Mitochondrial (mt) genomes are an evolutionary paradox. There are many reasons why genomes should not have persisted in mitochondria - and yet they have indeed persisted (Saccone et al. 2002). Mitochondrial genomes also exhibit features not seen, or not as pronounced, in nuclear genomes. Among these are biases in base composition that must have an influence on the protein subunits for which they code. Studies on mitochondrial codon usage investigating this phenomenon have mainly focused on vertebrates.

We have an ongoing program of sequencing and characterising mitochondrial genomes from parasitic flatworms (Le et al., 2000a, 2000b). The major classes of parasitic flatworms, Trematoda, Monogenea and Cestoda, belong to a larger monophyletic taxon, the Neodermata. This taxon is distinct from other members of the Platyhelminthes (Littlewood and Bray 2001). Arising largely from our previous work, it is now clear that mt genomes of neodermatans resemble those of other metazoans in their organisation (Le et al., 2000a, 2000b). We have previously noted striking differences in base composition among these genomes (Le et al., 2002b). Here, we report codon usage and associated phenomena for as many of these genomes as are currently available.

Available to us for analysis were DNA sequences encompassing all protein-coding genes of the mitochondria of 11 species of neodermatans, 5 trematodes and 6 cestodes. For an additional trematode species, Schistosoma malayensis, we had sequence for most of the protein-coding genes. Further information on the taxa, GenBank accession numbers and reference sources, are in the footnote to Table 1.

Sequences were aligned using AssemblyLIGN v 1.9c and analysed by MacVector 6.5.3 package (Oxford Molecular Group). Pairwise comparisons of nucleotide and amino acid (aa) sequences of individual genes were undertaken using ClustalW as incorporated into the MacVector 6.5.3 package. Base composition and codon usage was calculated with MacVector 6.5.3, the DNA Strider program (Douglas 1995) and MEGA v2.1 (Kumar et al., 2001 - Arizona State University, Tempe, Arizona, USA).

Translations were done using the neodermatan mt genetic code most recently discussed in Blair et al. (1999), Nakao et al. (2000) and Telford et al. (2000). This code differs from the universal code in that TGA specifies tryptophan, AGA and AGG specify serine, ATA specifies isoleucine and AAA specifies asparagine. Initiation and termination codons will be discussed further below.

The program Tree-Puzzle v5 (Strimmer and von Haeseler, 1996) was used to explore base composition variation and amino-acid composition variation among the mt genomes examined. This program uses a chi-square test to determine whether the base composition of each sequence is identical to the average base composition of the whole alignment.

Mitochondrial genes of neodermatans are all encoded on the same strand. All calculations of base composition, skew etc used the strand reported in GenBank which is equivalent in sequence to the mRNAs of the various genes.

Skew is the unequal representation on a single strand of complementary bases such as G and C, something frequently reported from mt genomes (e.g. Saccone et al., 2002). Skew was estimated using the AT and GC-skew indices (Perna and Kocher, 1995) where:

AT skew = (A-T)/(A+T) and

GC skew = (G-C)/(G+C)

Values for the skew indices can range from -1 to +1. A value of zero indicates that A = T or G = C in frequency on the strand being analysed. A negative value for AT skew implies that T occurs more frequently than A, and so on.

A first requirement in characterising genes is to determine where they start and end. ATG and TAG are regarded as the typical mt stop and start codons respectively in neodermatans. However, we have presented evidence (Blair et al., 1999; Le et al., 2000a) that TAA can also act as a stop codon and GTG as an alternative start codon in neodermatans (as reported for other metazoans: Wolstenholme, 1992). In this study, we have confirmed that these two codons (GTG and TAA) can act respectively to initiate and terminate a gene.

Using a similar approach, we have inferred that the initiation codon of cox1 in the cestode H. diminuta is GTT (Le et al., 2002b) (Table 1) thus agreeing with von Nickisch-Rosenegk et al. (2001). In the latter paper, the initiation codon for nad4 is stated to be ATT. However, an in-frame ATG is situated 6 codons further upstream and we regard this as the correct start codon.

Pairs of genes may overlap, leading to interpretive difficulties. For example, in T. crassiceps, the last A of the stop codon (TAA) of atp6 is shared with the ATG start codon in the following nad2 gene (Fig. 1A). The former could be interpreted as a truncated stop codon or as an actual overlap between two genes. Abbreviated stop codons (T or TA) are known from some metazoans (Wolstenholme, 1992). There is only one example of this among the neodermatans. In T. solium, nad1 ends with a T (Nakao, unpublished). Von Nickish-Rosenegk et al. (2001) suggested that cox1 in H. diminuta is terminated with such a codon (T). However, our later analysis including sequences from several other cestodes indicate that a normal stop codon (TAG) is present here in cestodes but this requires overlap with the downstream trnT (Le et al., 2002a).

