The First Complete Mitochondrial Genome Sequences of Amblypygi (Chelicerata: Arachnida) Reveal Conservation of the Ancestral Arthropod Gene Order

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456 The first complete mitochondrial genome sequences of Amblypygi (Chelicerata: Arachnida) reveal conservation of the ancestral arthropod gene order

Kathrin Fahrein, Susan E. Masta, and Lars Podsiadlowski

Abstract: Amblypygi (whip spiders) are terrestrial chelicerates inhabiting the subtropics and tropics. In morphological and rRNA-based phylogenetic analyses, Amblypygi cluster with Uropygi (whip scorpions) and Araneae (spiders) to form the taxon Tetrapulmonata, but there is controversy regarding the interrelationship of these three taxa. Mitochondrial genomes provide an additional large data set of phylogenetic information (sequences, gene order, RNA secondary structure), but in arachnids, mitochondrial genome data are missing for some of the major orders. In the course of an ongoing project con- cerning arachnid mitochondrial genomics, we present the first two complete mitochondrial genomes from Amblypygi. Both genomes were found to be typical circular duplex DNA molecules with all 37 genes usually present in bilaterian mitochondrial genomes. In both species, gene order is identical to that of Limulus polyphemus (Xiphosura), which is assumed to reflect the putative arthropod ground pattern. All tRNA gene sequences have the potential to fold into structures that are typical of metazoan mitochondrial tRNAs, except for tRNA-Ala, which lacks the D arm in both amblypygids, suggest- ing the loss of this feature early in amblypygid evolution. Phylogenetic analysis resulted in weak support for Uropygi being the sister group of Amblypygi. Key words: Arachnida, Megoperculata, mitochondrial genome, phylogony, mitochondrial control region. Re´sume´ : Les amblypyges (araigneés a` fouet) sont des che´lice´rates terrestres habitant les re´gions tropicales et subtropica- les. Dans les analyses phylogeńe´tiques fondeés sur les caracte`res morphologiques ou l’ARNr, les amblypyges se groupent avec les uropyges (scorpions a` fouet) et les araneés (araigneés) pour former le taxon Tetrapulmonata, mais il existe une controverse quant aux relations entre ces trois taxons. Les geńomes mitochondriaux fournissent une importante quantite´ d’informations phylogeńe´tiques additionnelles (se´quences, ordre des ge`nes, structure secondaire des ARN), mais les don- neés manquent sur les geńomes mitochondriaux chez certains des ordres majeurs au sein des arachnides. Dans le cadre d’un projet en cours sur la geńomique mitochondriale chez les arachnides, les auteurs pre´sentent les deux premiers geńo- mes mitochondriaux complets chez les amblypyges. Les deux geńomes pre´sentent une molećule d’ADN bicateńaire circu- laire typique comprenant les 37 ge`nes habituellement pre´sents chez les geńomes mitochondriaux des bilate´riens. Chez les deux espe`ces, l’ordre des ge`nes est identique a` celui rencontre´ chez Limulus polyphemus (Xiphosura), ce qui est pre´sume´ refle´ter la disposition basale chez les arthropodes. Tous les ge`nes codant pour des ARNt ont des se´quences ayant le poten- tiel de se replier pour former des structures qui sont typiques des ARNt-mt chez les me´tazoaires, a` l’exception de l’ARNt- Ala auquel il manque le bras D chez les deux amblypyges. Cela sugge`re que la perte de cette caracte´ristique serait surve- nue toˆt dans l’e´volution des amblypyges. Une analyse phylogeńe´tique supporte faiblement le positionnement des uropyges comme groupe fre`re des amblygypes. Mots-cle´s:Arachnides, Megoperculata, geńome mitochondrial, phylogeńie, re´gion de controˆle mitochondriale. [Traduit par la Re´daction]

Introduction Taxonomically, they are divided into two suborders: the Pa- leoamblypygi are represented by a single extant species, the Whip spiders (Amblypygi) are a small order of terrestrial small (7 mm) and blind Paracharon caecus (Hansen 1921), chelicerates common in humid regions of the tropics and and the larger (6–36 mm) Euamblypygi (Weygoldt 1996) subtropics all over the world, with some species also occur- comprise four families, 16 genera, and at least 157 described ring in more temperate to arid regions (Weygoldt 2000). species (Harvey 2002, 2003, 2007). Amblypygi are bizarre

