Rare Genomic Changes and Mitochondrial Sequences Provide Independent Support for Congruent Relationships Among the Sea Spiders (Arthropoda, Pycnogonida)

Molecular Phylogenetics and Evolution 57 (2010) 59–70

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Molecular Phylogenetics and Evolution

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

Susan E. Masta *, Andrew McCall, Stuart J. Longhorn 1

Department of Biology, Portland State University, P.O. Box 751, Portland, OR 97207, USA article info abstract

Article history: Pycnogonids, or sea spiders, are an enigmatic group of arthropods. Their unique anatomical features have Received 15 September 2009 made them difﬁcult to place within the broader group Arthropoda. Most attempts to classify members of Revised 15 June 2010 Pycnogonida have focused on utilizing these anatomical features to infer relatedness. Using data from Accepted 25 June 2010 mitochondrial genomes, we show that pycnogonids are placed as derived chelicerates, challenging the Available online 30 June 2010 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 lin- Keywords: eages. Phylogenetic analyses based on all 13 mitochondrial protein-coding genes yield well-resolved rela- Chelicerata tionships among the sea spider lineages. Gene order and tRNA secondary structure characters provide Arachnida Mitochondrial genomics independent lines of evidence for these inferred phylogenetic relationships among pycnogonids, and tRNA secondary structure show a minimal amount of homoplasy. Additionally, rare changes in three tRNA genes unite pycnogonids Lys Ser(AGN) Gene rearrangements as a clade; these include changes in anticodon identity in tRNA and tRNA and the shared loss of D-arm sequence in the tRNAAla 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. Ó 2010 Elsevier Inc. All rights reserved.

1. Introduction data have each yielded results that support both of these hypotheses, and therefore, it is still not known which hypothesis, if either, Pycnogonida, or sea spiders, are arthropods with anatomical is correct. More data, including data from different sources, is features so unique that their relationships to other members of necessary to resolve the placement of Pycnogonida within Arthropoda have been enigmatic. Pycnogonids possess a proboscis Arthropoda. for feeding, extreme abdominal reduction, reproductive organs on Pycnogonids typically possess anterior appendages termed their legs, and ventral appendages termed ovigers with which chelifores, and the lack of clear homology of these appendages males carry eggs. There are two main hypotheses regarding pycno- with other arthropod appendages has fueled debate over the clos- gonid relationships to other arthropods (reviewed in Dunlop and est relatives of pycnogonids. If sea spiders are chelicerates, then Arango, 2005). One hypothesis is that Pycnogonida is the sister chelifores should be homologous to the chelicerae of other cheli- group to all other Arthropoda. Under this hypothesis the arthro- cerates, as possession of chelicerae is one of the deﬁning traits pods, excluding pycnogonids, have been named Cormogonida for the Chelicerata. However, if chelifores are not directly homolo- (Zrzavy´ et al., 1998). A second hypothesis is that Pycnogonida are gous to chelicerae, then this calls into question whether sea spiders members of Chelicerata, and are the sister group to the Xiphosura are truly chelicerates. Some analyses based on developmental fea- (horseshoe crabs) plus Arachnida. Anatomical and developmental tures have found evidence that these appendages are not direct homologues of chelicerae, and support the hypothesis that sea spiders are sister to all other arthropods (Maxmen et al., 2005). How- * Corresponding author. Fax: +1 503 725 3888. ever, more recent data based on developmental expression E-mail address: [email protected] (S.E. Masta). patterns and neuroanatomy have shown that chelifores are likely 1 Current address: Department of Biology, National University of Ireland, Mayno- oth. County Kildare, Ireland. chelicerae homologues (Brenneis et al., 2008; Jager et al., 2006;

