Partial Sequence of the Mitochondrial Genome of Littorina saxatilis: Relevance to Gastropod Phylogenetics*

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1 J Mol Evol (1999) 48: Springer-Verlag New York Inc Partial Sequence of the Mitochondrial Genome of Littorina saxatilis: Relevance to Gastropod Phylogenetics* Craig S. Wilding, Peter J. Mill, John Grahame School of Biology, The University of Leeds, Leeds LS2 9JT, UK Received: 4 June 1998 / Accepted: 20 August 1998 Abstract. A 8022 base pair fragment from the mitochondrial DNA of the prosobranch gastropod Littorina saxatilis has been sequenced and shown to contain the complete genes for 12 transfer RNAs and five protein genes (CoII, ATPase 6, ATPase 8, ND1, ND6), two partial protein genes (CoI and cyt b), and two ribosomal RNAs (small and large subunits). The order of these constituent genes differs from those of other molluscan mitochondrial gene arrangements. Only a single rearrangement involving a block of protein coding genes and three trna translocations are necessary to produce identical gene orders between L. saxatilis and K. tunicata. However, only one gene boundary is shared between the L. saxatilis gene order and that of the pulmonate gastropod Cepaea nemoralis. This extends the observation that there is little conservation of mitochrondrial gene order amongst the Mollusca and suggests that radical mitochondrial DNA gene rearrangement has occurred on the branch leading to the pulmonates. Key words: Littorina saxatilis Mitochondrial genome Gastropod phylogenetics Gene order Mollusca * The nucleotide sequence data reported in this paper will appear in the DDBJ/EMBL/GenBank nucleotide sequence databases with accession number AJ Correspondence to: C.S. Wilding Introduction With few exceptions, the circular mitochondrial genome of the Metazoa contains 13 protein coding genes, 2 ribosomal RNAs (rrna genes), and 22 transfer RNAs (trna genes). Although the number and nature of constituent genes remain constant, their organization around the circle does not, resulting in considerable differences in mitochondrial genome organization between taxa (Wolstenholme 1992). However, because of the immense number of possible arrangements of the constituent genes, it is unlikely that taxa will converge on identical arrangements independently, and thus identical gene order, or the presence of shared gene boundaries, is likely to indicate common ancestry. Thus analysis of mtdna gene order is expected to provide important data for understanding deep phylogenies (Boore and Brown 1994b; Boore et al. 1995). The elucidation of complete mitochondrial sequences from a range of taxa has allowed limited testing of this theory, and indeed it seems that closely related taxa typically do display similar gene orders, and within a phylum, gene order is typically conserved. Thus the 37 constituent genes of the mtdna are arranged in the same order for most vertebrates examined thus far (see Wolstenholme 1992, and references therein). The exceptions concern the marsupials, where there has been some rearrangement of trna genes (Pääbo et al. 1991); birds, where ND6 and trna glu have been transposed relative to cytochrome b; trna thr and trna pro (Desjardins and Morais 1990; Desjardins et al. 1990); and minor rearrangements in certain reptiles argued to result from movement of the light strand origin of replication (Macey et al. 1997). This tendency for a consensus gene order within a phylum also holds for the Echinodermata, where echinoids differ from asteroids in only a single transversion (Smith et al. 1993). Even within the largest phylum, the arthropods, gene orders

