BACKGROUND
pycnogonids, or sea spiders, comprising about  <dig> extant species  <cit> , 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  <cit> .
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  <cit> .
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  <cit> , a transitional form between crustaceans and arachnids  <cit> , an isolated group unrelated to other arthropods  <cit> , a sister group of aquatic arachnids or mites within chelicerata  <cit> , a sister group of euchelicerates  <cit> , or a sister group of all other extant euarthropods   <cit> .
because of rapid methodological improvements in phylogenetic systematics and related fields such as developmental biology, molecular biology and computational biology , 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 , combined morphological and molecular data, hox expression data and immunohistochemical data  <cit> .
on the other hand, the hypothesis that the pycnogonids are a sister group of euarthropods  was initially formally proposed by zrzavy et al.
on the basis of all the available morphological and gene sequence data  <cit> .
they named the clade of euchelicerates and mandibulates  "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  <cit> , the presence of gonopores on the trunk in cormogonida has been doubted as a plesiomorphic character  <cit> .
interestingly, a detailed neuroanatomical study of the anoplodactylus sp.
protonymphon lava  <cit>  suggested that the chelifore of euchelicerates is not homologous to the chelicerae of pycnogonids, but rather that it is homologous to the protocerebral appendages  proposed for ancestral arthropods  <cit> .
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  <cit>  refuted the result of the neuroanatomical study  <cit> .
the hox expression data suggested that all the anterior-most appendages of all extant arthropods  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  <cit> .
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  <cit> .
typical metazoan mitochondrial genomes are circular, 14– <dig> kb in size, and encode  <dig> proteins, large and small subunit ribosomal rnas  and  <dig> trnas  <cit> .
the  <dig> polypeptides are involved in atp synthesis coupled to electron transfer during o <dig> consumption .
approximately  <dig> complete mitochondrial genome sequences have been determined to date from metazoa including  <dig> protostomes  and  <dig> deuterostomes  .
of the metazoan mitochondrial genomes sequenced,  <dig> were from arthropods .
in pycnogonids, a partial mitochondrial genome for e. spinosa  was reported by hassanin  <cit>  and a complete mitochondrial genome sequence for n. gracile  by podsiadlowski and braband  <cit> .
according to these recent reports, phylogenetic analyses of protein-coding genes show that pycnogonids may be associated with acari , 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  <dig> trna genes, which are not typically found in arthropods  <cit> .
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  <cit>  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  <cit> .
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.
RESULTS
genome organization
the mitochondrial genome of a. bituberculata is  <dig>  bp in length, which is similar to those of arthropod mitochondrial genomes .
it includes  <dig> protein-coding genes ,  <dig> trna genes , two ribosomal rna genes  and one large non-coding region , as typically found in metazoa .
the components are compactly juxtaposed.
there are even some overlapping nucleotides between trnc and trnw , nad <dig> and trna , and trne and trnf  .
the order and orientation of the gene arrangement pattern is identical to that of a horseshoe crab, limulus polyphemus, except for the position of the trnq gene .
a the number of control regions within each mitochondrial genome
?
: it is impossible to estimate the a+t content owing to the large number of unidentified sequences 'n'.
a indicates gap nucleotides  or overlapped nucleotides  between two adjacent genes.
* incomplete termination codon, which is probably extended by post-transcriptional adenylation.
comparison of gene arrangements
as shown in fig.
<dig>  the mitochondrial gene arrangement of a. bituberculata was compared with that of l. polyphemus, which is considered a putative ground pattern for arthropods  <cit> , and with those of two sea spiders, n. gracile and e. spinosa.
the identical gene arrangement pattern shown in a. bituberculata and l. polyphemus except for the trnq gene is an interesting feature because the other sea spider, n. gracile, has an extensive inversion  and ten trna gene translocations .
the partial genome of e. spinosa has the same gene arrangement pattern as those of l. polyphemus and a. bituberculata.
the trnq gene in a. bituberculata is located between rrns and the control region .
it is found between trniand trnm in most arthropods  <cit> .
comparisons among the trnq positions in arthropods  show that the rrns-trnq-cr arrangement in pycnogonids is also found in the jumping spider habronattus oregonensis   <cit>  and in a collembolan, tetrodontophora bielanensis  <cit> .
a similar pattern  is also observed in the chinese earth tiger spider ornithoctonus huwena   <cit> .
however, collembolans and spiders are not closely related, and another terrestrial spider  and another collembolan  have the limulus-like arthropod ground pattern .
thus, the rrns-trnq-cr arrangement found in a pycnogonid , a collembolan  and two terrestrial spiders  is likely to be homoplastic.
further intensive comparative studies are needed to examine whether the rrns-trnq-cr pattern is indeed a homoplastic feature or an important phylogenetic marker.