Received 4 November 2008. Accepted 25 February 2009. Published on the NRC Research Press Web site at genome.nrc.ca on 14 April 2009. Corresponding Editor: L. Bonen. K. Fahrein and L. Podsiadlowski.1 Department of Biology, Koenigin-Luise-Str 1-3, D-14195 Berlin, Germany. S.E. Masta. Department of Biology, P.O. Box 751, Portland State University, Portland, OR 97207, USA. 1Corresponding author (e-mail: [email protected]).

Genome 52: 456–466 (2009) doi:10.1139/G09-023 Published by NRC Research Press Fahrein et al. 457 animals owing to their strong and spinous raptorial pedi- Weygoldt (2000). Total DNA was extracted from one leg palps and their thin and multisegmented first walking legs, by using Qiagen extraction kits (Qiagen, Hilden, Germany) which serve as sensory and communicatory organs. Thus, following the manufacturer’s protocol. amblypygids show functional hexapody. Phylogenetically amblypygids are well characterized as a monophylum by PCR various apomorphies from morphology: a pretarsal depressor The whole mt genome of D. diadema was amplified in muscle without a patella head, a vestigial labrum, large ante- two overlapping fragments by using the primer pairs Art- rior coxal apodemes on all walking legs, divided tibiae, and HPK16SA/B (Simon et al. 1994; Kambhampati and Smith nearly immovable patellotibial joints owing to the fusion of 1995) and Art-HPK16Saa/bb (Hwang et al. 2001). Long- these two segments (Shultz 1990). Based on morphological range PCR with primers Art-HPK16Saa/bb was performed characters, most arachnologists agree that there is likely a with a Takara LA Taq kit (Takara) in 50 mL volumes (5 mL close phylogenetic relationship among Amblypygi, Araneae, of buffer, 8 mL of dNTP solution, 0.5 mL of Takara LA Taq, and Uropygi (e.g., Weygoldt and Paulus 1979; Shultz 1989, 1 mL of DNA, 1 mL of primer mix (10 mmol/L), 34.5 and mL 1990; Van der Hammen 1989). This group (Tetrapulmonata, of water). This yielded a PCR fragment of about 15 kb size. respectively Megoperculata sensu Weygoldt and Paulus Conserved primers for crustaceans (Yamauchi et al. 2004) 1979) is supported by the existence of two-segmented cheli- were used to amplify smaller mitochondrial fragments from cerae hinged ventrolaterally and an unusual microtubule ar- the long PCR product. Successful amplification was per- rangement in their sperm axonemes. The phylogenetic formed with primer pairs S1, S2, S5, S7–S11, S13, S15, relationships among these three taxa are more controversial, S24, S25, S29, S30, S35, S36, S42, S46, and S48. Finally, with two major competing hypotheses. Many authors favour additional primer pairs were designed to amplify larger frag- Uropygi as sister group to Amblypygi (‘‘Pedipalpi’’ hypoth- ments to bridge the gaps between S13/S15, S15/S24, S25/ esis) owing to the presence of raptorial pedipalps and anten- S29, S30/S35, S35/S42, and S48/S2 (for primer sequences niform first walking legs in both taxa (Shear et al. 1987; and annealing temperatures, see Supplementary Table S12). Shultz 1989, 1990, 1999, 2007). In contrast, a sister group Secondary PCRs were performed in an Eppendorf Mastercy- relationship between Amblypygi and Araneae (‘‘Labellata’’ cler and Mastercycler gradient using the Eppendorf 5-prime- hypothesis) is recognized by other authors (Petrunkevitch Taq kit (Eppendorf, Germany) in 50 mL volumes (41.75 mL 1955; Weygoldt and Paulus 1979; Van der Hammen 1989), of molecular-grade water, 5 mL of buffer; 1 mL of dNTP mix with support provided by a postcerebral pharynx and a ped- (10 mmol/L), 1 mL of template DNA (= 1:100 dilution of the icel in both taxa (Ax 1996). long PCR fragment), 1 mL of primer mix (10 mmol/L each), and 0.25 of mL Taq polymerase). PCR products were visual- The difficulties in evaluating phylogenetic relationships ized on 1% agarose gels and purified using a Bluematrix within the Tetrapulmonata based on morphological data are probably caused by homoplasy or reduction of anatomical DNA purification kit (EURx, Gdansk, Poland). If extra characters. Controversial results from nuclear sequence data bands were present, a gel extraction was performed follow- and from combined analyses (Wheeler and Hayashi 1998; ing the manufacturer’s protocol (Qiagen). The mt genome sequence of Phrynus was amplified with taxon-specific pri- Giribet et al. 2002) hint for the need for additional data sets 2 for phylogenetic reconstructions, such as mitochondrial ge- mers (see Supplementary Table S1 ), which were designed nomes (mt genomes). In animals, these circular double- based on a region of the cob gene that was amplified with stranded DNA molecules are about 16 kb long and contain the primers CobF and CobR (Boore and Brown 2000). For 37 genes plus one AT-rich noncoding region (Wolstenholme details on long PCR amplification, sequencing, and se- 1992; Boore 1999). In this article, we provide the first two quence assembly, see Masta and Boore (2008). complete mt genome sequences covering two families of the Amblypygi, Damon diadema (Phrynichidae) and Phry- Sequencing and genome assemblage nus sp. (Phrynidae). We discuss general features of the ge- Sequencing of D. diadema was performed on a CEQ 8000 nomes, compare inferred secondary structures of tRNAs and capillary sequencer using a CEQ DCTS kit (both Beck- rRNAs, nucleotide frequency bias, and codon usage, and mann-Coulter). Sequencing reactions were performed in an provide a phylogenetic analysis of arachnid interrelation- Eppendorf Mastercycler and Mastercycler gradient. The ships. quality of the sequences was checked with CEQ software. Sequence assembly was performed with BioEdit version Materials and methods 7.0.1. (Hall 1999). Protein-coding and ribosomal genes were identified by BLAST searches on NCBI databases. To deter- Animals mine boundaries, the sequences were also compared with A specimen of D. diadema was obtained from a commer- alignments from other chelicerate species. We assumed the cial source. Species determination was done with the mor- start and ending of the rRNA genes and the control regions phological key and in comparison with cox1 sequences to be extended to the boundaries of flanking genes. The according to Prendini et al. (2005). A gift from M. Hedin of boundary of the 12S rRNA gene to the control region was a specimen of Phyrnus sp. that was collected in Baja Cali- inferred by comparison with 12S rRNA genes of other fornia, Mexico, was identified using the key provided in arachnids. Hairpin structures in the control regions were