Manuel et al., 2006), consistent with the placement of sea spiders number of non-canonical tRNA structures have been found within as sister to other chelicerates. arachnid mt genomes (Masta and Boore, 2008), but their structures Analyses of molecular data are likewise inconsistent in their have also proven to be phylogenetically informative within some support for these opposing two hypotheses. Some analyses have groups, such as ticks (Murrell et al., 2003) and spiders (Masta found support for pycnogonids being sister to all arthropods (Giri- and Boore, 2008). bet et al., 2001), or sister to all arthropods except chelicerates (Re- Besides potentially providing new types of genome structure gier et al., 2008), or sister to the myriapod arthropods (Mallatt and characters for making phylogenetic inferences, mitochondrial gen- Giribet, 2006; Mallatt et al., 2004). However, most molecular anal- omes provide a rich source of sequences for analysis. Typical meta- yses recover phylogenetic trees that are congruent with pycnogo- zoan mitochondria encode the same 37 genes, consisting of 13 nids as a sister group to all other chelicerates, although often protein-coding genes, 22 tRNA genes, and 2 ribosomal RNA genes. with low bootstrap support (Bourlat et al., 2008; Mallatt and Giri- Therefore, it is clear that each of these genes is homologous among bet, 2006; Regier et al., 2008). Phylogenomic analyses with ex- metazoans, making full-genome sequence comparisons possible pressed sequence tags yielded a tree with more support for among animal taxa. However, while mt genome data have many pycnogonids as sister to other chelicerates, although taxon sam- useful attributes, there are limits on which genes or gene regions pling was more limited (Dunn et al., 2008). Analyses of mitochon- are useful. In part, this depends on the divergence times of the taxa drial genome sequences have suggested a third hypothesis, that used in the analysis. The rate of evolution in mitochondrial genes sea spiders are derived arachnids. The mitochondrial genomes of tends to be more rapid than in many nuclear loci, and therefore two species of sea spiders have previously been sequenced, and a mt gene comparisons tend to show the effects of phylogenetic sat- third has been partially sequenced. In phylogenetic analyses of uration sooner than other loci. Additionally, arthropod mt genomes the protein-coding genes from these genomes, along with those tend to possess a pronounced A + T bias, exacerbating the effects of of other arthropods, pycnogonids have been recovered as derived saturation. Because saturation can mislead phylogeny reconstruc- arachnids (Hassanin, 2006; Park et al., 2007; Podsiadlowski and tion, it is necessary to try to correct for the possible misleading ef- Braband, 2006). Given the contradictory results from different fects of saturation. Possible corrective measures for protein-coding sources of data, and the often sparse taxon sampling, it is necessary genes include omitting saturated third- and first-position nucleo- to further evaluate the phylogenetic placement of sea spiders. tides of codons, or using only translated amino acid data. Alterna- Despite the growing number of studies aimed at understanding tively, coding third and first positions of codons as purines or the relatedness of pycnogonids to other arthropods, very few stud- pyrimidines (RY coding) has been found to help reduce the mis- ies have attempted to infer relationships among the major pycno- leading effects of saturation found in mtDNA phylogenetic analyses gonid lineages. Hedgpeth and Fry classified sea spider taxa into (Phillips and Penny, 2003). families (Fry, 1978; Hedgpeth, 1947), but did not use modern cla- Another potential challenge when using mtDNA sequences in distic methods. More recent work by Arango has utilized both mor- phylogenetic analyses is uneven base composition among taxa in- phological and molecular characters to infer phylogenetic cluded in the study. Genome-wide biases are present in metazoan relationships within Pycnogonida (Arango and Wheeler, 2007; mitochondrial genomes in that genes encoded on the strand asso- Arango, 2002, 2003). These phylogenetic studies have lent support ciated with initiating replication tend to have a pronounced A + C to some of the previously defined families of sea spiders, but have bias (Reyes et al., 1998). However, within arthropods, and arach- also found relationships among pycnogonids that differ from the nids in particular, this bias is reversed in some taxa (Hassanin traditional taxonomy. Additionally, although many relationships et al., 2005; Masta and Boore, 2004; Masta et al., 2009), so that among sea spiders were similar using morphological and molecu- mitochondrial genes of phylogenetically disjunct taxa can share lar analyses, some relationships were supported only by molecular similar compositional bias. This has the effect of causing the re- data. Arango proposed that more characters were necessary to versed-biased taxa to cluster together in phylogenetic analyses, clearly delineate the pycnogonid lineages (Arango and Wheeler, unless measures are taken to ameliorate the misleading impact 2007; Arango, 2002, 2003). of such biases (Hassanin et al., 2005; Jones et al., 2007; Masta Using genomic-level characters to infer relatedness among taxa et al., 2009). Such measures include recoding some of the nucleo- has become possible with the increased capability of sequencing tide sites in the codons of the protein-coding genes as purines or large contiguous stretches of DNA. The use of data on gene pyrimidines (Hassanin et al., 2005; Jones et al., 2007; Masta arrangements to help deduce phylogenetic relationships has been et al., 2009), or recoding amino acids into categories corresponding particularly useful for inferring relationships among some meta- to their physiochemical properties (Masta et al., 2009). zoan taxa. Much of this work was pioneered with comparisons of In this manuscript, we present new data to bear on the relation- mitochondrial genome sequences, as the small size of mitochon- ship of sea spiders to other arthropods, and on their relationships dria made them an early target for studying genome evolution to one another. We have sequenced the complete suite of 13 mt and making phylogenomic inferences (Boore, 2006; Boore and protein-coding genes from three divergent sea spiders. This boosts Brown, 1998; Boore et al., 1995; Larget et al., 2005). the taxonomic coverage of pycnogonids with complete mt protein- Moreover, as well as changes in primary sequence and genome coding genes to 5 taxa. We conduct phylogenetic analyses using arrangements, the structures of molecules can evolve, subject to the entire suite of 13 protein-coding genes, and include represen- the selective constraints imposed on them to function within the tative taxa from 8 arachnid orders. Previous analyses that con- cell. One of the most highly conserved molecular structures is that cluded that pycnogonids were derived arachnids had of transfer RNA, which maintains a canonical cloverleaf shape representatives from only three arachnid orders. We compare gene across the three domains of life, Eukarya, Bacteria, and Archaea. arrangement of our three new pycnogonid mt genomes (one com- However, metazoan mitochondrial tRNAs are typically much smal- plete, and two missing sequence near the control region) to the ler than their nuclear counterparts, and in some cases may lack two published pycnogonid mt genomes and infer the phylogenetic either the DHU or TWC arms present in the canonical tRNA struc- history of gene arrangements in this group. Gene order provides ture (see reviews in Soll and RajBhandary, 1995; Wolstenholme, independent phylogenetic characters that support our findings 1992a). Even among metazoans, however, the evolutionary loss based on sequences. We also compare secondary structures of of one of the paired stem regions in a mt tRNA gene is rare. There- the transfer RNA genes in these 5 mt genomes, and use these struc- fore, when organisms share similar non-canonical tRNA structures, tures as phylogenetic characters. All three types of data are in it is likely due to shared evolutionary history. An unprecedented agreement. Together, our data provide strong support for relation- S.E. Masta et al. / Molecular Phylogenetics and Evolution 57 (2010) 59–70 61 ships among the pycnogonids that have not been inferred based on and Boore, 2008). Inferred secondary structures of tRNAs were anatomical characters. made by searching for conserved structural features. Anticodon stems always consist of 5 pairs of nucleotides while acceptor stems always consist of 7 pairs of nucleotides. However, the stems of the 2. Materials and methods TWC or DHU arms (also termed T-arm and D-arms) can vary in length in the mitochondrial genomes of arthropods. These arms 2.1. Specimens used, DNA extraction, amplification, and sequencing were determined to be present if there were potential stems consisting of at least 2 nucleotides that could pair with the nucleotides Specimens of the pycnogonids Tanystylum orbiculare (collected on the adjacent stem, and if the T- or D-loops consisted of at least 3 from the coast of Massachusetts; gift from Jeffrey Shultz), Colossen- nucleotides. If either of these two conditions were not met, the deis sp. (collected from Antarctica; gift from Jeffrey Shultz), and nucleotides present in these regions were determined to form TV Ammothea hilgendorfi (collected from the coast of California; gift or D replacement loops, structures common in other chelicerates from Bonnie Bain), were used in this study. DNA was extracted (Masta and Boore, 2008). The secondary structures of the tRNAs from a leg, or part of a leg, from each specimen, following the pro- from the published mt genome sequences of the sea spiders Achelia tocol from the Qiagen™ DNAeasy kit. bituberculata (GenBank accession # NC_009724) and Nymphon Initially, short fragments were PCR amplified from both the CO1 gracile (GenBank accession # NC_008572) were also inferred and and Cytb genes with the primers LCO/HCO and CobF/CobR, respec- compared to those from the three newly sequenced sea spider tively (see Masta and Boore, 2008 for further details). Then, taxon mt genomes. specific outward-facing primers (see Appendix) were developed Nucleotide composition was inferred with the aid of the pro- from these resulting fragments, and used for long-PCR amplifica- gram MacVector version 10.0.2. Strand asymmetry or skew was tion. Long-PCR amplification was performed using the Takara™ calculated following Perna and Kocher (1995), where AT LA Taq DNA polymerase kit. A 100 ll reaction contained final con- skew = (A T)/(A + T) and CG skew = (C G)/(C + G). Skew was centrations of 0.16 mM of each dNTP, 0.4 mM of each primer, 1X calculated for the strand encoding the majority of the protein-cod- Takara™ polymerase buffer, 1 ll of mitochondrial DNA (concentra- ing genes in most arthropods. This strand is termed the alpha tion not determined) and 2.5 Units of Takara™ polymerase. The strand in this manuscript, while the opposite strand is termed reaction was cycled at 92 °C for 30 s, 50–58 °C (depending on the the beta strand. primers) for 25 s, and 68 °C for 12 min, for 37 cycles, followed by We compared the gene order of the mt genomes of these three a final extension at 72 °C for 15 min. The PCR products were elec- sea spiders to those of the published sequences of the sea spiders A. trophoresed in a 0.8% agarose gel to estimate size and concentra- bituberculata and N. gracile, and to the gene arrangement ancestral tion, then cleaned by ethanol precipitation, and resuspended in for chelicerates, as found in horseshoe crabs, mesothele spiders, water. amblypygids, and some scorpions and ticks (see Fahrein et al., The mitochondrial genome for Tanystylum was amplified in one 2009; Masta, 2010). complete 15 kb piece using primers facing out from the Cytb amplicon. The majority of protein-coding genes for Colossendeis 2.3. Sequence alignment and phylogenetic analyses and Ammothea (about 9 kb) between CO1 and Cytb were amplified in one fragment using specific primers from the two alternate Sequences from 34 additional complete arthropod mitochon- genes. These long-PCR fragments were sheared into 1.5–2 kB drial genomes that span the diversity of arthropods were selected pieces, cloned into plasmid libraries, and subject to automated for phylogenetic analyses, with the onychophoran Epiperipatus sequencing at the DOE Joint Genome institute (as in Masta and used as an outgroup (see Table 1 for a list of taxa). We purposefully Boore, 2008). This resulted in approximately 15 coverage depth excluded candidate mitogenomic data for several mites (e.g., Lepto- for Tanystylum and Colossendeis, and 5 coverage depth for trombidium, Steganacarus; Metaseiulus), as these taxa show many Ammothea. substitutions that may cause branch attraction artifacts (e.g., Pods- We were unable to amplify across the control region and rRNA iadlowski and Braband, 2006, plus exploratory analyses not segments for Colossendeis and Ammothea with long-PCR. Instead, shown). All 13 mt protein-coding genes from these 40 taxa were for these two taxa remaining genomic segments of protein-coding translated with the Drosophila mitochondrial genetic code and genes were amplified by primer-walking. In both cases, a 2 kb re- the amino acids initially aligned with MacClade (Maddison and gion containing ND2 was amplified in a single reaction using com- Maddison, 2000). Amino acid sequences were then aligned in Clus- plementary primers that targeted tRNAMet and CO1. Additional tal X version 1.83 (Thompson et al., 1997) using the PAM350 pro- taxon specific primers were designed within this amplicon for tein matrix, gap opening equal to 10, and gap extension equal to internal sequencing across ND2. To amplify between CytB and 0.1. To increase confidence that our alignment accurately reflected ND1, we first amplified a short piece of the 16S rRNA in both taxa, homologous amino acids, poorly conserved sites were subse- and then designed taxon specific primers amplicon to sequence quently excluded with the program Gblocks (Castresana, 2000) internally. In addition, we sequenced between 12S rRNA and 16S version 0.91b. Regions of low sequence similarity were removed rRNA in Ammothea using taxon specific primers. by using the half gap setting and conserving alignment regions with at least five consecutive conserved amino acids. The 2.2. Sequence annotation, analyses, and tRNA secondary structure Gblocks-trimmed amino acid sequences from the 13 genes were determination then concatenated into a single sequence. For analysis of nucleotide sequences, we used only nucleotides Protein-coding genes were identified by similarity of inferred that correspond to the amino acids kept by Gblocks for phyloge- amino acid sequences to those of other arthropod mtDNAs. Ribo- netic analyses. The output files from Gblocks (that list which amino somal RNA genes were annotated based on alignments with other acids in the alignment were kept) were used to compare to the arachnid rRNA genes. After the protein-coding gene boundaries nucleotide alignments in MacClade, using the ‘‘Nucleotide with had been determined, the remaining regions were searched for Amino Acid Translation” display option. The regions that were ex- tRNAs with the use of the program tRNAscan-SE 1.21 (Lowe and cluded from the GBlocks alignment were then manually excluded Eddy, 1997), and by aligning the candidate sequences to the sec- from the MacClade file. The nucleotides at the first and third posi- ondary structure-based alignments of other arachnids (see Masta tions of each codon were then recoded as either purines or pyrim- 62 S.E. Masta et al. / Molecular Phylogenetics and Evolution 57 (2010) 59–70