furthermore, a. bituberculata and n. gracile have two different anticodon-possessing trnl genes, marked l <dig> and l <dig>  which have the same orientations and positions as those in most other chelicerates and myriapods.
directly abutted tandem arrays of two trnls between rrnl and nad <dig>  are also observed in other metazoan phyla  <cit> .
in pancrustacea , l <dig> is generally located between rrnl and nad <dig>  and l <dig> between cox <dig> and cox <dig>  <cit> .
the rrnl-l1-nad <dig> and cox1-l2-cox <dig> arrangement patterns are considered to be critical evidence for a monophyletic origin of pancrustaceans other than the chelicerates and myriapods in arthropods.
therefore, the finding of the rrnl-l1-l2-nad <dig> arrangement in pycnogonids rejects the view that pycnogonids have crustacean affinities  <cit> , generally regarded as an old-fashioned hypothesis today.
base composition and codon usage pattern
as shown in table  <dig>  the overall a+t content in the mitochondrial genome of a. bituberculata is  <dig> % .
this a+t content is much higher than those in l. polyphemus  and the scorpions , but is similar to those in acari and araneae , including n. gracile .
metazoan mitochondrial genomes usually present a clear strand bias in nucleotide composition  <cit> .
table  <dig> shows the at- and cg-skews of each of the  <dig> protein-coding and  <dig> ribosomal rna genes and those of the whole genome  in a. bituberculata mitochondria.
the results show no marked strand-bias in nucleotide composition.
the at-skew for all genes is negative whether they are on the  or the  strand.
the cg-skew is positive for four genes and negative for five on the  strand.
the cg-skew for all six genes on the  strand is negative.
this is totally different from the clear strand-bias observed in n. gracile.
at skew = /; cg skew = /; bold letters mark the values of the genes on the  strand.
investigation of the anticodon and codon usage patterns in the protein-coding genes of the a. bituberculata mitochondrial genome indicates that the nucleotides in the 3rd codon position are primarily 'a' or 't' rather than 'c' or 'g'.
codons composed of only 'a' and 't' are predominantly used, which seems to reflect the high a+t bias composition of the entire genome.
a strong preference for a+t-rich codons is found in a. bituberculata as in mitochondrial protein-coding genes in general .
for example, the most frequently-used codons are l  and i .
additional file  <dig> shows the usage of leucine and serine codons in chelicerates and pycnogonids and reveals some interesting features in the usage of l and l. totals of  <dig> and  <dig> l codons are used in a. bituberculata and n. gracile, respectively, which are the highest usage patterns of these codons in the chelicerates observed to date.
a total of  <dig> l codons are used in a. bituberculata , similar to the usage patterns in araneae and acari  but different from those in l. polyphemus  and scorpions .
that is, the l codon frequency in a. bituberculata is similar to that in araneae and acari.
* the number of codons used in  <dig> mitochondrial protein-coding genes
protein-coding genes
the aggregate length of all the  <dig> protein-coding genes is  <dig>  bp .
there was no overlap between these genes.
they account for 76% of the entire mitochondrial genome length.
in terms of the lengths of individual protein-coding genes, a. bituberculata has relatively short atp <dig>  and nad <dig>  compared to other chelicerates  and myriapods .
in contrast, the nad <dig> gene of a. bituberculata  and n. gracile  and nad <dig> of a. bituberculata  are larger than those in the other species.
while nad <dig> is dramatically reduced in n. gracile , that in a. bituberculata is similar in size  to the other chelicerates .
amino acid usages and a+t contents of mitochondrial protein-coding genes were compared with those of chelicerates .
greater numbers of asn  and met  are used than in the other chelicerates.
the a+t content of the third codon positions of the  <dig> protein-coding genes is  <dig> %, which is lower than in n. gracile  and higher than in l. polyphemus .
the start and stop codons of the  <dig> protein-coding genes of the a. bituberculata mitochondrial genome were identified by comparison with those of other known arthropods; particular attention was given to the amino acid sequence alignment in order to identify the most likely codon in ambiguous situations.
as shown in table  <dig>  the start codons of  <dig> mitochondrial of the protein-coding genes  are 'atn', which is typical of most metazoan mitochondria  <cit> : 'atg' is the start codon for nad <dig>  nad4l, nad <dig>  cox <dig>  cox <dig> and cob; 'att' is the start codon found in nad <dig> and atp6; and 'ata' is the start codon for nad <dig>  nad <dig>  nad <dig> and atp <dig>  a less typical 'gat' start codon appears in the remaining cox <dig> gene.