2 349 for the protein coding and ribosomal rrna genes are constant (Staton et al. 1997) and only the position of transfer RNAs varies (Boore et al. 1995; Staton et al. 1997). In contrast, within the Mollusca, there are divergences in mitochondrial gene order. This is the second largest phylum, comprising seven classes: Aplacophora (e.g., solenogasters), Monoplacophora (Neoplina), Polyplacophora (chitons), Scaphopoda (tusk shells), Bivalvia (clams and mussels), Gastropoda (slugs and snails), and Cephalopoda (octopods and squids). The phylogeny of the Mollusca is ambiguous. A number of alternative phylogenies have been proposed by various authors (Boore and Brown 1994b; Ponder and Linberg 1996, 1997), with even some debate still ongoing concerning monophyly of the phylum (Boore and Brown 1994b). Mitochondrial gene order may provide valuable information for phylogeny construction or resolution of polytomies. Despite the diversity of form within the phylum and the evidence that mtdna gene order can provide valuable information for phylogeny reconstruction, few molluscan mitochondrial gene orders are known. The only available mitochondrial gene arrangements are for the polyplacophoran Katharina tunicata (Boore and Brown, 1994a), the bivalve mollusk Mytilus edulis (Hoffmann et al. 1992), and three pulmonate gastropods, Albinaria coerulea (Hatzoglou et al. 1995), Cepaea nemoralis (Terrett et al. 1996), and Euhadra herklotsi (Yamazaki et al. 1997). These three groupings, the Polyplacophora, mytilid bivalves, and pulmonate gastropods, all display substantial differences in mitochondrial gene order, suggesting that mitochondrial gene order may provide useful characters for phylogeny analysis in this phylum. However, no information is yet available on the extent of gene order rearrangements within a class [although Yamazaki et al. (1997) describe limited differences within the pulmonates] and such information is vital to understand the great differences already seen between classes. Here we report on sequence information of approximately half the mitochondrial DNA of the prosobranch gastropod Littorina saxatilis (Olivi). The availability of further mtdna gene orders such as this is essential for examining the usefulness of mtdna gene organisation for the study of mollusk relationships. PCR Amplification of Mitochondrial DNA Amplification of mtdna was initially undertaken in an effort to obtain sequence to enable primers to be designed for the C terminus of the cytochrome oxidase gene for use in population-level sequencing studies (Wilding et al. unpublished data) of this region, which is known to be highly polymorphic in insects (Lunt et al. 1996). Preliminary sequence of the N terminus of the gene was obtained using the universal CoI primers of Folmer et al. (1994). From this a new primer (CoI-long) was designed and used in conjunction with each of the universal cyt b primers of Kocher et al. (1989). Because we had no prior information on gene order, CoI-long was used with each cyt b primer. The Expand Long Template PCR System (Boehringer Mannheim) was employed to reduce PCR errors to an absolute minimum. This uses Taq enzyme as the main polymerase with a low-concentration proofreading Pwo enzyme. Approximately 8 kb of mtdna was PCR amplified from genomic DNA using the primers CoI-long (5 -GCT CAT GCT GGG GGC TC-3 ) and cyt b-rev (5 -AGG GAA CTT TTT CTC CAT CTC TGT- 3 ). Two fragments of 5614 and 2237 bp were amplified. From sequence information these were shown to be near-contiguous and the interim sequence was then amplified using primers (ATPF 5 -ATG CCT CAA CTA TCC CCT-3 and ATPR 5 -CTA ATG AGG ATG TTC GTC TGA-3 ) designed from the original sequence data. This PCR fragment was then sequenced using the amplification primers and one internal primer. Both larger fragments were cloned into PCR-script SK+ (Stratagene). Subcloning of the two larger fragments was performed through religation following restriction digestion with a variety of enzymes recognizing sites within the inserts and vector MCS (Fig. 1). The original and subclones were sequenced using standard sequencing primers (M13R and M13-20) by MWG Biotechnology (Milton Keynes, UK). Where these subclones did not adequately cover the sequence, internal sequencing primers were designed and sequencing was performed on an ABI377. The sequencing strategy is depicted in Fig. 1. Sequence Analysis Open reading frames were detected using MAP in the GCG suite of programs (Devereux et al. 1984), initially using the Drosophila yakuba genetic code (Clary and Wolstenholme 1985), and genes identified using BLASTX (Altschul et al. 1995). Open reading frames with limited homology to mitochondrial proteins were identified through comparison of hydropathy profiles with those of the chiton Katharina tunicata (Boore and Brown 1994a) and Drosophila yakuba (Wolstenholme and Clary 1985). Hydropathy profiles (Kyte and Doolittle 1982) were constructed using PEPPLOT in the GCG package. Transfer RNA genes were identified using the trna scan program (Lowe and Eddy 1997). Alignment of amino acid sequences was achieved using CLUSTALW (Thompson et al. 1994). Results and Discussion Materials and Methods DNA Extraction Total genomic DNA was extracted from the head foot region of two Littorina saxatilis collected from the Gann estuary, Pembrokeshire, Wales, using the protocol of Ashburner (1989). Two large filaments (5614 and 2237 bp) of L. saxatilis mtdna were PCR amplified and sequenced. Paradoxically each had been primed by a single (but different) primer the larger fragment by CoI-long and the smaller by cyt b-rev. This self-priming was probably mediated by the low annealing temperature used in the PCR amplification. Bridging sequence was obtained to yield a