2 Supplementary data for this article are available on the journal Web site (http://genome.nrc.ca) or may be purchased from the Depository of Unpublished Data, Document Delivery, CISTI, National Research Council Canada, Building M-55, 1200 Montreal Road, Ottawa, ON K1A 0R6, Canada. DUD 3913. For more information on obtaining material, refer to http://cisti-icist.nrc-cnrc.gc.ca/cms/unpub_e.html.

Published by NRC Research Press 458 Genome Vol. 52, 2009 identified by eye inspection. Genomic position and secon- Control region dary structure of tRNAs were identified using tRNAscan-SE In both amblypygid mt genomes, the largest noncoding (Lowe and Eddy 1997) and ARWEN (Laslett and Canba¨ck sequence (364 bp in D. diadema and 359 bp in Phrynus sp.) 2008). Secondary structures of the rRNAs were made in is located between rrnS and trnI. The location of the putative comparison with published structure models of the honeybee control region in invertebrates shows great variability, but in Apis mellifera (Gillespie et al. 2006). Complete mt genome many arthropods showing a conserved mitochondrial gene sequences were deposited at the NCBI database (GenBank order, it has the same relative location as in Amblypygi NC_011293/FJ204233 (D. diadema) and NC_010775/ (Wolstenholme 1992; Zhang and Hewitt 1997; Saito et al. EU520641 (Phrynus sp.)). Nucleotide frequency and relative 2005). Part of the putative control region can be folded into synonymous codon usage were determined using DAMBE stable stem-loop structures owing to inverted repeat sequen- version 4.2.13 (Xia and Xie 2001). ces. In D. diadema, the stem is composed of 23 paired nucleotides without any mismatches and the loop consists of Phylogenetic analysis 15 nucleotides (Fig. 1). The stem-loop formation in the con- Phylogenetic analysis was performed with concatenated trol region of Phrynus sp. shows a larger loop consisting of amino acid alignments of 11 protein-coding genes (omitting 35 nucleotides, and the stem consists of 22 paired nucleoti- the shortest and least conserved genes atp8 and nad4L). Se- des (one mismatch and a side loop). While their exact func- quences were retrieved from the Mitome database (www. tion is unclear, inverted repeat sequences often occur in mitome.info) (Lee et al. 2008). Alignments were done with arthropod mitochondrial control regions (Kilpert and Podsia- ClustalW (Chenna et al. 2003) under default conditions and dlowski 2006; Fahrein et al. 2007; Masta et al. 2008) and are in some cases corrected after inspection by eye. Phyloge- probably located at or near the replication origin of the L netic analysis of this data set was performed in two ways. strand (Zhang and Hewitt 1997). Conserved motifs in the (i) Maximum likelihood analysis with RAxML version 7.0 flanking sequences around the stem-loop structure are also (Stamatakis et al. 2008) was performed using the found in the two amblypygids: both species exhibit a TATA mtREV+G+I model with data partitions according to the 11 motif in the 5’ flanking sequence whereas a G(A)nT motif genes. In addition to a search for the best tree, 100 bootstrap only appears in the 3’ flanking sequence of Phrynus sp. replicates were performed making use of the CIPRES Portal (Fig. 1). The latter motif also occurs in the origin of L-strand web server (www.phylo.org/sub_sections/portal/). (ii) Maxi- replication in vertebrates and the plant Petunia hybrida mum likelihood analysis with Treefinder version Oct. 2008 (Zhang et al. 1995). Both motifs are presumed to play an im- (Jobb et al. 2004) was performed using the mtART+G+I portant role in the initiation of transcription and (or) replica- model with individual optimization of data partitions (ac- tion of the mitochondrial genome (Zhang et al. 1995; Zhang cording to the 11 genes). Edge support (= an approximation and Hewitt 1997). of bootstrapping) was computed with 1000 replicates. Secondary structure of tRNAs Results and discussion In the mt genomes of both amblypygids, all 22 tRNAs typical for bilaterian mt genomes were found (see Supple- Genome organization and gene order mentary Fig. S12). As in many other animals, the inferred Both mt genomes have a typical circular organization secondary structure of tRNA-Ser(AGN) lacks a dihydrouri- with a length of 14 764 bp in Phrynus sp. and 14 786 bp in dine stem (Wolstenholme 1992). While most of the other D. diadema. For both genomes, we identified all 37 genes tRNA genes have inferred secondary structures with a can- usually present in bilaterians: 13 protein-coding genes, two onical cloverleaf shape, in both Phrynus (Masta and Boore genes for rRNA subunits, and 22 tRNA genes (Fig. 1; see 2008) and Damon (Fig. 1), tRNA-Ala lacks the dihydrouri- Supplementary Tables S2 and S32). The gene order in both dine stem. This loss is not shared by other chelicerates but species is identical to that found in the mt genome of the instead is probably a synapomorphy shared by members of horseshoe crab Limulus polyphemus (Xiphosura), which is Amblypygi. thought to represent the putative arthropod ground pattern (Staton et al. 1997; Lavrov et al. 2000). Between rrnS and rRNA genes trnI, we detected one major noncoding region in the mt ge- Both rRNA genes of Amblypygi have lengths similar to nome of both species (see next section). This region has a those found in the xiphosuran L. polyphemus. The inferred higher A+T content than the rest of the genome. In verte- lengths of the rrnL genes in these two amblypigid taxa are brates, a similar region bears signal sequences that initiate 1218 nucleotides for Phrynus and 1294 nucleotides for H-strand synthesis in replication as well as H- and L-strand Damon, while Limulus possesses an rrnL gene of 1296 nu- transcription (Clayton 1991) and thus is referred to as the cleotides (Lavrov et al. 2000). In contrast, the jumping spi- ‘‘mitochondrial control region’’. Beside the putative control der Habronattus oregonensis has a considerably shorter rrnL region, only much smaller noncoding sequences occur, ex- gene, with a length of only 1018 nucleotides (Masta 2000). tending up to 21 bp (between rrnL and trnV)inPhrynus sp. Likewise, the inferred sizes for the rrnS genes are similar in and 23 bp (between trnS-UGA and nad1)inD. diadema (see the two amblypygids (786 nucleotides in Phrynus and 750 Supplementary Tables S2 and S32). Both mt genomes show nucleotides in Damon) and in Limulus (799 nucleotides) overlap at 11 gene boundaries with up to 20 shared nucleo- (Lavrov et al. 2000). The rrnS gene in Habronattus is much tides (between nad4 and nad4L)inPhrynus sp. and up to 15 shorter, with a length of only 693 nucleotides (Masta and nucleotides (between trnH and nad4)inD. diadema (Fig. 1; Boore 2004). Together, these data suggest that the RNA see Supplementary Tables S2 and S32). components of the ribosome became reduced in size in spi-

