Adam D. Miller, Thuy T.T. Nguyen, Christopher P. Burridge, Christopher M. Austin*

Gene 331 (2004) 65 72 www.elsevier.com/locate/gene Complete mitochondrial DNA sequence of the Australian freshwater crayfish, Cherax destructor (Crustacea: Decapoda: Parastacidae): a novel gene order revealed Adam D. Miller, Thuy T.T. Nguyen, Christopher P. Burridge, Christopher M. Austin* School of Ecology and Environment, Deakin University, P.O. Box 423, Warrnambool, Victoria 3280, Australia Received 7 October 2003; received in revised form 29 December 2003; accepted 26 January 2004 Received by G. Pesole Abstract The complete mitochondrial DNA sequence was determined for the Australian freshwater crayfish Cherax destructor (Crustacea: Decapoda: Parastacidae). The 15,895-bp genome is circular with the same gene composition as that found in other metazoans. However, we report a novel gene arrangement with respect to the putative arthropod ancestral gene order and all other arthropod mitochondrial genomes sequenced to date. It is apparent that 11 genes have been translocated (ND1, ND4, ND4L, Cyt b, srrna, and trnas Ser(UGA), Leu(CUN), Ile, Cys, Pro, and Val), two of which have also undergone inversions (trnas Pro and Val). The duplication/random loss mechanism is a plausible model for the observed translocations, while intramitochondrial recombination may account for the gene inversions. In addition, the arrangement of rrna genes is incompatible with current mitochondrial transcription models, and suggests that a different transcription mechanism may operate in C. destructor. D 2004 Elsevier B.V. All rights reserved. Keywords: Astacidae; Inversion; Translocation; Duplication/random loss; Intramolecular recombination; Drosophila 1. Introduction The typical metazoan mitochondrial genome is a covalently closed circular molecule, approximately 16 kb in size, containing 37 genes: 13 protein coding genes (ATP6 and 8, CO1 3, Cyt b, ND1 6 and 4L), two rrna genes (lrrna and srrna), and 22 trna genes (one for each amino acid except for leucine and serine, which have two genes) (Boore, 1999). In addition, the mtdna molecule contains one major non-coding region that is thought to play a role in the initiation of transcription and replication (Wolstenholme, 1992). Abbreviations: ATP6 and 8, ATPase subunits 6 and 8; bp, base pair(s); CO1 3, cytochrome c oxidase subunits 1 3; CR, control region; Cyt b, cytochrome b; kb, kilobase; mt, mitochondria(l); ND1 6 and 4L, NADH dehydrogenase subunits 1 6 and 4L; PCR, polymerase chain reaction; srrna and lrrna, small and large ribosomal RNA subunits; trna, transfer RNA; a, strand encoding the majority of genes; h, strand encoding the minority of genes. * Corresponding author. Tel.: +61-35563-3518; fax: +61-35563-3462. E-mail address: cherax@deakin.edu.au (C.M. Austin). Due to its presumed lack of recombination, maternal inheritance, and relatively rapid mutation rate, mitochondrial DNA sequences have been extensively used for the investigation of population structures and phylogenetic relationships at various taxonomic levels (Avise, 1994). In addition, mitochondrial gene arrangements have proven useful for studying deep metazoan divergences (Sankoff et al., 1992; Smith et al., 1993; Boore et al., 1995; Boore and Brown, 1998; Curole and Kocher, 1999; Le et al., 2000; Roehrdanz et al., 2002). Mitochondrial gene order rearrangements appear to be unique, generally rare events that are unlikely to arise independently in separate evolutionary lineages as a result of convergence (Boore, 1999). However, our limited knowledge of the mechanisms responsible for the rearrangement of mtdna genes limits their broader acceptance for phylogenetic research (Curole and Kocher, 1999). Complete mtdna sequences have been determined for approximately 370 species, although the majority (approximately 75%) represent vertebrates. By comparison, the most diverse taxon on earth, the Arthropoda, is poorly 0378-1119/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2004.01.022