Table 1 Taxonomic sampling used in phylogenetic analyses.

Taxonomic lineage Species GenBank No. Chelicerata Pycnogonida Ammotheidaea Tanystylum orbiculare GU370074 Ammothea hilgendorfi GU370075 Achelia bituberculata NC_009724 Colossendeidae Colossendeis sp. GU370076 Nymphonidae Nymphon gracile NC_008572 Xiphosura Limulidae Limulus polyphemus NC_003057 Arachnida Acari, Ixodida Ornithodoros moubata NC_004357 Haemaphysalis flava NC_005292 Ixodes hexagonus NC_002010 Rhipicephalus sanguineus NC_002074 Carios capensis NC_005291 Acari, Mesostigmata Varroa destructor NC_004454 Amblypygi Phrynus sp. NC_010775 Araneae, Mygalomorphae Calisoga longitarsisb NC_010780 Ornithoctonus huwena NC_005925 Araneae, Araneomorphae Habronattus oregonensis NC_005942 Nephila clavata NC_008063 Hypochilus thorelli NC_010777 Araneae, Mesothelae Heptathela hangzhouensis NC_005924 Ricinulei Pseudocellus pearsii NC_009985 Scorpiones, Buthidae Buthus occitanus NC_010765 Centruroides limpidus NC_006896 Scorpiones, Chactidae Uroctonus mordax NC_010782 Solifugae Eremobates palpisetulosus NC_010779 Nothopuga sp. NC_009984 Thelyphonida Mastigoproctus giganteus NC_010430 Opiliones Phalangium opilio NC_010766 Myriapoda Chilopoda Lithobiidae Lithobius forficatus NC_002629 Scutigeridae Scutigera coleoptrata NC_005870 Diplopoda Spirobolidae Narceus annularus NC_003343 Harpagophoridae Thyropygus sp. DVL-2001 NC_003344 Pancrustacea Hexapoda Collembola, Poduridae Podura aquatica NC_006075 Insecta, Diptera Drosophilidae Drosophila yakuba NC_001322 Insecta, Diptera Anopheles gambiae NC_002084 Neoptera, Hemiptera Triatoma dimidiata NC_002609 Crustacea Maxillopoda, Cirripedia, Pollicipes polymerus NC_005936 Malacostraca, Paguridae Pagurus longicarpus NC_003058 Branchiopoda, Anostraca Artemia franciscana NC_001620 Diplostraca Daphnia pulex NC_000844 Onychophora Peripatidae Epiperipatus biolleya NC_009082