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Fig. 1. Mitochondrial genomes and control region loop secondary structure of Amblypygi. tRNA genes are depicted by their one-letter code abbreviations. Numbers reflect noncoding (positive) or overlapping (negative) nucleotides between two adjacent genes; small arrows indicate the orientation of the genes. Above the circular maps, stem-loop structures found in the largest noncoding part (= the putative control region (CR)) are shown. Small boxes in sequence highlight putative signal motifs. Below the circular maps, plots of inferred secondary structures of tRNA-Ala and tRNA-Ser(AGN) are shown. These are the only tRNAs in Amblypygi lacking the dihydrouridine stem. For all Phrynus sp. tRNAs, see Masta and Boore (2008), and for all Damon diadema tRNAs, see Supplementary Fig. S12. Dots illustrate the base pairing of the pyrimidine base uracil with the purine base guanine. The depicted animal is Phrynus sp.

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Fig. 2. Plot of inferred secondary structure of SSU rRNA (12S) from the mitochondrial genome of Damon diadema. Helix numbering according to Gillespie et al. (2006).

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Fig. 3. Plot of inferred secondary structure of LSU rRNA (16S) from the mitochondrial genome of Damon diadema. Helix numbering according to Gillespie et al. (2006).

ders after their divergence from their common ancestor with are in Damon (196 nucleotides) and Apis (187 nucleotides). amblypygids and Limulus. Secondary structures of 12S and Likewise, helix H837 of rrnL is shorter in the two mites (36 16S rRNAs were inferred for D. diadema (Figs. 2 and 3). nucleotides in Steganacarus and 31 nucleotides in Lepto- There is much more similarity to the secondary structures trombidium) than in Damon (55 nucleotides) or in Apis (52 proposed for the insect A. mellifera (Gillespie et al. 2006) nucleotides). The rrnS gene also exhibits noticeable length than to the other published rRNA secondary structure ana- differences between Damon and the two mites. This is most lyses of mites, the chigger mite Leptotrombidium pallidum apparent in the 5’ end and in the helices between H769 and (Shao et al. 2006) and the oribatid mite Steganacarus mag- H885. Together, this suggests that amblypygids have re- nus (Domes et al. 2008). The differences in length between tained a ribosome structure that is typical for arthropods but Damon and the mites are primarily due to differences in the that secondary reductions in helix sizes have occurred in number of nucleotides that comprise certain helices. In par- mites and spiders. Further comparative work is necessary to ticular, the 5’ end of rrnL appears to be truncated in mites more fully understand the evolution of structural differences such that they have lost helices H183, H235, and H461 among arachnids. whereas these regions are present in Apis and amblypygids. The termination signal for rrnL transcription in animal Other differences in size can be attributed to the fact that the mitochondria is believed to be the motif TGGCAGA helices between H2043 and H2455, which are flanked by (Valverde et al. 1994). In insects and crustaceans, this hep- highly conserved sequence motifs in all four species, are tamer is located downstream of the large rRNA gene in the substantially shorter in mites (115 nucleotides in Stegana- tRNA-Leu(CUN) gene, while in vertebrates, it is found carus and 114 nucleotides in Leptotrombidium) than they within the downstream tRNA-Leu(UUR) gene (Valverde et

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Table 1. Relative synonymous codon usage of assorted amino acids in different arachnid species.