66 A.D. Miller et al. / Gene 331 (2004) 65 72 Specimens of C. destructor were collected from Dwyers Creek in the Grampian Ranges, located in southwestern Victoria (37jS, 142jE). Mitochondrion-enriched DNA extracts were obtained from frozen specimens following Tamura and Aotsuka (1988). Using species-specific primers designed from partial lrrna and CO1 sequence data (Gen- Bank Accession numbers AY191769 and AY153891) the entire mitochondrial genome for C. destructor was amplified by long-pcr in two overlapping fragments. The PCR fragments, approximately 6.8 and 9.0 kb in size, were amplified using the primer pairs Cherax.co1.F (5V-GGG ACT TTA GGG ATA ATC TAT GCC ATG ACA-3V) with Cherax.rrnL.R (5V-GTT TGC GAC CTC GAT GTT GAA TTA AAA TTG -3V), and Cherax.rrnl.F (5V-AAA TTT TAA TTC AAC ATC GAG GTC GCA AAC-3V) with Cherax.co1.R (5V- GCT GTC ATG GCA TAG ATT ATC CCT AAA GT-3V), respectively, and High Fidelity Platinum Taq DNA Polymerase (Invitrogen), following the supplier s instructions. 2.2. Cloning, sequencing, and gene identification Fig. 1. A phylogeny of the Decapoda, partially derived from Crandall et al. (2000) and Martin and Davis (2001), indicating species for which complete mtdna sequences have been determined to date. The Pagurus longicarpus mtdna sequence is not complete, lacking approximately 300 bp of the control region. *Denotes species displaying mt gene rearrangements. GenBank accession numbers are given. represented with complete mt genome sequences for only 41 species available on GenBank. Further, taxonomic bias is also evident within the Arthropoda: 25 of the 41 sequenced mtdnas are from the subphylum Hexapoda, 7 from the Crustacea, 6 from the Chelicerata, and only 3 from the Myriapoda. In this study, we report the complete nucleotide sequence of the mitochondrial genome from the Australian freshwater crayfish Cherax destructor (Crustacea: Decapoda: Parastacidae). This is the fourth decapod crustacean to have its complete mtdna sequence determined (Fig. 1). Our data not only represent the first complete nucleotide sequences for the majority of mtdna genes in freshwater crayfish (Infraorder Astacidea), but have also revealed a novel gene order, unlike that reported for any other arthropod species. This finding makes C. destructor only the second decapod crustacean and one of nine arthropod taxa to display a gene order rearrangement (excluding trnas) relative to the typical arthropod mitochondrial genome. 2. Materials and methods 2.1. Sample, DNA extraction, and PCR PCR products from a single individual were gel purified, ligated into pcrrxl plasmid vector using the TOPO XL cloning kit (Invitrogen), and DNA sequence data from both strands was generated from single clones representing each of the PCR fragments using the primer walking approach (Yamauchi et al., 2003). All automated sequencing was performed with ABI PRISM BigDye terminator chemistry, version 3, and analysed on an ABI 3700 automated sequencer. Chromatograms were visually inspected using the computer software EditView 1.0.1 (Perkin Elmer) and DNA sequences were aligned using SeqPup (Gilbert, 1997). Protein-coding and rrna gene sequences were initially identified using BLAST searches on GenBank, and then subsequently by alignment with Penaeus monodon and Drosophila yakuba (GenBank accession numbers; NC_002184 and NC_001322, respectively) mitochondrial DNA and amino acid sequences. The amino acid sequences of C. destructor protein-coding genes were inferred from the Drosophila translation code. The majority of the trna genes were identified using trnascan-se 1.21 (Lowe and Eddy, 1997), using the default search mode and specifying mitochondrial/chloroplast DNA as the source and using the invertebrate mitochondrial genetic code for trna structure prediction. Remaining trna genes were identified by inspecting sequences for trna-like secondary structures and anticodons. The resulting sequences were deposited in GenBank under the accession number AY383557. 