3 350 Fig. 1. Sequencing strategy of cloned L. saxatilis mtdna fragments. Two fragments (A and B) were PCR amplified and cloned (see text) and the intervening fragment amplified using primers complementary to the ends of the cloned regions. Subcloning was performed with EcoRI, HindIII, KpnI, PstI, SacI, and SalI. Arrows below fragments depict the orientation and length of sequences obtained using universal sequencing primers. Arrows above the sequence depict the orientation and length of sequences obtained using primers designed from the Littorina sequence. Below: Inferred gene order. CoI and CoII, subunits I and II of cytochrome c oxidase. A6 and A8, subunits 6 and 8 of mitochondrial ATP synthase. s-rrna and l-rrna, small (12S) and large (16S) subunit ribosomal RNA. ND1 and ND6, subunits 1 and 6 of NADH dehydrogenase. Cyt b, cytochrome b apoenzyme. Transfer RNA genes are denoted by their single-letter amino acid codes. All genes are transcribed from left to right except those that are underlined. total of 8022 bp of mitochondrial sequence (Fig. A1). The exact length of L. saxatilis mtdna has not been determined. However, Crossland (1994) crudely estimated the size at 14.5 kbp from a clone library probed with an L. littorea clone, suggesting that there is approximately 55% of the L. saxatilis mtdna cloned in the present study. Gene content and order The 8 kb of L. saxatilis partial mtdna sequence reported here contains genes for 7 proteins (ATPase 6, ATPase 8, CoI, CoII, cytochrome b, ND1, and ND6), 2 ribosomal RNAs (large and small subunits), and 12 trnas (Fig. 1). All genes are transcribed from the same strand with the exception of a single block of eight trnas. No protein coding genes overlap, although trna trp and trna gln havea4bpoverlap and trna gly and trna glu overlap by 2 bp. Only 78 bp of the 8022 bp sequence (0.97%) have not been ascribed a function; 48 bp of this apparent noncoding sequence is represented by small (<10 bp) intergenic regions. The remaining 30 bp is situated between the CoI and the CoII genes. The polyplacophoran K. tunicata (Boore et al. 1994) and the four gastropods for which mtdna gene order information is available [A. coerulea (Hatzoglou et al. 1995), C. nemoralis (Terrett et al. 1996), E. herklotsi (Yamazaki et al. 1997), and L. saxatilis (this study)] all contain an ATPase 8 gene. However, this gene is absent in the bivalve M. edulis (Hoffmann et al. 1992). As in K. tunicata but unlike for the pulmonates, the L. saxatilis ATPase 8 gene follows on directly from ATPase 6. Terrett et al. (1996) suggested that, as ATPase 8 has been lost independently from the nematode lineage and from M. edulis, a broader depth of sampling was needed to clarify where the loss had occurred. It seems likely that the gene is universally present in the gastropods but a wider range of sampling throughout the Mollusca is necessary to understand the distribution of ATPase 8 in this phylum. The gene arrangement of L. saxatilis mtdna resembles that of the chiton (Boore and Brown 1994a) far more closely than it does the gene orders of pulmonate gastropods, e.g., C. nemoralis (Fig. 2). Disregarding trna translocations, only one rearrangement is necessary to produce identical gene orders (of the seven protein coding genes and two rrna genes sequenced in the present study) between L. saxatilis and K. tunicata, while at least seven separate translocations of protein coding regions are necessary to produce an arrangement similar to that of C. nemoralis. The 12 trna portions are also more similar between L. saxatilis and K. tunicata; only three movements are necessary to produce identical orders. Comparisons of the trna orders between L. saxatilis and C. nemoralis reveal a radically different gene order, with only one (trna glu ; E, Fig. 2) at the same gene boundary (A6 s-rrna) in both organisms. One trna rearrangement is particularly interesting. In L. saxatilis the arrangement 1-rRNA/tRNA leu(uur) /