on the other hand, only four of the protein-coding genes are terminated with a complete stop codon: 'taa' in nad4l and cox <dig> and 'tag' in nad <dig> and nad <dig>  the remaining eight genes are terminated using truncated stop codons that are presumably completed by post-transcriptional polyadenylation  <cit> : 'ta' for nad <dig> and nad <dig>  and 't' for nad <dig>  nad <dig>  cox <dig>  cox <dig>  cob, atp <dig> and atp <dig>
transfer rna genes
all  <dig> of the trnas typically found in metazoan mitochondrial genomes were identified in a. bituberculata.
all these genes are located among protein- and/or rrna-coding genes .
twenty-two putative secondary structures were predicted from the trna gene sequences .
most of the trnas are capable of forming the typical clover-leaf structure, with the exception of trnaala , trnaser, trnatyr and trnaval .
the lengths of the tψc and dhu arms in the  <dig> trnas with a stable clover-leaf shape range from  <dig> to  <dig> bp, and the variable loops range in size from  <dig> to  <dig> bp.
trnaser with an unpaired dhu arm has been reported in the mitochondrial genomes of a number of metazoa including the sea spider n. gracile  <cit> , the centipede scutigera coleoptrata  <cit> , the jumping spider habronattus oregonensis  <cit> , the nematodes caenorhabditis elegans and ascaris suum  <cit>  and the annelid lumbricus terrestris  <cit> .
the dhu arm of trnaala in n. gracile does not form a stable stem; however, the other two trnas, trnatyr and trnaval, are folded into typical clover-leaf shapes  <cit> .
twenty of the  <dig> trnas have a completely matched 7-bp-long amino-acyl stem, and the remaining two, trnaglu and trnatyr, have a single mismatch within the stem.
while  <dig> of the trnas have the typical 5-bp-long anticodon stem, the remaining three  have a single mismatch within this stem.
it is not uncommon for some mitochondrial trnas to have stem mismatches, which are probably repaired by a post-transcriptional editing process  <cit> .
the trna anticodon sequences in a. bituberculata  are identical to those of the corresponding trnas of other metazoans with the exception of trnalys and trnaser.
trnalys and trnaser in both a. bituberculata and n. gracile possess 'uuu' and 'ucu' anticodons, respectively, instead of 'cuu' and 'gcu', which are more commonly found in invertebrates and some vertebrates  <cit> .
ribosomal rna genes
the trnv gene separates rrns from rrnl, as in most arthropods.
the sizes of rrns and rrnl are approximately  <dig>  bp and  <dig> bp, respectively .
these are similar to the centipedes lithobius forficatus  and scutigera coleoptrata , but are slightly shorter than those of l. polyphemus .
compared with that in arachnids, the length of rrnl in a. bituberculata is similar to those in four species of acari  and larger than those in three terrestrial spiders .
on the other hand, rrns in a. bituberculata is larger than those in four species of acari , and three terrestrial spiders .
the a+t contents of rrnl and rrns are  <dig> %  and  <dig> % , respectively.
these account for over 77% of the entire a. bituberculata mitochondrial genome.
the rrnl gene of a. bituberculata has the highest similarities with those of n. gracile  and l. polyphemus , and the rrns gene of a. bituberculata with that of l. polyphemus .
control region
a large noncoding region  is found between trnq and trni.
it includes three tandem repeats, the first and second of which are completely identical in length  and sequence .
however, the third repeat  is  <dig> bp shorter than the other two at the 3'-end, and one nucleotide is different in the 315-bp repeat .
the repeats have unique stem and loop structures that may play key roles in controlling the replication and transcription of the mitochondrial genome .
the a+t content of the cr is 79%, which is similar to those of most chelicerates .
the inclusion of pycnogonida within arachnida
as shown in fig.
<dig>  phylogenetic analyses based on the amino acid sequences of  <dig> protein-coding genes for  <dig> arthropods and  <dig> outgroups  as shown in table  <dig> indicate that pycnogonida, including a. bituberculata and n. gracile, are clustered together , and located within the monophyletic chelicerata .
l. polyphemus appears to be a sister of all the other chelicerates .
although a monophyletic arachnida  including pycnogonida is supported by node confidence values that are very low , scorpiones are placed as the most basal arachnids and pycnogonida, araneae and acari form a monophyletic group ; pycnogonida is identified as a sister group of the clade of araneae and acari within arachnida.
when the onychophoran epiperipatus biolleyi is employed as a single outgroup, the resultant tree  is the same as fig.