Nephila Habronattus Ornithoctonus Heptathela Mastigoproctus Phrynus Damon Codon Amino acid (Araneae) (Araneae) (Araneae) (Araneae) (Uropygi) (Amblypygi) (Amblypygi) GC skew 0.242 0.301 0.344 –0.235 –0.297 –0.453 –0.473 AT skew –0.053 –0.113 –0.083 –0.023 –0.014 –0.029 0.102 AT (%) 76 74.4 69.8 72.2 70.6 67.5 63.2 Plus strand UUG L 0.983 1.413 2.588 0.247 0.301 0.053 0.145 UUA L 4.198 3.993 2.679 3.152 2.589 1.567 1.745 CUA L 0.246 0.287 0.229 1.215 1.043 0.979 1.455 CUC L 0.02 0 0.023 0.285 0.542 1.175 1.364 CUG L 0.041 0.061 0.115 0.171 0.161 0.089 0.2 CUU L 0.512 0.246 0.366 0.93 1.365 2.136 1.091 Minus strand UUG L 0.19 0.03 0.145 0.882 1.053 2.038 2.941 UUA L 3.714 3.149 2.754 3.796 3.658 3.538 2.525 CUA L 0.698 1.99 1.507 0.416 0.553 0.113 0.089 CUC L 0.159 0.238 0.348 0.024 0 0 0.03 CUG L 0.032 0.03 0.058 0.171 0.132 0.057 0.149 CUU L 1.206 0.564 1.188 0.71 0.605 0.255 0.267 Plus strand AGC S 0 0.122 0.077 0.214 0.134 0.335 0.652 AGU S 1.089 0.975 0.423 0.286 0.504 0.502 0.326 AGG S 0.749 0.244 0.462 0 0 0.033 0 AGA S 2.077 2.315 2.692 1.25 1.076 0.837 0.688 UCG S 0.102 0.244 0.423 0.071 0.067 0.033 0.109 UCU S 2.689 3.168 3.115 2.5 1.916 2.343 1.484 UCC S 0.034 0.325 0.115 1.286 1.445 1.941 2.172 UCA S 1.26 0.609 0.692 2.393 2.857 1.975 2.57 Minus strand AGC S 0.133 0.127 0.123 0.105 0.26 0.125 0.2 AGU S 0.133 0.254 0.205 0.737 0.715 1 1.6 AGG S 0.133 0.042 0 0.053 0.195 1 1.467 AGA S 1.156 1.439 1.19 2.526 3.187 2.5 1.4 ulse yNCRsac Press Research NRC by Published UCG S 0 0.085 0.164 0.263 0.325 0.313 0.333 UCU S 2.711 1.693 1.559 2.684 2.146 1.5 2

UCC S 0.667 0.974 1.518 0.684 0 0.188 0.067 2009 52, Vol. Genome UCA S 3.067 3.386 3.241 0.947 1.171 1.375 0.933 Note: Bold numbers indicate values above 1 (= predominantly used codons). GC and AT skew is for complete genomes (plus strand). For accession numbers, see Fig. 4. Fahrein et al. 463

Fig. 4. Phylogenetic analysis of arachnid relationships based on mitochondrial sequence data (concatenated amino acid alignment of protein- coding genes). The best tree from RAxML analysis (mtREV+G+I) is shown. Numbers next to nodes reflect bootstrap percentages from RAxML analysis (mtREV+G+I, partitioned optimization, left number) and edge support percentages from Treefinder analysis (mtART+G+I, partitioned optimization, right number). Two asterisks depict maximal support from all three methods. Accession numbers of GenBank en- tries are given after the species name. Scale bar reflects amino acid substitutions per site.