4 351 Fig. 2. Comparison of mitochondrial gene order in three species of mollusk: Littorina saxatilis (this study), Katharina tunicata (Boore and Brown 1994a), and Cepaea nemoralis (Terrett et al. 1996). All genes are transcribed from left to right except where underlined. Transpositions of protein coding genes are denoted by lines connecting genes or blocks of genes when marked by a bar, and inversions are denoted by lines with encircling arrows. trna leu(cun) /ND1 is found. In K. tunicata (Boore and Brown 1994a) and also, and more pertinently, in the prosobranch gastropod Plicopurpurata columellaris (Boore et al. 1995), the arrangement is 1-rRNA/ trna leu(cun) /trna leu(uur) /ND1. Littorina and the muricid Plicopurpurata are both members of the Caenogastropoda, to which the Littorinidae form the most basal group (Winnepenninckx et al. 1998), and therefore since Plicopurpurata appears to have the ancestral gene arrangement, the trna translocation seen in Littorina is presumably limited to the Littorinidae or part of this group. As in most mtdnas the partial L. saxatilis mtdna is strongly AT rich (66.94% AT). This AT bias does seem to affect codon usage. For the third position of codons, 1210 (76.5%) end in A or T and the 3 most common codons, TTT (F), TTA (L), and ATT (I), are composed solely of A and T. Translation Initiation and Termination Prediction of translation initiation and termination sites can be undertaken with absolute certainty only through transcript mapping. Here we infer these sites through the presence of apparent stop and start codons bounding open reading frames of a length comparable to those of other mtdnas. The six protein coding genes for which we have sequenced the initiation sites all have methionine as an initiation codon. Acceptance of this results in genes of lengths comparable to those of other mollusks (K. tunicata and C. nemoralis; Table 1). All genes identified end in complete (TAA or TAG) termination codons. There appear to be no incomplete (TA or T) codons as in C. nemoralis and K. tunicata. There is an alternative ATA start codon four codons upstream of the predicted ATG start of ATPase 6. This would reduce the noncoding region between ATPase 8 and ATPase 6 from 13 to 1 bp. We have chosen the ATG codon, as this results in an ATPase 6 gene of standard length with good alignment with the initial amino acid sequence of the K. tunicata sequence (K. tunicata, MMMDIFSSFDDN; L. saxatilis, MLVDIFSSFDDN; differences boldfaced). Genetic Code The mitochondrial genetic code differs from the universal genetic code for all mtdnas (Wolstenholme 1992). The genetic code used here is that used to translate Drosophila, Katharina, and Cepaea mitochondrial genomes. The differences between this code and the standard genetic code are discussed with reference to L. saxatilis.