<dig> in the position of pycnogonids within arachnida, although arthropod monophyly is not supported.
the two pycnogonids examined, a. bituberculata and n. gracile, are clustered together  within the monophyletic chelicerata .
pycnogonida again appears as a sister group of the clade of araneae and acari .
two previous studies based on the mitochondrial genome sequences of e. spinosa  <cit>  and n. gracile  <cit>  indicated that the pycnogonida-acari affinity might have resulted from biased features such as the strand-biased nucleotide contents and long-branch attraction.
interestingly, the a. bituberculata mitochondrial genome shows no marked strand-bias nucleotide composition .
since most of the other chelicerates and n. gracile have strand-biased nucleotide contents  <cit> as well as extremely long branches on the phylogenetic tree , the possibility remains that the appearance of pycnogonida within arachnida may result from artifacts of analysis due to the strand-biased and long-branching patterns.
to test whether the inclusion of pycnogonida within arthropoda results from long-branch attraction artifacts, we removed  <dig> long-branch taxa – all  <dig> acari members and  <dig> terrestrial spiders  – from the dataset for the tree in additional file  <dig>   <dig>  and then conducted a new phylogenetic analysis with  <dig> ingroup and  <dig> outgroup taxa .
we then removed n. gracile as well because of its long branching pattern, and reconstructed a new phylogenetic tree with only  <dig> ingroup and  <dig> outgroup taxa .
in addition, we removed the  <dig> pancrutacean taxa and  <dig> onychophoran from the data sets including the  <dig> and  <dig> taxa, and then reconstructed additional phylogenetic trees with only  <dig> and  <dig> taxa, respectively .
in the subsequent ml analyses, while onychophora was used as an outgroup for the first and second reduced datasets including  <dig> and  <dig> taxa, the  <dig> remaining pancrustaceans were used as outgroups for the analyses with only  <dig> and  <dig> taxa.
although the long-branch taxa were removed, the ml trees inferred from the four different reduced alignment data sets showed that the pycnogonids are consistently clustered with the terrestrial spider h. langzhouensis , except for the ml tree with the  <dig> taxa in which a. bituberculata appears as a sister group of euchelicerates .
the present findings conflict with the two major hypotheses that pycnogonida is a sister group of the monophyletic euchelicerata  or that it is a sister group of the monophyletic cormogonida .
nevertheless, the inclusion of pycnogonida within arachnida is compatible with some previous analyses based on combinations of  <dig> morphological characteristics and molecular data   <cit> , and the mitochondrial protein-coding genes  <cit> , which indicated that pycnogonida appears within arachnida.
more recently, mallatt and giribet  <cit>  on the basis of nearly-complete 18s and 28s rdna sequences, and giribet et al.
<cit>  on the basis of parsimony analyses of combined data for  <dig> nuclear genes,  <dig> mitochondrial genes and  <dig> morphological characters, both showed that pycnogonida is united with chelicerata with very weak support and cormogonida is strongly rejected.
the present result is consistent with theirs in that cormogonida is rejected and pycnogonida has an affinity with chelicerata.
however, they did not suggest that pycnogonida may possibly be included within arachnida; many morphological taxonomists may not agree with this point of view.
thus, it is likely that the inclusion of pycnogonida within arachnida, deduced only from mitochondrial genome data  <cit> , will remain an open question.
in addition, scorpiones are often considered to be "one of the most primitive arachnids" because they possess a number of plesiomorphic character states, even though their phylogenetic position remains controversial.
recent publications based on mitochondrial protein-coding genes  <cit>  show that scorpiones is a sister group of all other arachnids .
the present result  is consistent with previous reports  <cit>  showing that scorpiones is a sister group of the clade of pycnogonida plus araneae plus acari.
the tree in fig.
<dig> shows the paraphyly of myriapoda  <cit> ; the placement of collembola as a sister of all other pancrustacean taxa  <cit> ; the monophyly of myriapoda and chelicerata   <cit> ; and the monophyly of pancrustacea  <cit> .
complete mitochondrial genome sequences need to be determined from another important sister group  of the arthropods in order to elucidate the deep arthropod phylogeny, including the phylogenetic position of pycnogonids.
CONCLUSIONS
this study presents the complete sequence of the mitochondrial genome from a pycnogonid, a. bituberculata .
this complete sequence and a previously-reported partial sequence of endeis spinosa show the typical gene arrangement patterns of arthropods , but they differ from that of n. gracile.
thus, it is most likely that the peculiar arrangements found in n. gracile are only present in the family nymphonidae or even in the genus nymphon.
phylogenetic trees based on amino acid sequences deduced from mitochondrial protein-coding genes showed that pycnogonida may be authentic arachnids  within chelicerata, as indicated by the name 'sea spider', and suggest that the cormogonida theory, which considers the pycnogonids a sister of all other arthropods, should be rejected.
however, since node confidence values supporting the pycnogonida inclusion within arachnida are slightly low and the possibility of long-branch attraction artifact still remains, further more intensive studies seem necessary to resolve the problem.

