Pycnogonids, or sea spiders, comprising about 1163 extant species , are a special group of exclusively marine arthropods that are distributed from the intertidal zone to the abyssal depths in all the seas around the world. The pycogonids are traditionally classified as a sister group or even an ingroup taxon of euchelicerates, which comprises the arachnids, xiphosurans and extinct eurypterids [2, 3]. However, the phylogenetic position of pycnogonids has been vigorously debated during the last two centuries, as summarized in a recent review of pycnogonid affinities by Dunlop and Arango . According to this review, there have been a number of controversial hypotheses related to this issue. The pycnogonids have been regarded as either degraded crustaceans [5, 6], a transitional form between crustaceans and arachnids [7–9], an isolated group unrelated to other arthropods [10–12], a sister group of aquatic arachnids or mites within Chelicerata [13–15], a sister group of euchelicerates [3, 4, 16, 17], or a sister group of all other extant euarthropods (encompassing chelicerates, myriapods, crustaceans and hexapods) [18–20]. Because of rapid methodological improvements in phylogenetic systematics and related fields such as developmental biology, molecular biology and computational biology (bioinformatics), recent phylogenies have continuously attempted to resolve this issue and have eliminated many of the possible hypotheses, leaving only the two most plausible to be examined: namely that pycnogonids are either a sister group of euchelicerates or a sister group of euarthropods.
Pycnogonids have continually been recovered as a sister group of euchelicerates not only by a convincing autapomorphy – 'chelicerae/chelifore'- between the pycnogonids and euchelicerates, but also by a number of cladistic and phylogenetic studies based on molecular data (DNA or amino acid sequences), combined morphological and molecular data, Hox expression data and immunohistochemical data [4, 21–32].
On the other hand, the hypothesis that the pycnogonids are a sister group of euarthropods (all extant arthropods except for pycnogonids) was initially formally proposed by Zrzavy et al. on the basis of all the available morphological and gene sequence data . They named the clade of euchelicerates and mandibulates (excluding Pycnogonida) "Cormogonida;" they share the putative autapomorphic characteristic of a gonopore on the trunk. Pycnogonids have multiple gonopores on the bases of their legs. Although some authors have recovered this relationship [14, 19, 20, 33, 34], the presence of gonopores on the trunk in Cormogonida has been doubted as a plesiomorphic character .
Interestingly, a detailed neuroanatomical study of the Anoplodactylus sp. protonymphon lava  suggested that the chelifore of euchelicerates is not homologous to the chelicerae of pycnogonids, but rather that it is homologous to the protocerebral appendages ("great appendages") proposed for ancestral arthropods [35, 36]. These authors suggested that the pycnogonids are a sister group of all the euarthropods. However, more recent studies of Hox expression patterns in Endeis spinosa [29, 30] refuted the result of the neuroanatomical study . The Hox expression data suggested that all the anterior-most appendages of all extant arthropods (chelifore of pycnogonids, chelicerae of euchelicerates and the first antennae of mandibulates) are homologous and deutocerebral, indicating that protocerebral appendages have been lost in all extant arthropods.
In recent years, comparison of complete mitochondrial genomes has become a very powerful tool for reconstructing arthropod phylogeny [15, 37–41]. Mitochondrial genomes contain a variety of useful phylogenetic information, such as gene orders and orientations, alternative start codons of protein-coding genes, transfer RNA and ribosomal RNA secondary structures, genetic code variations, and features of the control region for genome replication and transcription . Typical metazoan mitochondrial genomes are circular, 14–16 kb in size, and encode 13 proteins, large and small subunit ribosomal RNAs (rrnL and rrnS) and 22 tRNAs [38, 43]. The 13 polypeptides are involved in ATP synthesis coupled to electron transfer during O2 consumption [ATP synthetase subunits (atp6 and atp8), cytochrome C oxidase subunits (cox1-cox3), apocytochrome b (cob) and NADH dehydrogenase subunits (nad1-nad6 and nad4L)].
Approximately 1014 complete mitochondrial genome sequences have been determined to date from metazoa including 243 protostomes (126 arthropods, 21 nematodes, 4 annelids, 33 mollusks, 16 platyhelminthes, 3 brachiopods, 1 echiuran, 1 bryozoan, 1 acanthocephalan and 2 chaetognaths, 24 cnidarian, 1 onychophoran, 4 placozoan, 5 poriferan, 1 priapulidan) and 771 deuterostomes (752 chordates, 16 echinoderms and 2 hemichordates, 1 xenoturbella) (GenBank status on June, 2007). Of the metazoan mitochondrial genomes sequenced, 126 were from arthropods (65 hexapods, 34 crustaceans, 4 myriapods and 23 chelicerates). In pycnogonids, a partial mitochondrial genome for E. spinosa (family Endeidae) was reported by Hassanin  and a complete mitochondrial genome sequence for N. gracile (family Nymphonidae) by Podsiadlowski and Braband . According to these recent reports, phylogenetic analyses of protein-coding genes show that pycnogonids may be associated with Acari (ticks and mites), although the authors acknowledged that the result may be due to a long-branch attraction artifact and higher A+T content.
The N. gracile mitochondrial genome has a peculiar gene order with extensive inversion of protein-coding genes and translocations of 10 tRNA genes, which are not typically found in arthropods . These peculiar characteristics are probably restricted to the N. gracile lineage, since the partial E. spinosa mitochondrial genome possesses the arthropod ground pattern of mitochondrial gene arrangements. Furthermore, the N. gracile branch on the phylogenetic trees presented by Podsiadlowski and Braband  was very long, as was also the case for Acari. This may have given rise to an artifactual relationship between Acari and Pycnogonida, which suggests that N. gracile may not be an appropriate representative of the pycnogonids. Thus, additional pycnogonid mitochondrial genomes must be sequenced if such information is to be used to address the problem of the phylogenetic position of pycnogonids [45, 46].
In this study, we present a complete new pycnogonid mitochondrial genome from a sea spider, Achelia bituberculata, belonging to the family Ammotheidae, which combines a number of anatomical features considered plesiomorphic with respect to other pycnogonids. The genome is characterized and compared to those of other arthropods, including two pycnogonids, N. gracile and E. spinosa. We attempt to use the data obtained in this study to address the long-standing and hotly-debated issue of the phylogenetic position of pycnogonids.