5 352 Table 1. Protein Inferred sizes, a initiation and termination codons, and calculated identity b of seven genes in three mollusk species Percentage amino acid identity Number of amino acids Initiation and termination codons Littorina/ Littorina/ Littorina/ Littorina Katharina Cepaea Katharina Cepaea Drosophila Littorina Katharina Cepaea ATP ATG TAG ATG TAA TA ATP ATG TAG ATG TAG TAA CoI 371 (partial) ? TAA ATG T TAA CoII ATG TAA ATG TAG TAA Cyt b 211 (partial) ATG? ATG TAA TAA ND ATG TAA ATG TAA TA ND ATG TAG ATG T TA a CoI and cyt b in L. saxatilis are incomplete. b Identities calculated following alignment with CLUSTALW (Thompson et al. 1994) and dividing the number of identical amino acids by the average length of the compared sequences. Table 2. Codon usage in L. saxatilis mtdna a Amino acid Codon N % Amino acid Codon N % Amino acid Codon N % Amino acid Codon N % Phe TTT Ser TCT Tyr TAT Cys TGT (GAA) TTC (UGA) TCC (GUA) TAC (GCA) TGC Leu TTA TCA TER TAA Trp TGA (UAA) TTG TCG TAG (UCA) TGG Leu CTT Pro CCT His CAT Arg CGT (UAG) CTC (UGG) CCC (GUG) CAC (UCG) CGC CTA CCA Gln CAA CGA CTG CCG (UUG) CAG CGG Ile ATT Thr ACT Asn AAT Ser AGT (GAU) ATC (UGU) ACC (GUU) AAC (GCU) AGC Met ATA ACA Lys AAA AGA (CAU) ATG ACG (UUU) AAG AGG Val GTT Ala GCT Asp GAT Gly GGT (UAC) GTC (UGC) GCC (GUG) GAC (UCC) GGC GTA GCA Glu GAA GGA GTG GCG (UUC) GAG GGG a N, number of occurrences; %, percentage of total amino acids. ATA Codes for Methionine. ATA encodes isoleucine in the standard genetic code but methionine in the mtdna of many organisms. The ATA codon occurs 47 times in the L. saxatilis sequence (Table 2). Of the corresponding positions in the K. tunicata sequence, 28 encode methionine (21 ATA, 7 ATG), 8 encode leucine (6 TTA, 2 TTG), 2 encode isoleucine (ATT), 2 phenylalanine (TTT), and 7 for other amino acids. The much higher proportion of corresponding amino acids corresponding to methionine as opposed to isoleucine suggests ATA encodes methionine in L. saxatilis. TGA Specifies Tryptophan, Not Termination. TGA is a common codon in the L. saxatilis sequence, accounting for 2.6% of all codons (Table 2). Specification of TGA as a termination codon would severely truncate all proteins. Of the 41 occurrences of TGA, the homologous position in the sequence of K. tunicata was tryptophan in 37 cases (also 2 phenylalanine TTT, 1 serine TGT, and 1 leucine TTA). We therefore accept TGA as specifying tryptophan as in Drosophila. AGA and AGG Specify Serine. AGA and AGG encode a variety of amino acids in various taxa. In the universal code, AGA specifies arginine but in D. yakuba codes for serine. In L. saxatilis both AGA and AGG codons are represented, although AGG is rare, occurring only twice (0.13%). When aligned to the K. tunicata amino acid sequence the two AGG codons align with serine (AGC and AGA), while, for the 19 AGA codons, seven correspond to serine. The remaining 12 align with a variety of other amino acids but none with arginine. We believe this to confirm that AGA/AGG specifies serine in L. saxatilis.

6 353 Fig. 3. Kyte and Doolittle (1982) hydropathy profiles for three genes (ND6, ATPase 6, and ATPase 8) in three species, Littorina saxatilis (this study), Katharina (Boore and Brown 1994a), and Drosophila yakuba (Clary and Wolstenholme 1985). Profiles were computed using PEPPLOT with a search window of 7. Values along the x-axis are the amino acid position, while the y-axis represents hydrophobicity: values above zero are hydrophobic, and negative values are hydrophilic. Protein Coding Genes Seven protein coding genes were identified. We have no information on the presence/absence of the other six genes usually found in metazoan mtdna. CoI, CoII, ND1, and cyt b could all be identified unambiguously from amino acid similarity to other mitochondrial genomes. ATPase 6, ATPase 8, and ND6 showed only weak similarity to the corresponding sequences of other animals. However, hydropathy profiles (Fig. 3) provide strong evidence that these genes are correctly identified. The lengths of the five fully sequenced genes appear more similar to K. tunicata than to C. nemoralis (Table 1). The similarity of lengths for L. saxatilis, K. tunicata, and D. yakuba but dissimilarity to C. nemoralis suggests that the coding sequences of Cepaea are unusually short.

7 354 Fig. 4. Predicted secondary structure of 12 trna genes in L. saxatilis.