3. Results and discussion 3.1. Genome composition The mitochondrial genome of C. destructor is circular and consists of 15,895 bp, containing the same 13 proteincoding, 22 trna, and 2 rrna genes as found in other metazoans (Fig. 2; Table 1). The majority-strand encodes 26 genes, whereas the minority-strand encodes 11 genes. These strands will be referred to as a and h, respectively. We found eight gene pairs overlapping by up to 10 bp (Table 1), a characteristic which has been reported for other animal mtdnas (Kumazawa et al., 1998; Boore, 2001; Delarbre et al., 2002; Nishibori et al., 2002). Notable gene length

A.D. Miller et al. / Gene 331 (2004) 65 72 67 Fig. 2. Linearized representation of the mitochondrial gene arrangement for the Australian freshwater crayfish C. destructor (Decapoda: Parastacidae) and the putative ancestral arthropod. Protein-coding and rrna genes are transcribed from left to right except those indicated by underlining, which are transcribed from right to left. trna genes are designated by single-letter amino acid codes except those encoding leucine and serine, which are labelled L 1 (trna Leu(UAG) ), L 2 trna Leu(UAA),S 1 (trna Ser(UCU) ), and S 2 (trna Ser(UGA) ). Arrows indicate differences in gene locations between C. destructor and the putative ancestral arthropod. The circling arrows indicate inversions. The two primer pairs indicated above the C. destructor gene arrangement (A= Cherax.CO1.F, B = Cherax.rrnL.R, C = Cherax.rrnL.F, and D = Cherax.CO1.R) were used to amplify the entire mitochondrial genome. discrepancies were not observed when compared with those reported for other crustaceans (Table 1). The overall A + T content of the h-strand was 62.4% (A= 30.3%; C = 13.5%; G = 24.1%; T = 32.1%), significantly less ( p < 0.001) than that reported for any other decapod (Table 2), although comparable to other crustaceans (Daphnia pulex = 62.3%; Artemia franciscana = 64.5%) (Valverde et al., 1994; Crease, 1999; Yamauchi et al., 2002). This pattern of base composition held for the protein-coding, rrna, and trna genes, as well as the control region (Table 2). A total of 1166 non-coding nucleotides are evident, with 190 bp in 13 intergenic regions and 977 bp in a single noncoding region. We propose that the latter represents the control region, identified on the basis of its position between the lrrna and trna Gln genes, and sequence characteristics (A + T rich, non-coding, polythymine-stretch). 3.2. Gene order Numerous differences in gene order are apparent in the mt genome of C. destructor compared with the putative ancestral arthropod gene arrangement demonstrated by Drosophila melanogaster (Lewis et al., 1995) and Pen. monodon (Wilson et al., 2000) (Fig. 2). The arrangement of genes indicates a number of unique gene boundaries that have not been reported for any other crustacean species. Further, the differences between the mt gene orders of C. destructor and its closest marine relative Homarus (Superfamily Nephropoidea) (Boore et al., 1995) allow us to speculate that novel gene order observed in the C. destructor mt genome maybe restricted to the freshwater members of the infraorder Astacidea (Crandall et al., 2000), since Homarus appears to have retained the ancestral arthropod mt gene arrangement based on information from 10 gene boundaries (Boore et al., 1995). However, until further taxon sampling is performed, the exact phylogenetic distribution of the C. destructor gene order remains yet to be determined. Eleven gene