al. 1994). We find this same motif downstream from the There are alternative models for rrnL termination signals large rRNA genes in both of these amblypygid mt genomes. (Cameron and Whiting 2008), but in our case, we favour However, in both amblypygids, an identical motif is located the abovementioned model (Valverde et al. 1994) as being in the tRNA-Leu(UUR) gene, while in Damon, the motif is more parsimonious owing to the highly conserved motif, its also present in the tRNA-Leu(CUN) gene. Phrynus pos- location closer to the boundary rrnL/trnL(CUN), and a com- sesses a modification of this motif, TGACAGA, in its parable situation in other chelicerates. tRNA-Leu(CUN) gene. We find that amblypygids, Limulus, and Habronattus (Masta 2000) share this same gene location Protein-coding genes whereby the rrnL termination signal is located within the Except for one gene, in both amblypygids, all of the 13 tRNA-Leu(CUN) gene. It is known that the gene in which identified protein-coding genes start with one of the usual the rRNA transcription termination motif is located has start codons for arthropod mtDNA (ATA, ATC, ATG, or changed over evolutionary time (Valverde et al. 1994), but ATT). Only cox1 from Phrynus sp. starts with the excep- we conclude that in amblypygids, the location of the rRNA tional codon TTA (see Supplementary Table S32). Most transcription termination motif represents the ancestral loca- genes possess the stop codon TAA, except for the single oc- tion for chelicerates. currence of the stop codon TAG, which terminates nad4L

