The effective number of codons (N_c) is a widely used index for characterizing codon usage bias because it does not require a set of reference genes as does codon adaptation index (CAI) and because of the freely available computational tools such as CodonW. However, N_c, as originally formulated has many problems. For example, it can have values far greater than the number of sense codons; it treats a 6-fold compound codon family as a single-codon family although it is made of a 2-fold and a 4-fold codon family that can be under dramatically different selection for codon usage bias; the existing implementations do not handle all different genetic codes; it is often biased by codon families with a small number of codons. We developed a new N_c that has a number of advantages over the original N_c. Its maximum value equals the number of sense codons when all synonymous codons are used equally, and its minimum value equals the number of codon families when exactly one codon is used in each synonymous codon family. It handles all known genetic codes. It breaks the compound codon families (e.g., those involving amino acids coded by six synonymous codons) into 2-fold and 4-fold codon families. It reduces the effect of codon families with few codons by introducing pseudocount and weighted averages. The new N_c has significantly improved correlation with CAI than the original N_c from CodonW based on protein-coding genes from Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, Escherichia coli, Bacillus subtilis, Micrococcus luteus, and Mycoplasma genitalium. It also correlates better with protein abundance data from the yeast than the original N_c.

We here report the first complete mitochondrial (mt) genome of a skipper,Ctenoptilum vasavaMoore, 1865 (Lepidoptera: Hesperiidae: Pyrginae). The mt genome of the skipper is a circular molecule of 15,468 bp, containing 2 ribosomal RNA genes, 24 putative transfer RNA (tRNA), genes including an extra copy of trnS (AGN) and a tRNA-like insertion trnL (UUR), 13 protein-coding genes and an AT-rich region. All protein-coding genes (PCGs) are initiated by ATN codons and terminated by the typical stop codon TAA or TAG, except for COII which ends with a single T. The intergenic spacer sequence between trnS (AGN) and ND1 genes also contains the ATACTAA motif. The AT-rich region of 429 bp is comprised of nonrepetitive sequences, including the motif ATAGA followed by an 19 bp poly-T stretch, a microsatellite-like (AT)₃(TA)₉element next to the ATTTA motif, an 11 bp poly-A adjacent to tRNAs. Phylogenetic analyses (ML and BI methods) showed that Papilionoidea is not a natural group, and Hesperioidea is placed within the Papilionoidea as a sister to ((Pieridae+Lycaenidae)+Nymphalidae) while Papilionoidae is paraphyletic to Hesperioidea. This result is remarkably different from the traditional view where Papilionoidea and Hesperioidea are considered as two distinct superfamilies.

The complete mitochondrial genome sequences of the two butterfly species Euploea mulciber (Lepidoptera: Nymphalidae: Danainae) and Libythea celtis (Lepidoptera: Nymphalidae: Libytheinae) were determined in this study, comprising 15,166 bp and 15,164 bp, respectively. The orientation and the gene order of the two mitogenomes are identical to those of most of the other lepidopteran species. All protein-coding genes of Euploea mulciber and Libythea celtis mitogenomes start with a typical ATN codon with the exception of COI gene which uses CGA as its initial codon. All tRNA genes possess the typical cloverleaf secondary structure except for tRNA^Ser (AGN), which has a simple loop with the absence of the DHU stem. There are short microsatellite-like repeat regions, but no conspicuous macrorepeats scattered throughout the A + T-rich regions. Phylogenetic analysis among the available butterfly species suggests that Libythea celtis (Libytheinae) is closely related to Calinaga davidis (Calinaginae), indicating that the subfamily Libytheinae may not represent a basal lineage of the Nymphalidae as previously suggested, and that Euploea mulciber stands at the base of the nymphalid tree as a sister to all other nymphalids.