a Tanystylum was formerly placed in the family Tanystylidae, but found by Arango (2002) to possess morphological characters that placed it within Ammotheidae, and was subsequently considered to be in the family Ammotheidae (Arango and Wheeler, 2007). b This mygalomorph spider was formerly labeled as Aphonopelma sp. in GenBank, but it has now been identiﬁed as Calisoga longitarsis (although it is currently placed in the genus Brachythele, until the nomenclature is revised).

idines (RY coding), following Phillips and Penny (2003) as this has following six groups: sulfhydryl (cysteine); small/neutral (serine, been found to increase the phylogenetic signal in analyses of mito- threonine, alanine, proline, and glycine); acidic/amides (aspartate, chondrial genomes (Delsuc et al., 2003; Phillips and Penny, 2003). glutamate, asparagine, and glutamine); basic (histidine, arginine, Phylogenetic analyses of amino acids and nucleotides were per- and lysine); hydrophobic (valine, leucine, isoleucine, and methio- formed using maximum likelihood with RaxML, version 7.0.4 (Sta- nine); and aromatic (phenylalanine, tyrosine, and tryptophan). This matakis, 2006). For analyses with amino acids, the MtREV model of six-character-state data matrix was then analyzed by parsimony evolution was used, with gamma plus invariant sites model of rate using PAUP* (Swofford, 2000) with a heuristic search with 10 rep- heterogeneity. The MtREV model of evolution is the best model for licates, ACCTRAN, and tree-bisection-reconnection branch-swap- mtDNA analyses that is available in RaxML, as determined by the ping. One thousand bootstrap replicates were performed using use of ProtTest (Abascal et al., 2005). For analyses of nucleotides, these same parameters. the GTR with gamma plus invariant sites model of evolution was used on the RY recoded dataset. One thousand bootstrap replicates were performed for both of these analyses using the same models, 3. Results except using the CAT approximation instead of gamma for the amino acid analysis. RaxML using the CAT approximation has been 3.1. Genome composition shown to perform well on large datasets (Stamatakis, 2006). Fig- ures of the recovered phylogenetic trees were made with the aid We obtained the entire mt genome sequence of T. orbiculare,a of FigTree v1.2.3. circular genome of 15,251 nt (GenBank accession number Because some of the pycnogonid mt genomes were found to GU370074). For the mt genome of A. hilgendorﬁ, we obtained possess a reversed nucleotide strand bias, as were those of some 14,026 nt of sequence (GenBank accession number GU370075), arachnids (Masta et al., 2009), we also recoded amino acids into and for Colossendeis we sequenced 12,776 nt (GenBank accession six physiochemical groups. We categorized amino acids into the number GU370076). The regions not sequenced in Ammothea and S.E. Masta et al. / Molecular Phylogenetics and Evolution 57 (2010) 59–70 63

Colossendeis are located near the where the control region is typi- ossendeis also lacks sequence for the SSU rRNA gene, and has only cally found in arthropods. It is not uncommon to have difﬁculty the 30 end of the LSU rRNA and tRNAMet genes. Sequence coding for amplifying and sequencing across this region in arthropods, possi- the putative control region is also missing. bly due to the high A + T content, or the stem-forming stretches of Surprisingly, there are pronounced differences in the nucleotide nucleotides that create secondary structures and can inhibit func- skews in these pycnogonid mt genomes. Ammothea and Nymphon tion of polymerase enzymes. As a result, some of the mt tRNA and have a biased composition with positive AT and CG skews on the rRNA genes were not sequenced for Ammothea and Colossendeis; alpha strand, while Tanystylum and Colossendeis have the opposite however, we obtained full coverage of all 13 of the mt protein-cod- pattern, with negative AT and CG skews (Table 2). Achelia shows a ing genes for each of the three pycnogonids. third pattern, with a negative AT skew and a positive CG skew. The mt genome of Tanystylum contains all 22 tRNA genes and the SSU and LSU rRNA genes that are typically found in metazoan 3.2. Gene order comparisons mt genomes. The partial mt genome of Ammothea lacks sequences for the genes for tRNAIle and tRNAGln and has only the 30 ends of the The mitochondrial gene arrangements of all 5 pycnogonid taxa SSU rRNA and tRNAMet genes. The sequence for the putative control with complete protein-coding data from the mt genome are de- region is lacking. Presumably the control region plus the two miss- picted in Fig. 1. Tanystylum has a gene order identical to that of ing tRNA genes are present in the unsequenced portion of the mt Achelia, which differs from the ancestral chelicerate gene order genome. The partial mt genome of Colossendeis possesses 16 tRNA only in the location of a single tRNA gene. In these mt genomes, genes, as we did not obtain sequence for tRNAPro, tRNAAla, tRNAAsn, the tRNAGln gene is located between the SSU gene and the large tRNASer(AGN), tRNAGln, and tRNAVal genes. The partial genome of Col- noncoding region, instead of between the genes coding for tRNAIle and tRNAMet as it is in the ancestral chelicerate genome. With the exception of the missing region, the gene order for the Ammothea Table 2 mt genome is identical to that of the ancestral chelicerate, or Tany- Nucleotide composition and skew of the alpha strand of pycnogonid mt genomes. stylum, and Achelia. Taxon Proportion of nucleotides AT skew CG skew The genome of Colossendeis shows a gene order quite different ACGT from that of the ammotheid sea spiders, in several ways (Fig. 1). Ala Asn Tanystylum 0.389 0.097 0.103 0.410 0.0263 0.03 First, four of the six unsequenced tRNA genes (tRNA , tRNA , Ammothea 0.414 0.117 0.081 0.388 0.0324 0.1818 tRNASer(AGN), and tRNAPro) should have been obtained had they Achelia 0.379 0.119 0.110 0.391 0.0156 0.0393 been in the same location as in the ancestral chelicerate; thus, Nymphon 0.454 0.141 0.085 0.319 0.1746 0.2478 these must have been translocated. Second, the relative positions Colossendeis 0.363 0.116 0.127 0.388 0.0333 0.0453 of tRNAGlu and tRNAArg in Colossendeis are opposite to their posi-