 pycnogonids, or sea spiders, comprising about 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. 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, a transitional form between crustaceans and arachnids, an isolated group unrelated to other arthropods, a sister group of aquatic arachnids or mites within chelicerata, a sister group of euchelicerates, or a sister group of all other extant euarthropods. because of rapid methodological improvements in phylogenetic systematics and related fields such as developmental biology, molecular biology and computational biology, 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, combined morphological and molecular data, hox expression data and immunohistochemical data. on the other hand, the hypothesis that the pycnogonids are a sister group of euarthropods 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 "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, 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 proposed for ancestral arthropods. 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 refuted the result of the neuroanatomical study. the hox expression data suggested that all the anterior-most appendages of all extant arthropods 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. 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– kb in size, and encode proteins, large and small subunit ribosomal rnas and trnas. the polypeptides are involved in atp synthesis coupled to electron transfer during o consumption. approximately complete mitochondrial genome sequences have been determined to date from metazoa including protostomes and deuterostomes. of the metazoan mitochondrial genomes sequenced, were from arthropods. in pycnogonids, a partial mitochondrial genome for e. spinosa was reported by hassanin and a complete mitochondrial genome sequence for n. gracile by podsiadlowski and braband. according to these recent reports, phylogenetic analyses of protein-coding genes show that pycnogonids may be associated with acari, 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 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. 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.  genome organization the mitochondrial genome of a. bituberculata is bp in length, which is similar to those of arthropod mitochondrial genomes. it includes protein-coding genes, trna genes, two ribosomal rna genes and one large non-coding region, as typically found in metazoa. the components are compactly juxtaposed. there are even some overlapping nucleotides between trnc and trnw, nad and trna, and trne and trnf. the order and orientation of the gene arrangement pattern is identical to that of a horseshoe crab, limulus polyphemus, except for the position of the trnq gene. a the number of control regions within each mitochondrial genome?: it is impossible to estimate the a+t content owing to the large number of unidentified sequences'n'. a indicates gap nucleotides or overlapped nucleotides between two adjacent genes. * incomplete termination codon, which is probably extended by post-transcriptional adenylation. comparison of gene arrangements as shown in fig. the mitochondrial gene arrangement of a. bituberculata was compared with that of l. polyphemus, which is considered a putative ground pattern for arthropods, and with those of two sea spiders, n. gracile and e. spinosa. the identical gene arrangement pattern shown in a. bituberculata and l. polyphemus except for the trnq gene is an interesting feature because the other sea spider, n. gracile, has an extensive inversion and ten trna gene translocations. the partial genome of e. spinosa has the same gene arrangement pattern as those of l. polyphemus and a. bituberculata. the trnq gene in a. bituberculata is located between rrns and the control region. it is found between trniand trnm in most arthropods. comparisons among the trnq positions in arthropods show that the rrns-trnq-cr arrangement in pycnogonids is also found in the jumping spider habronattus oregonensis and in a collembolan, tetrodontophora bielanensis. a similar pattern is also observed in the chinese earth tiger spider ornithoctonus huwena. however, collembolans and spiders are not closely related, and another terrestrial spider and another collembolan have the limulus-like arthropod ground pattern. thus, the rrns-trnq-cr arrangement found in a pycnogonid, a collembolan and two terrestrial spiders is likely to be homoplastic. further intensive comparative studies are needed to examine whether the rrns-trnq-cr pattern is indeed a homoplastic feature or an important phylogenetic marker. furthermore, a. bituberculata and n. gracile have two different anticodon-possessing trnl genes, marked l and l which have the same orientations and positions as those in most other chelicerates and myriapods. directly abutted tandem arrays of two trnls between rrnl and nad are also observed in other metazoan phyla. in pancrustacea, l is generally located between rrnl and nad and l between cox and cox. the rrnl-l1-nad and cox1-l2-cox arrangement patterns are considered to be critical evidence for a monophyletic origin of pancrustaceans other than the chelicerates and myriapods in arthropods. therefore, the finding of the rrnl-l1-l2-nad arrangement in pycnogonids rejects the view that pycnogonids have crustacean affinities, generally regarded as an old-fashioned hypothesis today. base composition and codon usage pattern as shown in table the overall a+t content in the mitochondrial genome of a. bituberculata is %. this a+t content is much higher than those in l. polyphemus and the scorpions, but is similar to those in acari and araneae, including n. gracile. metazoan mitochondrial genomes usually present a clear strand bias in nucleotide composition. table shows the at- and cg-skews of each of the protein-coding and ribosomal rna genes and those of the whole genome in a. bituberculata mitochondria. the results show no marked strand-bias in nucleotide composition. the at-skew for all genes is negative whether they are on the or the strand. the cg-skew is positive for four genes and negative for five on the strand. the cg-skew for all six genes on the strand is negative. this is totally different from the clear strand-bias observed in n. gracile. at skew = /; cg skew = /; bold