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(see Supplementary Tables S2 and S32). Truncated stop co- by NaFo¨G (Berlin). S.E.M. was funded by National Science dons consisting only of a T or a TA are observed in three Foundation award No. DEB-0416628. genes from Phrynus sp. and five genes from D. diadema. Such incomplete stop codons are also reported from the mt References genomes of many animal species and it is assumed that these partial stop codons are completed by post-transcriptional pol- Ax, P. 1996. Multicellular animals. A new approach to the phylo- yadenylation (Ojala et al. 1981). genetic order in nature. Springer, Berlin, Heidelberg, New York. pp. 1–225. Boore, J.L. 1999. Animal mitochondrial genomes. Nucleic Acids Nucleotide frequency and codon usage Res. 27: 1767–1780. doi:10.1093/nar/27.8.1767. PMID:10101183. In both species, genes coded on the plus strand have a neg- Boore, J.L., and Brown, W.M. 2000. Mitochondrial genomes of ative GC skew, while genes coded on the minus strand have a Galathealinum, Helobdella, and Platynereis: sequence and gene positive GC skew (see Supplementary Tables S2 and S32). arrangement comparisons indicate that Pogonophora is not a We compared nucleotide frequencies and strand bias among phylum and Annelida and Arthropoda are not sister taxa. Mol. species from Amblypygi, Uropygi, and Araneae (Table 1). Biol. Evol. 17: 87–106. PMID:10666709. Except for Heptathela, all spiders have a reversed GC skew Cameron, S.L., and Whiting, M F. 2008. The complete mitochon- compared with Amblypygi and Uropygi. Damon is the only drial genome of the tobacco hornworm, Manduca sexta (Insecta: species with a slightly positive AT skew (0.102), while all Lepidoptera: Sphingidae), and an examination of mitochondrial other species considered have a slightly negative one, with gene variability within butterflies and moths. Gene, 408: 112– the maximal value in Habronattus (–0.113). AT content of 123. doi:10.1016/j.gene.2007.10.023. the complete genomes is lower in Amblypygi (63.2% in Chenna, R., Sugawara, H., Koike, T., Lopez, R., Gibson, T.J., Hig- Damon and 67.5% in Phrynus) than in Uropygi and Araneae gins, D.G., and Thompson, J.D. 2003. Multiple sequence align- (69.8%–76%). The reversal in GC skew is also reflected in ment with the clustal series of programs. Nucleic Acids Res. 31: codon usage. As an example, Table 1 shows the relative syn- 3497–3500. doi:10.1093/nar/gkg500. PMID:12824352. onymous codon usage values for leucine and serine in differ- Clayton, D.A. 1991. Replication and transcription of vertebrate mi- ent arachnids (Amblypygi, Uropygi, and Araneae). In the tochondrial DNA. Annu. Rev. Cell Biol. 7: 453–478. doi:10. 1146/annurev.cb.07.110191.002321. PMID:1809353. case of leucine, the most frequently used codon in all cases Domes, K., Maraun, M., Scheu, S., and Cameron, S.L. 2008. The is UUA, but besides this, in plus-strand-encoded genes, there complete mitochondrial genome of the sexual oribatid mite Ste- is UUG second-most often in use in taxa with a positive GC ganacarus magnus: genome rearrangements and loss of tRNAs. skew, while in taxa with a negative GC skew, it is CUA or BMC Genomics, 9: 532. doi:10.1186/1471-2164-9-532. PMID: CUU that is more often found. Genes encoded on minus 18992147. strand show a reversed