Fig. 1. Mitochondrial genome arrangement of 5 pycnogonids and ancestral chelicerate arrangement. The circular mt genomes are illustrated as linear by depicting CO1 as the beginning of each linearized genome (except the highly rearranged Nymphon). The putative control regions are designated with ‘‘lnr” (large noncoding region), while tRNA genes are abbreviated with their one-letter amino acid code. The two leucine and two serine tRNA genes are further differentiated by numerals, where L1 = tRNALeu(CUN), L2 = tRNALeu(UUR)), S1 = tRNASer(AGN), and S2 = tRNASer(UCN). Shaded regions indicate that the genes present in that location differ from those present in that location in the ancestral chelicerate. Genes are not shown proportional to size. 64 S.E. Masta et al. / Molecular Phylogenetics and Evolution 57 (2010) 59–70 tions in the ancestral chelicerate and in ammotheid sea spiders. tRNALeu(CUN) both possess mispaired nucleotides in their acceptor Third, the tRNATyr gene is located next to the tRNAPhe gene on stem regions in Nymphon and Colossendeis, whereas the other pyc- the beta strand of Colossendeis. The gene order between the ND3 nogonids have well-paired stems. The anticodon stems of the and tRNAPhe genes in Colossendeis is therefore the same as in Nym- tRNACys genes have mispaired nucleotides in Nymphon and Colos- phon, except that the control region of Nymphon is located between sendeis (Fig. 3). the tRNAArg and tRNATyr genes (Fig. 1). Likewise, Colossendeis and Nymphon have identical gene arrangements between ND4L and 3.4. Phylogenetic analyses of amino acids ND6, as both lack the gene for tRNAPro that is located here in the other pycnogonids. Finally, a unique gene arrangement found in There were 3894 amino acids in our initial alignments of the Colossendeis but not other sea spiders, is the location of the tRNAMet protein-coding genes. Of these, 2313 amino acids from all 13 con- gene immediately upstream of the tRNAIle gene. catenated protein-coding genes were retained after GBlocks trim- ming of the dataset. This reduced dataset was used in 3.3. tRNA structure comparisons subsequent phylogenetic analyses. The maximum likelihood analysis of amino acids yielded a phy- Most of the 22 tRNA genes in the mt genomes of these 5 pyc- logenetic tree in which a clade of chelicerates was recovered with nogonids have inferred secondary structures that are typical of 95% bootstrap support (Fig. 4A). Nested within the chelicerates, a metazoan mt tRNAs. In some cases, we reached different interpre- clade of pycnogonids was recovered with 100% bootstrap support. tations of secondary structure for Achelia and Nymphon than had While there is not strong support to infer which group of arachnids been previously published (Park et al., 2007; Podsiadlowski and may be the sister group to pycnogonids, there is ample support to Braband, 2006). While most of the tRNAs possess cloverleaf struc- place them within the clade Micrura, which traditionally consists tures, some tRNA genes lack the sequence to form the DHU or D- of the orders Acari, Ricinulei, Araneae, Thelyphonida, Schizomida, arm that is part of the canonical tRNA (see Fig. 2). All pycnogonids and Amblypygi (see review in Shultz, 2007). lack D-arm sequences in their tRNAAla genes, with the most ex- Relationships among the other major lineages of arthropods are treme nucleotide reductions occurring in Tanystylum, Ammothea, similar to those that have been recovered in other analyses of and Achelia (Fig. 3), although Colossendeis was unsampled. Nym- molecular data, with high support for a clade of Pancrustacea. Myr- phon and Colossendeis lack well-paired D-arm stem sequences in iapoda was not recovered as a clade, but instead the centipedes their tRNAArg genes. Like other metazoans (Wolstenholme, and millipedes were each found to be monophyletic, with very 1992b), all pycnogonids lack D-arm sequence in their tRNASer(AGN) weak support for a sister-group relationship between the Diplo- genes. All 22 of the tRNAs present in the mitochondrial genomes of poda (millipedes) and the chelicerates. these pycnogonids possess T-arms (Fig. 3). The interrelationships among the pycnogonids were all strongly Another structural difference is present in the tRNAIle gene in supported, with two divergent lineages recovered. One lineage Nymphon and Colossendeis. These taxa, but not the ammotheids, consists of Tanystylum, Ammothea and Achelia, while the other con- possess a variable loop that is only 4 nt long, instead of the 5-nt- sists of Nymphon and Colossendeis. long loop found in Tanystylum and Achelia. The parsimony tree that we recovered from analyses of amino Differences also exist in the anticodons present in two tRNA acids that had been recoded into 6 functional groups yielded a tree genes within sea spiders. The anticodon for tRNALys is UUU in all with unresolved relationships among the major lineages of arthro- ﬁve pycnogonids, whereas other arthropods possess a CUU antico- pods, and therefore is not shown. However, this tree yielded iden- don. The anticodon for tRNASer(AGN) is UCU in all four pycnogonids tical relationships among the pycnogonids, with 100% bootstrap for which this tRNA gene was sequenced. This differs from the typ- support for a clade consisting of Tanystylum, Ammothea and Achelia, ical GCU anticodon found in most other chelicerates. and 100% bootstrap support for a clade consisting of Nymphon and Some mispaired nucleotides are present in the anticodon and Colossendeis. There was 76% bootstrap support for a sister-group acceptor stem regions of 8 tRNA genes within pycnogonids. While relationship between Tanystylum and Ammothea. most of these are conﬁned to only a single individual, tRNAIle and 3.5. Phylogenetic analyses of RY-coded nucleotides