letters mark the values of the genes on the strand. investigation of the anticodon and codon usage patterns in the protein-coding genes of the a. bituberculata mitochondrial genome indicates that the nucleotides in the 3rd codon position are primarily'a' or't' rather than'c' or'g'. codons composed of only'a' and't' are predominantly used, which seems to reflect the high a+t bias composition of the entire genome. a strong preference for a+t-rich codons is found in a. bituberculata as in mitochondrial protein-coding genes in general. for example, the most frequently-used codons are l and i. additional file shows the usage of leucine and serine codons in chelicerates and pycnogonids and reveals some interesting features in the usage of l and l. totals of and l codons are used in a. bituberculata and n. gracile, respectively, which are the highest usage patterns of these codons in the chelicerates observed to date. a total of l codons are used in a. bituberculata, similar to the usage patterns in araneae and acari but different from those in l. polyphemus and scorpions. that is, the l codon frequency in a. bituberculata is similar to that in araneae and acari. * the number of codons used in mitochondrial protein-coding genes protein-coding genes the aggregate length of all the protein-coding genes is bp. there was no overlap between these genes. they account for 76% of the entire mitochondrial genome length. in terms of the lengths of individual protein-coding genes, a. bituberculata has relatively short atp and nad compared to other chelicerates and myriapods. in contrast, the nad gene of a. bituberculata and n. gracile and nad of a. bituberculata are larger than those in the other species. while nad is dramatically reduced in n. gracile, that in a. bituberculata is similar in size to the other chelicerates. amino acid usages and a+t contents of mitochondrial protein-coding genes were compared with those of chelicerates. greater numbers of asn and met are used than in the other chelicerates. the a+t content of the third codon positions of the protein-coding genes is %, which is lower than in n. gracile and higher than in l. polyphemus. the start and stop codons of the protein-coding genes of the a. bituberculata mitochondrial genome were identified by comparison with those of other known arthropods; particular attention was given to the amino acid sequence alignment in order to identify the most likely codon in ambiguous situations. as shown in table the start codons of mitochondrial of the protein-coding genes are'atn', which is typical of most metazoan mitochondria:'atg' is the start codon for nad nad4l, nad cox cox and cob;'att' is the start codon found in nad and atp6; and'ata' is the start codon for nad nad nad and atp a less typical'gat' start codon appears in the remaining cox gene. on the other hand, only four of the protein-coding genes are terminated with a complete stop codon:'taa' in nad4l and cox and'tag' in nad and nad the remaining eight genes are terminated using truncated stop codons that are presumably completed by post-transcriptional polyadenylation:'ta' for nad and nad and't' for nad nad cox cox cob, atp and atp transfer rna genes all of the trnas typically found in metazoan mitochondrial genomes were identified in a. bituberculata. all these genes are located among protein- and/or rrna-coding genes. twenty-two putative secondary structures were predicted from the trna gene sequences. most of the trnas are capable of forming the typical clover-leaf structure, with the exception of trnaala, trnaser, trnatyr and trnaval. the lengths of the tψc and dhu arms in the trnas with a stable clover-leaf shape range from to bp, and the variable loops range in size from to bp. trnaser with an unpaired dhu arm has been reported in the mitochondrial genomes of a number of metazoa including the sea spider n. gracile, the centipede scutigera coleoptrata, the jumping spider habronattus oregonensis, the nematodes caenorhabditis elegans and ascaris suum and the annelid lumbricus terrestris. the dhu arm of trnaala in n. gracile does not form a stable stem; however, the other two trnas, trnatyr and trnaval, are folded into typical clover-leaf shapes. twenty of the trnas have a completely matched 7-bp-long amino-acyl stem, and the remaining two, trnaglu and trnatyr, have a single mismatch within the stem. while of the trnas have the typical 5-bp-long anticodon stem, the remaining three have a single mismatch within this stem. it is not uncommon for some mitochondrial trnas to have stem mismatches, which are probably repaired by a post-transcriptional editing process. the trna anticodon sequences in a. bituberculata are identical to those of the corresponding trnas of other metazoans with the exception of trnalys and trnaser. trnalys and trnaser in both a. bituberculata and n. gracile possess'uuu' and'ucu' anticodons, respectively, instead of'cuu' and'gcu', which are more commonly found in invertebrates and some vertebrates. ribosomal rna genes the trnv gene separates rrns from rrnl, as in most arthropods. the sizes of rrns and rrnl are approximately bp and bp, respectively. these are similar to the centipedes lithobius forficatus and scutigera coleoptrata, but are slightly shorter than those of l. polyphemus. compared with that in arachnids, the length of rrnl in a. bituberculata is similar to those in four species of acari and larger than those in three terrestrial spiders. on the other hand, rrns in a. bituberculata is larger than those in four species of acari, and three terrestrial spiders. the a+t contents of rrnl and rrns are % and %, respectively. these account for over 77% of the entire a. bituberculata mitochondrial genome. the rrnl gene of a. bituberculata has the highest similarities with those of n. gracile and l. polyphemus, and the rrns gene of a. bituberculata with that of l. polyphemus. control region a large noncoding region is found between trnq and trni. it includes three tandem repeats, the first and second of which are completely identical in length and sequence. however, the third repeat is bp shorter than the other two at the 3'-end, and one nucleotide is different in the 315-bp repeat. the repeats have unique stem and loop structures that may play key roles in controlling the replication and transcription