codon usage for leucine. Similar dif- Fahrein, K., Talarico, G., Braband, A., and Podsiadlowski, L. 2007. ferences in usage of C- and G-containing codons is seen for The complete mitochondrial genome of Pseudocellus pearsei serine. Genes that are positively GC skewed (plus-strand (Chelicerata: Ricinulei) and a comparison of mitochondrial gene genes in those taxa with positive GC bias and minus-strand rearrangements in Arachnida. BMC Genomics, 8: 386. doi:10. genes in those with negative GC bias) use predominantly 1186/1471-2164-8-386. PMID:17961221. AGA (but also UCU), while those genes with a negative GC Gillespie, J.J., Johnston, J.S., Cannone, J.J., and Gutell, R.R. 2006. skew predominantly use UCU, UCC, and UCA codons. Characteristics of the nuclear (18S, 5.8S, 28S and 5S) and mitochondrial (12S and 16S) rRNA genes of Apis mellifera (Insecta: Phylogenetic interrelationships Hymenoptera): structure, organization, and retrotransposable ele- ments. Insect Mol. Biol. 15: 657–686. doi:10.1111/j.1365-2583. Gene order is conserved in Amblypygi, so therefore does 2006.00689.x. PMID:17069639. not help in resolving interrelationships of the Tetrapulmo- Giribet, G., Edgecombe, G.D., Wheeler, W.C., and Babbitt, C. nata. Our phylogenetic analysis of sequence data from mito- 2002. Phylogeny and systematic position of Opiliones: a com- chondrial protein-coding genes (Fig. 4) weakly supports a bined analysis of chelicerate relationships using morphological sister group relationship of Uropygi and Amblypygi as well and molecular data. Cladistics, 18: 5–70. PMID:14552352. as monophyly of Tetrapulmonata. Our result for the interre- Hall, T.A. 1999. BioEdit: a user-friendly biological sequence align- lationships of the Tetrapulmonata corresponds well to a pre- ment editor and analysis program for Windows 95/98/NT. Nu- vious analysis of a similar data set (Masta et al. 2009; cleic Acids Symp. Ser. 41: 95–98. without data from D. diadema), where some maximum like- Harvey, M.S. 2002. The neglected cousins: What do we know lihood and Bayesian analyses of amino acid data sets also about the smaller Arachnid orders? J. Arachnol. 30: 357–372. found support for a clade of Uropygi and Amblypygi, doi:10.1636/0161-8202(2002)030[0357:TNCWDW]2.0.CO;2. although support varied with the model of evolution and Harvey, M.S. 2003. Catalogue of the smaller arachnid orders of the type of analysis performed. World: Amblypygi, Uropygi, Schizomida, Palpigradi, Ricinulei and Solifugae. CSIRO Publishing, Collingwood, Victoria, Aus- Acknowledgements tralia. Harvey, M.S. 2007. The smaller arachnid orders: diversity, descrip- We thank Peter Adam (FU Berlin) for the line drawing of tions and distributions from Linnaeus to the present (1758 to Phrynus sp. K.F. and L.P. would like to thank Professor 2007). Zootaxa, 1668: 363–380. Thomas Bartolomaeus (FU Berlin) for his kind support of Hwang, U.W., Park, C.J., Yong, T.S., and Kim, W. 2001. One-step our work and the German Science Foundation for financial PCR amplification of complete arthropod mitochondrial gen- support through grant DFG Ba 1520/10-1,2 (priority pro- omes. Mol. Phylogenet. Evol. 19: 345–352. doi:10.1006/mpev. gramme ‘‘Deep Metazoan Phylogeny’’). K.F. was supported 2001.0940. PMID:11399145.

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