The maximum likelihood analysis of nucleotides with the ﬁrst ABand third positions recoded as purines or pyrimidines yielded a tRNA-Arginine tRNA-Alanine phylogenetic tree that was largely congruent with the amino- 3' 3' acid-based tree. A clade of chelicerates was recovered with 81% 5' T 5' A bootstrap support, with a clade of pycnogonids nested within aminoacyl T A A T A T G C Chelicerata (Fig. 4B). Unlike the amino-acid-based tree, however, acceptor arm G T G T T A C G there was only very weak support for placing Pycnogonida within A T A T A T T-arm A T the micrurans. Additionally, all micrurans were not included in this G C G C A C C G A T A T clade, as Amblypygi fell outside of it. A A CTGA T D-arm T A G G T T T The RY-coded analysis recovered a high level of support (97% T G GT G T G C T A T A A T A A C AA A bootstrap) for a clade of Pancrustacea, as was also found in the T T T T T T T A T T T A T amino acid analysis. While there was generally lower support for T A variable T A most groupings in the recoded nucleotide based tree, there was T A T A G C loop A T high support for a monophyletic Myriapoda. Support for a sister- anticodon arm A T A T T C T A group relationship between Myriapoda and Chelicerata was weak. T A T A TCG TGC Pycnogonida was recovered with high support, as were the same two divergent lineages found in the amino acid analysis (a Fig. 2. Secondary structure drawing of the two types of tRNAs found in pycnogo- clade consisting of Tanystylum, Ammothea and Achelia, and a clade nids. The tRNA structures from Tanystylum orbiculare are illustrated. (A) A canonical consisting of Nymphon and Colossendeis). However, in the RY-re- tRNA, as illustrated with tRNAArg. Note the 5 regions of the tRNA: acceptor stem, D- arm, anticodon arm, variable loop, and T-arm. (B) A tRNA that lacks a D-arm, and coded nucleotide analysis, Tanystylum and Achelia were recovered instead has only a D-loop, as illustrated with tRNAAla. as sister taxa with 68% bootstrap support (Fig. 4B). S.E. Masta et al. / Molecular Phylogenetics and Evolution 57 (2010) 59–70 65

Fig. 3. Secondary structure alignments for the 22 tRNAs found in the mt genomes of pycnogonids. The 5 regions of the canonical tRNA structure are given at the top of the ﬁgure (see Fig. 2 for an illustration of these regions). Dashes separate these structural features in the alignments. Underlined nucleotides indicate they form base pairs with adjacent underlined nucleotides in the same region of the tRNA.

3.6. Rare genomic changes mapped onto the phylogenetic tree One is the translocation of tRNAGln between SSU and the large noncoding region, while the other is a reduction in the size of the D- The gene translocations and tRNA structure differences that we loop in tRNAAla. In contrast, the clade consisting of Nymphon and inferred were mapped onto the consensus phylogenetic tree of Colossendeis shows at least 12 changes in genome or tRNA struc- pycnogonids. The pycnogonids are united by three tRNA features ture that unite it as a monophyletic group. Five of these changes that distinguish them from other chelicerates: the loss of a D- occur within the tRNA genes, while the other seven changes are arm in tRNAAla, and changes in the anticodon identities of tRNALys based upon genome rearrangements (Fig. 5). and tRNASer(AGN) (Fig. 5). Within pycnogonids, the ammotheids At least seven mt tRNA translocations must have occurred in the Tanystylum and Achelia are united by two rare genomic changes. lineage leading to Nymphon and Colossendeis. These are mapped 66 S.E. Masta et al. / Molecular Phylogenetics and Evolution 57 (2010) 59–70

Fig. 3 (continued) onto the phylogenetic tree of pycnogonids (Fig. 5). An eighth trans- signed to overcome the effects of saturation and opposite patterns location, that of tRNAVal moving away from its ancestral location of mutational bias. Within pycnogonids, we found no evidence that between the LSU and SSU rRNA genes, cannot be placed with con- altered patterns of nucleotide bias affected phylogeny reconstruc- fidence onto the phylogeny, since we lack sequence data for this tion. Biased nucleotide composition may be expected to cause taxa region in Colossendeis. Four unique gene translocations distinguish with similar biases to group together. However, Tanystylum and Nymphon, and therefore must have occurred after its divergence Colossendeis show patterns of skew opposite to that of the other from a common ancestor with Colossendeis (Fig. 5). pycnogonids, and indeed to that of most arthropods. However, these taxa were not reconstructed as sister taxa, despite their shared biases. Likewise, other taxa with similar patterns of skew 4. Discussion (e.g., opisthothele spiders and scorpions; see Masta et al., 2009) also did not group closely with these pycnogonids. This gives us 4.1. Variable patterns of nucleotide bias confidence that our phylogenetic methods have adequately ac- counted for different mutational biases in this dataset. Arthropod mt genomes tend to have an AT nucleotide bias, with a positive AC skew on the major or alpha strand. The strength of the skew varies among taxa, and some arthropod taxa have been found 4.2. Pycnogonids as derived arachnids to show the opposite pattern; that is, a negative AC skew, or a positive TG skew (Hassanin, 2006). We found both of these two ex- Our analyses strongly support the placement of Pycnogonida treme patterns of skew present within the five pycnogonids we within Chelicerata, and therefore would reject the hypothesis that analyzed. Although reversals in skew can impact phylogenetic sea spiders are the sister group to all other arthropods. Our phylo- reconstruction and lead to erroneous conclusions (Hassanin, genetic analyses also consistently place sea spiders as derived 2006; Jones et al., 2007; Masta et al., 2009), we used methods de- chelicerates. These findings are in agreement with other recent S.E. Masta et al. / Molecular Phylogenetics and Evolution 57 (2010) 59–70 67

100 Ixodes A 100 Rhipicephalus 100 Haemaphysalis Acari 100 100 Ornithodoros Carios Varroa 57 80 Tanystylum 100 Ammothea 100 Achelia Pycnogonida 100 Nymphon Colossendeis 54 Pseudocellus Ricinulei Mastigoproctus Thelyphonida 69 Heptathela 73 100 100 Ornithoctonus Chelicerata 100 Calisoga Araneae 100 Habronattus 78 Nephila 52 Hypochilus Phrynus Amblypygi 100 Nothopuga Solifugae 95 Eremobates Phalangium Opiliones Limulus Xiphosura 56 100 Centruroides 100 Buthus Scorpiones 55 Uroctonus 100 Narceus Thyropygus 98 Scutigera Myriapoda Lithobius Pollicipes Pagurus 57 Podura 93 98 Artemia Pancrustacea Daphnia 58 97 Triatoma 100 Anopheles Drosophila Epiperipatus Onychophora