Tree-Puzzle always rejected the null hypothesis of equal base or amino-acid composition. A partial explanation for this might be that Tree-Puzzle requires a minimum of four species before it will run the analysis, but we did not have four sufficiently close relatives to include. When only four taeniid cestodes, E. granulosus G1 (sheep-dog strain) and G4 (horse-dog strain) genotypes, E. multilocularis and T. solium were included, all failed the test, although marginally in the case of the E. granulosus G4 genotype and E. multilocularis.

Associated with codon bias is the phenomenon of skew (unequal representation on a single strand of complementary bases). A well-developed theory is available to explain this situation, at least in the case of mammals (Saccone et al., 2002). The asymmetric nature of mt replication means that one of the strands remains in a single-stranded state for relatively long periods. During this time it is prone to particular mutational changes, specifically a reduction in C and A on that strand and a corresponding increase in G and T. Skew is likely to be most pronounced at third codon positions, and especially at four-fold degenerate sites, where any mutational change is synonymous and not subject to selection pressure. Although little is known about the mode of mt replication in phyla other than vertebrates, an echinoderm and a few insects, base composition bias and strong skew are observed in many phyla (Saccone et al., 1999) including flatworms, suggesting that similar mechanisms may operate. As predicted by the theory outlined above, base composition bias and skew are most evident in third codon positions among neodermatans. The pattern observed among neodermatans is similar to that seen in vertebrates, with four-fold redundant sites showing the most extreme skew (Fig. 2). The main difference is that, among vertebrates, GC-skew has a negative value and AT-skew a positive value (Perna and Kocher, 1995). Nematodes and at least some molluscs exhibit negative AT-skew values and positive GC-skew values, as in neodermatans (Perna and Kocher, 1995). The sign of the skew value reflects only the strand being investigated: each strand will have the same value for each skew statistic, but with the opposite sign (Perna and Kocher, 1995).

Reyes et al. (1999) found that, among mammals, bias and skew were greatest in the regions of the mt genomes where the heavy strand was exposed as single-stranded for the longest time during replication. If the mode of replication in neodermatans is similar to that in mammals, we might expect the same pattern. Given that the locations of the origin(s) of replication are unknown in neodermatan mt genomes, and that they probably differ among taxa (as evidenced by the differing location of long non-coding regions (Le et al., 2002a), we did not feel able to explore this in detail. However, marked differences in skew and bias were noted among genes in a single neodermatan genome (data not shown) as reported by Reyes et al. (1999) for mammals. Analysis of sequences from additional neodermatan taxa may make it possible to infer the nature of the replication process.

Deviations in base composition (and amino-acid composition) among species will violate a basic assumption implicit in many algorithms used to infer phylogenies and can lead to construction of incorrect topologies (Foster and Hickey, 1999). Despite the considerable differences among neodermatan species in nucleotide sequences, and corresponding differences in amino-acid sequences, phylogenetic trees (not shown) inferred from either class of data recover the topology expected from the known systematic relationships among these taxa. This should not be taken as evidence that base composition differences will not affect tree topologies when sequences from a wider array of neodermatan taxa are added. All but two of the species included here fall into two relatively narrow but well-separated systematic groups (trematodes of the family Schistosomatidae and cestodes of the order Cyclophyllidea). Base composition differences would presumably have to be extreme to fail to recover these two groups. The two remaining trematodes, P. westermani and F. hepatica, which have similar base compositions, form a group elsewhere in the tree.

Previous studies have revealed that mt genomes of neodermatans are similar in most respects to those of other bilateral metazoans (Le et al., 2000a). However, these genomes vary considerably in the location(s) of long non-coding regions presumed to have a role in replication, and in the length and structure of these regions (Le et al., 2000a). There is also variation in gene order which, in African schistosomes, is very different from that of all other taxa sequenced to date (Le et al., 2001). Here, we have shown that there can be convergence as well as divergence in nucleotide, and hence amino acid, composition among taxa. A pattern is thus emerging of diversity in the mt genome in neodermatans that may cast light on evolution of mt genomes generally. Certainly, there is a need of data from additional neodermatan taxa, and from flatworms generally. Once these patterns are better understood, mt genome data can be put to practical use in evolutionary and population/species-level studies of important parasites such as the schistosomes (Le et al., 2000b).