8 355 Fig. 5. Potential secondary structure of the noncoding region between CoI and CoII. Numbers refer to the position in the L. saxatilis mitochondrial sequence (Fig. A1). The bar represents the termination codon at the end of CoI. L. saxatilis sequences also appear more similar in amino acid sequence to K. tunicata than to C. nemoralis (Table 1). In fact, sequence identity is greater between L. saxatilis and D. yakuba than between L. saxatilis and C. nemoralis over all seven genes. Transfer RNAs Twelve sequences could be folded into typical trna structures (Fig. 4). Five of these have mismatches in the amino-acyl stem. Three of these (trna pro, trna cys, and trna val ) involve only 1 bp, while trna tyr and trna gln involve two and three, respectively. For trna gln a better match would be possible if the A at nucleotide 3135 were dropped. This would have a perfect trna. Sequencing of an alternative clone was undertaken to check if this represented a PCR error, but the sequence was identical. All of the trna secondary structures predicted here demonstrate both a variable loop and a T C arm. There is no evidence of TV replacement loops as have been demonstrated in gastropods (Yamazaki et al. 1997) but not in the chiton (Boore and Brown 1994a). Transfer RNA genes are believed to act as processing signals for cleavage of the polycistronic transcript into gene-specific RNAs (Ojala et al. 1981). However, three gene boundaries (CoI/CoII, A8/A6, and ND6/Cyt b) are not separated by a trna. At the CoI/CoII boundary there is stem-loop structure within the apparent noncoding region which may serve a similar purpose (see Fig. 5). Comparable structures for the other two boundaries would have to be folded from within coding sequence. Ribosomal RNAs Determination of the absolute 3 end of the small and large subunit RNAs can be reliably performed only using S1 hybridization-protection analysis, while 5 RACE can be used to locate the 5 terminus (e.g., see Pont-Kingdon et al. 1994). We did not determine these exactly. Instead the termini of these genes have been given as abutting the neighboring genes to produce a s-rrna of 964 bp and a 1-rRNA of 1382 bp. These rrna genes are larger than those of the chiton (826 and 1275 bp) but not uncharacteristically large when other organisms are considered, e.g., s-rrna and 1-rRNA are 945 and 1245 bp in Mytilus (Hoffmann et al. 1992), 955 and 1570 bp in Mus musculus (Bibb et al. 1981), and 880 and 1525 bp in echinoderms (Jacobs et al. 1988). Evolutionary Implications Substantial differences in gene order exist between three distantly related mollusk groups, the bivalves represented by M. edulis (Hoffmann et al. 1992), the chitons represented by K. tunicata (Boore and Brown 1994a), and the pulmonates (Hatzoglou et al. 1995; Terrett et al. 1996; Yamazaki et al. 1997). In this study we show that the considerable differences seen in mtdna gene order between classes of Mollusca are also seen within a class (the Gastropoda), with the mtdna of L. saxatilis having a radically different gene order from that of the pulmonate gastropods (Yamazaki et al. 1997). Indeed, the mtdna of L. saxatilis appears more similar to that of K. tunicata (Boore and Brown 1994a) but still differs due to a substantial inversion and three trna translocations. Even within the prosobranch gastropods there is evidence of mtdna gene rearrangement, with switching of the position of trna leu(cun) and trna leu(uur) when L. saxatilis mtdna is compared with that of Plicopurpurata (Boore et al. 1995). The most recent morphological phylogeny of the mollusks (Ponder and Linberg, 1997) places the Littorinidae in the caenogastropod clade, which in turn forms the apogastropod clade with the Pulmonata and Opisthobranchia. This clade is separated from the Polyplacophora by numerous groups including the Archaegastropoda, Neritopsina, Patellogastropoda, and monoplacophorans (Ponder and Linberg, 1996; Fig. 5). In this phylogeny, therefore, and indeed under any of the alternatives (see Ponder and Linberg 1996), the Littorinidae appear much more closely related to members of the Pulmonata than to the very distant Polyplacophora. Given that convergence on similar gene orders is unlikely, the resemblance of L. saxatilis mtdna to K. tunicata mtdna suggests that some of the gene order is plesiomorphic and maintained throughout most of the Gastropoda, the pulmonate branch being an important exception. It is unclear in the absence of additional genome organizations whether the translocations that distinguish pulmonate mtdna from prosobranch (or polyplacophoran) mtdna occurred on the branch leading to the Opisthobranchia and Pulmonata or on the pulmonate branch. To examine this hypothesis further requires data on additional molluscan mitochondrial genomes.

9 356 Appendix Fig. A1. Sequence of 8022 bp of L. saxatilis mtdna.

10 357 Fig. A1. Continued.

11 358 Fig. A1. Continued.

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