of the mitochondrial genome. the a+t content of the cr is 79%, which is similar to those of most chelicerates. the inclusion of pycnogonida within arachnida as shown in fig. phylogenetic analyses based on the amino acid sequences of protein-coding genes for arthropods and outgroups as shown in table indicate that pycnogonida, including a. bituberculata and n. gracile, are clustered together, and located within the monophyletic chelicerata. l. polyphemus appears to be a sister of all the other chelicerates. although a monophyletic arachnida including pycnogonida is supported by node confidence values that are very low, scorpiones are placed as the most basal arachnids and pycnogonida, araneae and acari form a monophyletic group; pycnogonida is identified as a sister group of the clade of araneae and acari within arachnida. when the onychophoran epiperipatus biolleyi is employed as a single outgroup, the resultant tree is the same as fig. in the position of pycnogonids within arachnida, although arthropod monophyly is not supported. the two pycnogonids examined, a. bituberculata and n. gracile, are clustered together within the monophyletic chelicerata. pycnogonida again appears as a sister group of the clade of araneae and acari. two previous studies based on the mitochondrial genome sequences of e. spinosa and n. gracile indicated that the pycnogonida-acari affinity might have resulted from biased features such as the strand-biased nucleotide contents and long-branch attraction. interestingly, the a. bituberculata mitochondrial genome shows no marked strand-bias nucleotide composition. since most of the other chelicerates and n. gracile have strand-biased nucleotide contents as well as extremely long branches on the phylogenetic tree, the possibility remains that the appearance of pycnogonida within arachnida may result from artifacts of analysis due to the strand-biased and long-branching patterns. to test whether the inclusion of pycnogonida within arthropoda results from long-branch attraction artifacts, we removed long-branch taxa – all acari members and terrestrial spiders – from the dataset for the tree in additional file and then conducted a new phylogenetic analysis with ingroup and outgroup taxa. we then removed n. gracile as well because of its long branching pattern, and reconstructed a new phylogenetic tree with only ingroup and outgroup taxa. in addition, we removed the pancrutacean taxa and onychophoran from the data sets including the and taxa, and then reconstructed additional phylogenetic trees with only and taxa, respectively. in the subsequent ml analyses, while onychophora was used as an outgroup for the first and second reduced datasets including and taxa, the remaining pancrustaceans were used as outgroups for the analyses with only and taxa. although the long-branch taxa were removed, the ml trees inferred from the four different reduced alignment data sets showed that the pycnogonids are consistently clustered with the terrestrial spider h. langzhouensis, except for the ml tree with the taxa in which a. bituberculata appears as a sister group of euchelicerates. the present findings conflict with the two major hypotheses that pycnogonida is a sister group of the monophyletic euchelicerata or that it is a sister group of the monophyletic cormogonida. nevertheless, the inclusion of pycnogonida within arachnida is compatible with some previous analyses based on combinations of morphological characteristics and molecular data, and the mitochondrial protein-coding genes, which indicated that pycnogonida appears within arachnida. more recently, mallatt and giribet on the basis of nearly-complete 18s and 28s rdna sequences, and giribet et al. on the basis of parsimony analyses of combined data for nuclear genes, mitochondrial genes and morphological characters, both showed that pycnogonida is united with chelicerata with very weak support and cormogonida is strongly rejected. the present result is consistent with theirs in that cormogonida is rejected and pycnogonida has an affinity with chelicerata. however, they did not suggest that pycnogonida may possibly be included within arachnida; many morphological taxonomists may not agree with this point of view. thus, it is likely that the inclusion of pycnogonida within arachnida, deduced only from mitochondrial genome data, will remain an open question. in addition, scorpiones are often considered to be "one of the most primitive arachnids" because they possess a number of plesiomorphic character states, even though their phylogenetic position remains controversial. recent publications based on mitochondrial protein-coding genes show that scorpiones is a sister group of all other arachnids. the present result is consistent with previous reports showing that scorpiones is a sister group of the clade of pycnogonida plus araneae plus acari. the tree in fig. shows the paraphyly of myriapoda; the placement of collembola as a sister of all other pancrustacean taxa; the monophyly of myriapoda and chelicerata; and the monophyly of pancrustacea. complete mitochondrial genome sequences need to be determined from another important sister group of the arthropods in order to elucidate the deep arthropod phylogeny, including the phylogenetic position of pycnogonids.  this study presents the complete sequence of the mitochondrial genome from a pycnogonid, a. bituberculata. this complete sequence and a previously-reported partial sequence of endeis spinosa show the typical gene arrangement patterns of arthropods, but they differ from that of n. gracile. thus, it is most likely that the peculiar arrangements found in n. gracile are only present in the family nymphonidae or even in the genus nymphon. phylogenetic trees based on amino acid sequences deduced from mitochondrial protein-coding genes showed that pycnogonida may be authentic arachnids within chelicerata, as indicated by the name'sea spider', and suggest that the cormogonida theory, which considers the pycnogonids a sister of all other arthropods, should be rejected. however, since node confidence values supporting the pycnogonida inclusion within arachnida are slightly low and the possibility of long-branch attraction artifact still remains, further more intensive studies seem necessary to resolve the problem. 
