Rare genomic changes and mitochondrial sequences provide independent support for congruent relationships among the sea spiders (Arthropoda, Pycnogonida).

Pycnogonids, or sea spiders, are an enigmatic group of arthropods. Their unique anatomical features have made them difficult to place within the broader group Arthropoda. Most attempts to classify members of Pycnogonida have focused on utilizing these anatomical features to infer relatedness. Using data from mitochondrial genomes, we show that pycnogonids are placed as derived chelicerates, challenging the hypothesis that they diverged early in arthropod history. Our increased taxon sampling of three new mitochondrial genomes also allows us to infer phylogenetic relatedness among major pycnogonid lineages. Phylogenetic analyses based on all 13 mitochondrial protein-coding genes yield well-resolved relationships among the sea spider lineages. Gene order and tRNA secondary structure characters provide independent lines of evidence for these inferred phylogenetic relationships among pycnogonids, and show a minimal amount of homoplasy. Additionally, rare changes in three tRNA genes unite pycnogonids as a clade; these include changes in anticodon identity in tRNA(Lys) and tRNA(Ser(AGN)) and the shared loss of D-arm sequence in the tRNA(Ala) gene. Using mitochondrial genome changes and tRNA structural changes is especially useful for resolving relationships among the major lineages of sea spiders in light of the fact that there have been multiple independent evolutionary changes in nucleotide strand bias among sea spiders. Such reversed nucleotide biases can mislead phylogeny reconstruction based on sequences, although the use of appropriate methods can overcome these effects. With pycnogonids, we find that applying methods to compensate for strand bias and that using genome-level characters yield congruent phylogenetic signals.