 357 were parsimony-informative. The mean character distance between Roboastra and Pupa is 0.27. The mean character distance between Albinaria and Cepaea is 0.43. The average mean character distance between opisthobranchs and pulmonates is 0.40.
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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To include the caenogastropodan L. saxatilis into the phylogenetic analyses, a smaller subset of protein-coding genes including cox1, cox2, atp8, atp6, nad1, nad6, and cob was analyzed with MP, ME, ML, and Bayesian methods of phylogenetic inference. The alignment of 1,789 positions was analyzed. Of these, 689 were excluded from the analyses because of ambiguity in the homology assignment, 495 were invariant, and 249 were parsimony-informative. All phylogentic analyses recovered a monophyletic opisthobranchia clade and Littorina as the most basal taxon of the in-group (fig. 6B ). In MP (one single tree of 1,287 steps; CI = 0.91), ME (score = 0.94), and ML (log likelihood = -9,451.60) phylogenetic analyses, pulmonates were paraphyletic (fig. 6B ). In Bayesian phylogenetic analyses, however, pulmonates were monophyletic with a 98% posterior probability (not shown).
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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Rearrangements of the trnV, trnL(cun), trnA, and trnP genes located between the cox1, rrnL, and nad6 genes were analyzed by mapping the relative positions of these tRNA genes onto the recovered phylogeny (fig. 8 ). The trnV gene is located between the cox1 and rrnL genes in all in-group taxa. Cepaea only presents the trnL(cun) and trnA genes between the rrnL and nad6 genes. Albinaria presents the trnP gene between the trnL(cun) and trnA genes. With this data set, it is not possible to infer which tRNA genes were located between the rrnL and nad6 genes in the ancestors of euthyneurans and of pulmonates. In all opisthobranchs, the trnP gene is located between the trnA and nad6 genes (fig. 8 ). This relative position of the trnP gene seems to be a synapomorphy of opisthobranchs.
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Discussion |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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The mitochondrial genome of R. europaea is one of the smallest known among Metazoa. The small size of the genome of Roboastra is the result of its compact gene organization, the absence of long noncoding regions, and the reduced size of its genes. This feature seems to be common to all Euthyneura because the size of the genome of Roboastra is within the range of variation in genome size found in Pupa (14.2 kb; Kurabayashi and Ueshima 2000a
) and in pulmonates (14.1–14.5 kb; Hatzoglou, Rodakis, and Lecanidou 1995
; Terrett, Miles, and Thomas 1996
; Yamazaki et al. 1997
). The mitochondrial genome of Roboastra shows an A+T content (66.1%) similar to that of other mollusks (Hoffman, Boore, and Brown 1992
; Boore and Brown 1994
; Hatzoglou, Rodakis, and Lecanidou 1995
; Terrett, Miles, and Thomas 1996
; Yamazaki et al. 1997
; Sasuga et al. 1999
; Kurabayashi and Ueshima 2000a,
2000b
).
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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Of the two largest noncoding regions, that located between the cox3 and trnI genes is the only one that contains a sequence with the potential to fold into a hairpin structure (fig. 4B ). In addition, this region shows a high A+T content (87%). Both features are typical of invertebrate mitochondrial control regions (e.g., Drosophila; Clary and Wolstenholme 1985
). Hence, this region seems to be a good candidate to contain the signals associated with the origin of replication and transcription of the Roboastra mitochondrial genome.
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 1929–1935
) 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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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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Guillermo San Martín and Xavier Turón provided some opisthobranch samples. Thanks to two anonymous reviewers for providing helpful suggestions on the manuscript. C.G. was sponsored by a predoctoral fellowship of the Ministerio de Ciencia y Tecnología. This work received financial support from projects of the Ministerio de Ciencia y Tecnología to J.T. (REN2000-0890/GLO) and to R.Z. (REN2001-1514/GLO).
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Footnotes |
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Ross Crozier, Reviewing Editor
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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