0.2

100 Ixodes B 100 Rhipicephalus 100 Haemaphysalis Acari 100 100 Ornithodoros Carios Varroa Pseudocellus Ricinulei 68 Tanystylum 100 Achelia 100 Ammothea Pycnogonida 100 Nymphon Colossendeis Mastigoproctus Thelyphonida Heptathela 92 100 Ornithoctonus Chelicerata 81 100 Calisoga 100 Habronattus Araneae 98 Nephila Hypochilus 100 Nothopuga Eremobates Solifugae Phrynus Amblypygi Limulus Xiphosura 100 Centruroides 100 Buthus Scorpiones Uroctonus Phalangium Opiliones 100 Narceus Thyropygus 84 98 Scutigera Myriapoda Lithobius Pollicipes Pagurus 93 97 Daphnia Artemia Pancrustacea 77 Podura Triatoma 98 100 Anopheles Drosophila Epiperipatus Onychophora 0.1

Fig. 4. (A) Maximum likelihood tree based on amino acids from 13 mt protein-coding genes, using the MtREV substitution matrix. Bootstrap values greater than 50% are shown above or below the corresponding branches. Likelihood = 86273.619981; Alpha = 0.857; invariant sites = 0.194572. (B) Maximum likelihood tree based on RY-coded nucleotides from 13 mt protein-coding genes, using the GTR substitution matrix with gamma and invariant sites. Bootstrap values greater than 50% are shown above or below the corresponding branches. For both trees, the bars colored medium gray highlight the three main arthropod groups: Chelicerata, Myriapoda, and Pancrustacea, while the other colored bars are used to highlight the chelicerate groups for which multiple taxa were sequenced. 68 S.E. Masta et al. / Molecular Phylogenetics and Evolution 57 (2010) 59–70

Tanystylum N A 9 Ammothea Achelia 1 2 3 I JKL Nymphon 4 5 6 7 8 B C DEF GH M Colossendeis