however, n. gracile appears to be a long-branch taxon on the phylogenetic tree and exhibits a number of peculiar features, such as trna translocations and even an inversion of several protein-coding genes. the complete mitochondrial genome sequence of a. bituberculata and the previously-reported partial sequence of endeis spinosa show the gene arrangement patterns typical of arthropods, but they differ markedly from that of n. gracile. maximum likelihood and bayesian inference trees inferred from the amino acid sequences of mitochondrial protein-coding genes consistently indicate that the pycnogonids may be closely related to the clade of acari and araneae. however, in view of the relatively weak node confidence, strand-biased nucleotide composition and long-branch attraction artifact, further more intensive studies seem necessary to resolve the exact position of the pycnogonids. the overall gene arrangement is completely identical to the arthropod ground pattern, with one exception: the position of the trnq gene between the rrns gene and the control region. this controversy has recently been rekindled by differences in the conclusions based on neuroanatomical data concerning the chelifore and the patterns of hox expression. phylogenetic analyses based on mitochondrial protein-coding genes showed that pycnogonida may be authentic arachnids within chelicerata sensu lato, as indicated by the name'sea spider,' and suggest that the cormogonida theory – that the pycnogonids are a sister group of all other arthropods – should be rejected. the mitochondrial genome of a sea spider, nymphon gracile, was recently reported in an attempt to address this issue. sequences of other pycnogonid mitochondrial genomes are needed if the position of pycnogonids is to be elucidated on this basis. the complete mitochondrial genome of a sea spider belonging to the family ammotheidae, which combines a number of anatomical features considered plesiomorphic with respect to other pycnogonids, was sequenced and characterized. the phylogenetic position of pycnogonids is a long-standing and controversial issue in arthropod phylogeny.
 the phylogenetic position of pycnogonids is a long-standing and controversial issue in arthropod phylogeny. this controversy has recently been rekindled by differences in the conclusions based on neuroanatomical data concerning the chelifore and the patterns of hox expression. the mitochondrial genome of a sea spider, nymphon gracile, was recently reported in an attempt to address this issue. however, n. gracile appears to be a long-branch taxon on the phylogenetic tree and exhibits a number of peculiar features, such as trna translocations and even an inversion of several protein-coding genes. sequences of other pycnogonid mitochondrial genomes are needed if the position of pycnogonids is to be elucidated on this basis.  the complete mitochondrial genome of a sea spider belonging to the family ammotheidae, which combines a number of anatomical features considered plesiomorphic with respect to other pycnogonids, was sequenced and characterized. the genome organization shows the features typical of most metazoan animal genomes. the overall gene arrangement is completely identical to the arthropod ground pattern, with one exception: the position of the trnq gene between the rrns gene and the control region. maximum likelihood and bayesian inference trees inferred from the amino acid sequences of mitochondrial protein-coding genes consistently indicate that the pycnogonids may be closely related to the clade of acari and araneae.  the complete mitochondrial genome sequence of a. bituberculata and the previously-reported partial sequence of endeis spinosa show the gene arrangement patterns typical of arthropods, but they differ markedly from that of n. gracile. phylogenetic analyses based on mitochondrial protein-coding genes showed that pycnogonida may be authentic arachnids within chelicerata sensu lato, as indicated by the name'sea spider,' and suggest that the cormogonida theory – that the pycnogonids are a sister group of all other arthropods – should be rejected. however, in view of the relatively weak node confidence, strand-biased nucleotide composition and long-branch attraction artifact, further more intensive studies seem necessary to resolve the exact position of the pycnogonids. 