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 mi- tochondrial 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 (araigne´es a` fouet) sont des che´lice´rates terrestres habitant les re´gions tropicales et subtropica- les. Dans les analyses phyloge´ne´tiques fonde´es sur les caracte`res morphologiques ou l’ARNr, les amblypyges se groupent avec les uropyges (scorpions a` fouet) et les arane´es (araigne´es) pour former le taxon Tetrapulmonata, mais il existe une controverse quant aux relations entre ces trois taxons. Les ge´nomes mitochondriaux fournissent une importante quantite´ d’informations phyloge´ne´tiques additionnelles (se´quences, ordre des ge`nes, structure secondaire des ARN), mais les don- ne´es manquent sur les ge´nomes mitochondriaux chez certains des ordres majeurs au sein des arachnides. Dans le cadre d’un projet en cours sur la ge´nomique mitochondriale chez les arachnides, les auteurs pre´sentent les deux premiers ge´no- mes mitochondriaux complets chez les amblypyges. Les deux ge´nomes pre´sentent une mole´cule d’ADN bicate´naire circu- laire typique comprenant les 37 ge`nes habituellement pre´sents chez les ge´nomes 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 phyloge´ne´tique supporte faiblement le positionnement des uropyges comme groupe fre`re des amblygypes. Mots-cle´s:Arachnides, Megoperculata, ge´nome mitochondrial, phyloge´nie, 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