Fig. 5. Phylogeny of pycnogonids with rare genomic changes depicted on the tree. Boxes that are shaded and with letters depict inferred gene translocation events, and unshaded boxes with numbers depict inferred changes in tRNA structure or anticodon identity. Letters for tRNA gene translocations are as follows, using the one-letter amino acid abbreviation: (A) Q translocated between SSU and the large noncoding region (lnr), on same beta strand as original location. (Not assessed in Ammothea); (B) I (or M) translocated between M and ND2, on same alpha strand as original location; (C) Y translocated next to F, on same beta strand as original location; (D) N translocated to near control region; (E) S1 translocated to near control region; (F) A translocated to near control region; (G) either R or E translocated such that E is directly upstream of R on alpha strand; (H) P translocated to near control region; (I) translocation and inversion of K and D to near control region; (J) translocation of region coding for I-ND2-W-C-CO1-CO2 onto opposite strand, upstream of ATP8; (K) M translocated to beta strand, between P and S1; (L) control region translocated to between R and Y; (M) translocation of I to between M and ND2, on same alpha strand and (N) V translocated away from location between LSU and SSU rRNA genes. Numbers for tRNA changes are as follows: (1) loss of the D-arm in tRNAAla; (2) anticodon for tRNALys changes to UUU; (3) anticodon for tRNASer(AGN) changes to UCU; (4) variable loop in tRNAIle decreases to 4 nt in Nymphon and Colossendeis, from 5 nt long in Tanystylum and Achelia; (5) loss of paired D-arm sequence in tRNAArg; (6) mispaired acceptor stems in tRNAIle; (7) Mispaired acceptor stems in tRNALeu(CUN); (8) mispaired anticodon stems of tRNACys and (9) reduced number of nucleotides in D-loop in tRNAAla. analyses of mitochondrial genome data (Jeyaprakash and Hoy, pions, solifuges, harvestmen). Under this scenario, the group 2009; Park et al., 2007; Podsiadlowski and Braband, 2006). How- Arachnida would be polyphyletic, with two separate origins. Fur- ever, unlike those analyses, which had representative taxa from ther fossil discoveries and developmental and molecular data with just three of the arachnid orders, our analyses included taxa from stronger phylogenetic signal will help elucidate if this is a likely 8 arachnid orders that span the diversity of Chelicerata. Our phylo- scenario. genetic analyses support Pycnogonida as members of micruran arachnids, a group that includes the Acari (mites and ticks) and 4.3. Rare genomic changes as phylogenetic markers Araneae (spiders). Pycnogonids have been found to be nested within a clade of micrurans in combined analyses of morphological and Although there are no gene arrangements that group sea spiders nuclear rRNA data (Giribet et al., 2002), but the authors cautioned with any single chelicerate lineage, pycnogonids are supported as that they did not find high support for these relationships. chelicerates by their similar gene order. The ammotheid sea spi- The possibility that sea spiders are derived arachnids or cheli- ders share the same basic gene arrangement as other chelicerates, cerates has not been supported by developmental or anatomical and differ only in the location of a single tRNA gene. Therefore, we studies. However, most studies that have tried to determine the can infer that the translocation of this tRNA occurred after the taxonomic placement of pycnogonids have not included a diversity divergence of sea spiders from their common ancestor with other of micruran arachnids in their analyses (but see Wheeler and Hay- chelicerates. Pycnogonids lack the derived mt gene order found ashi, 1998). Therefore, it is not clear which, if any, anatomical or in Pancrustacea, in which the tRNALeu(UUR) gene is located between developmental features may be shared among pycnogonids and CO1 and CO2, and not between ND1 and the LSU rRNA genes as in other arachnids. Indeed the idea that sea spiders are derived arach- chelicerates (Boore et al., 1995, 1998). Therefore, both our phyloge- nids may seem unlikely, not only because of sea spiders’ unique netic analyses and our genome arrangement data are consistent and highly derived anatomy, but also due to their strictly marine with pycnogonids being chelicerates. habitats. Most arachnids are strictly terrestrial, with anatomical The three tRNA structural characters that unite the pycnogonids features that allow them to live in terrestrial environments. (see Fig. 5) show some degree of homoplasy when evaluated across The ecology of sea spiders, however, does not necessarily help arthropods. A change in the anticodon from GCU to UCU in tRNA- us understand their phylogenetic affinities. Although they are re- Ser(AGN) has been found in some other arthropod groups. For exam- stricted to marine environments, pycnogonids are found in a di- ple, all beetles appear to possess tRNASer(AGN) genes with UCU verse array of ecological niches, from the intertidal zone to deep- anticodons, an apparent synapomorphy for this lineage of insects sea hydrothermal vents to the cold waters of Antarctica. Although (Sheffield et al., 2008). Likewise, a change in anticodon identity it is tempting to think that their marine habitats may imply a close for tRNALys has occurred multiple times in arthropods, and is cor- relationship to the (marine) horseshoe crabs, sea spiders are not related with changes in tRNASer(AGN) anticodon identity (Abascal the sole marine chelicerates. Some mites in the arachnid order et al., 2006). Changes in anticodon identity and remolding of tRNA- Acari inhabit marine environments, whereas others inhabit terres- Leu has occurred multiple times in the evolution of metazoan trial and freshwater environments. Clearly, transitions from sea to mtDNA (Rawlings et al., 2003), and can lead to incorrect inferences land and back again have occurred in the evolutionary history of about gene rearrangements if not correctly identified. However, the chelicerates. tRNA remolding cannot explain the translocations of whole blocks Paleontological data are consistent with an aquatic origin of of tRNA genes to new locations in the genome, as found in Nym- pycnogonids, and suggests they emerged during the Cambrian, be- phon and Colossendeis. The loss of the D-arm from tRNAAla has also fore the other extant chelicerate lineages (Dunlop and Selden, occurred independently in amblypygids (Fahrein et al., 2009). 2009). Fossils of horseshoe crabs (Rudkin et al., 2008) exist from Therefore, while these changes have occurred only rarely in evolu- the Ordovician, consistent with an early divergence of xiphosurans. tion, they do exhibit some degree of homoplasy within arthropods. Because these horseshoe crab fossils possess derived anatomical Searching for other rare evolutionary changes that pycnogonids features, it was suggested that xiphosurans likely diverged even share with other taxonomic groups may help to more definitively earlier, in the Cambro-Ordovician (Rudkin et al., 2008). The well- infer their closest relatives. supported nodes in our phylogenetic trees (e.g., >70% bootstrap In contrast, many of the genome rearrangements that we in- support) are not in conflict with this paleontological data. Instead, ferred to occur in the common ancestor of Nymphon and Colossen- they suggest a scenario of an ancestral chelicerate diversifying in deis are unique among arthropods. For example, the gene the Cambrian into at least two lineages. One of these lineages could arrangement of the genes for ND3, tRNAGlu, tRNAArg on the alpha then have subsequently diversified into pycnogonids and the strand, followed by the tRNATyr and tRNAPhe genes on the beta ancestor of micrurans, while the other lineage diversified into strand, is not found among other arthropods. This gene order re- xiphosurans and the ancestor of dromopodan arachnids (i.e., scor- quired multiple translocations of tRNA genes, and therefore, find- S.E. Masta et al. / Molecular Phylogenetics and Evolution 57 (2010) 59–70 69 ing evolution convergent with this same arrangement seems Both the analyses of Arango and Wheeler (2007) and Nakamura highly unlikely. However, the shared changes seen in the tRNA et al. (2007) consistently recovered Callipallenidae as sister to genes among Nymphon and Colossendeis are likely more evolution- Nymphonidae. Our findings would be consistent with these previ- arily labile, and may occur on a time scale that allows them to be ous analyses if our specimen were actually a member of the family useful for delimiting pycnogonid taxa, although not at deep phylo- Callipallenidae, and not Colossendidae. However, both our se- genetic levels. Together, though, the inferred gene translocation quence data and our rare genomic change data indicate that the and tRNA structural characters are synapomorphies that further specimen is quite divergent from the N. gracile mt genome, and support our phylogenetic analyses of amino acids and that group that rare genomic changes are useful for resolving sea spider Nymphon with Colossendeis. relationships. Our analyses provide strong support for the placement of sea 4.4. Phylogenetic relationships within Pycnogonida spiders as chelicerates, and moderate support for sea spiders as derived chelicerates. There are deep divergences within sea spiders, A traditional classification of pycnogonids divided them into 8 resulting in phylogenetic trees with lineages separated by long families (Hedgpeth, 1947), but did not make inferences as to how branches. Additionally, we find multiple cases of changes in mito- these may be interrelated. The taxa in our study included represen- chondrial strand bias among these lineages. Our analyses at- tatives from what have been considered to be quite divergent lin- tempted to overcome the misleading effects of changes in strand eages: Ammotheidae, Nymphonidae, and Colossendeidae. A bias by recoding data as amino acids or as purines and pyrimidines. cladistic analysis of morphological characters (Arango, 2002) found However, it is more difficult to overcome potential problems asso- that Ammotheidae was a diverse and paraphyletic group, with Col- ciated with long branches causing the recovery of artifactual rela- ossendeidae derived within the ammotheids. Taxa from the genera tionships. Future sampling of additional sea spider mitochondrial Achelia and Tanystylum were found to be more closely related to genomes may help break up the branch leading to sea spiders, as one another than they are to Ammothea. This paraphyletic group well as branches among the major lineages of sea spiders. Rare of ammotheids plus colossendeids was found to be sister to a clade changes in the mitochondrial tRNA genes and in mitochondrial that contained Nymphonidae. gene arrangements hold much promise for helping to resolve rela- Our analyses provide support for a group of ammotheids, but tionships among this enigmatic group of animals. only phylogenetic analyses of RY-coded nucleotide data give support for the closest relationship being between Achelia and Tanysty- Acknowledgments lum. Analyses of amino acids yield trees with high support for a closer relationship of Tanystylum with Ammothea. We do not find This work was funded by NSF award no. DEB-0416628 to S.E.M. rare genomic changes that support or refute either of these scenar- The manuscript was improved by the comments of Claudia Arango, ios. However, we do find very strong support for a close relation- Alfried Vogler, and an anonymous reviewer. ship between Colossendeis and Nymphon, in contrast to inferences made based upon morphological characters (Arango, 2002) and Appendix A. Supplementary data morphology plus six loci (Arango and Wheeler, 2007). We also find Nymphon to possess a highly derived gene arrangement that Supplementary data associated with this article can be found, in clearly shares its origins with those of Colossendeis. the online version, at doi:10.1016/j.ympev.2010.06.020. Our findings are consistent with those of Arango (2003), who found that 18S and 28S rRNA sequences yielded a tree with the References ammotheids sister to a clade that contains Nymphon and Colossen- deis (which were reconstructed as sister taxa). 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