translocations are evident in the C. destuctor mt genome, with two of these genes also involving inversions. For nine of the translocations, the duplication/ random loss mechanism is plausible. This involves the tandem duplication of gene regions, most widely considered a result of slipped-strand mispairing during replication, followed by the deletion of one of the duplicated gene regions (Levinson and Gutman, 1987; Moritz and Brown, 1987; Macey et al., 1997, 1998; Boore, 2000). A minimum of five independent duplication/random loss events are suggested for: (1) the translocation of the ND4 and ND4L gene cluster, (2) the translocation of the Cyt b, trna Ser(UGA), ND1 and trna Leu(CUN) gene cluster, (3) the translocation of trna Ile, and (4) the translocation of trna Cys and (5) the translocation of the srrna gene (Fig. 2). Deletion events seem to be incomplete at two sites with the presence of 22 and 77 unassignable intervening nucleotides at the trna Thr /ND6 and ND6/tRNA Pro gene boundaries. Although the intervening nucleotide fragments

68 A.D. Miller et al. / Gene 331 (2004) 65 72 Table 1 Mitochondrial gene profile of C. destructor (Decapoda: Parastacidae) Feature Position number a Size Codon Stop Intergenic start nucleotides From To b CO1 1 1535 1535 ACG TA* trna Leu(UAA) 1536 1600 65 0 CO2 1601 2296 696 ATG TAA 0 trna Lys 2312 2376 65 15 trna Asp 2379 2445 67 2 ATP8 2446 2604 159 ATG TAA 0 ATP6 2598 3272 675 ATG TAA 7 CO3 3276 4064 789 ATG TAA 3 trna Gly 4064 4126 62 1 ND3 4127 4478 352 ATC T* 0 trna Ala 4479 4538 60 0 trna Arg 4539 4598 60 0 trna Asn 4601 4663 63 2 trna Ser(UCU) 4663 4726 64 1 trna Glu 4726 4789 64 1 trna Phe (4791 4854) 64 1 ND5 (4855 6582) 1728 ATG TAA 0 trna His (6583 6650) 68 0 trna Thr 6659 6722 64 8 ND6 6745 7224 480 AAT TAA 22 trna Pro 7302 7367 66 77 16S (7368 8669) 1302 0 CR 8670 9646 977 0 trna Gln 9647 9715 68 0 trna Met 9719 9785 67 3 ND2 9786 10,790 1005 ATG TAA 0 trna Trp 10,789 10,857 69 2 trna Tyr (10,865 10,931) 67 7 ND4 (10,932 12,272) 1341 ATG TAG 0 ND4L (12,266 12,565) 300 ATG TAA 7 trna Val 12,556 12,623 68 10 Cyt b 12,616 13,750 1135 ATG T* 8 trna Ser(UGA) 13,751 13,817 67 0 ND1 (13,839 14,753) 915 ATG TAA 21 trna Leu(UAG) (14,780 14,847) 68 26 12S (14,848 15,764) 917 0 trna Ile 15,765 15,829 65 0 trna Cys (15,833 15,895) 63 3 a Brackets denote that the gene is encoded on the h-strand. b Numbers correspond to the nucleotides separating different genes. Negative numbers indicate overlapping nucleotides between adjacent genes. * Incomplete termination codon likely extended via post-transcriptional adenylation. Table 3 Base composition of the 13 protein-coding genes for the mitochondrial genome of C. destructor (Decapoda: Parastacidae) A C G T All genes 1st 27.5 18.0 23.8 30.7 2nd 18.1 21.7 16.1 44.1 3rd 29.3 22.4 18.0 30.3 Total 25.0 20.7 19.3 35.0 Genes encoded on a-strand a 1st 27.5 22.7 21.3 28.5 2nd 18.9 25.8 13.2 42.1 3rd 30.4 30.0 13.5 26.1 Total 25.6 26.2 16.0 32.2 Genes encoded on b-strand b 1st 28.1 9.7 28.8 33.4 2nd 17.2 17.4 19.9 45.5 3rd 25.5 11.0 25.3 38.2 Total 23.6 12.7 24.7 39.0 a COI, COII, CO111, ATP6, ATP8, Cyt b, ND2, ND3, and ND6 genes. b ND1, ND4, ND4L, and ND5 genes. Chi-square tests indicated that base compositions at each codon and across strands were heterogeneous ( p < 0.001). do not correspond to any gene that has possibly undergone a duplication/random loss event, homology may have been lost due to mutation events as a consequence of minimal or no selective pressure on the non-coding nucleotides. Therefore, it is likely that the unassignable intervening nucleotides represent degenerating vestiges of genes which have undergone duplication/random loss events, thus providing further support for the proposed rearrangement mechanism (Boore, 2000). However, this mechanism cannot entirely explain the translocation of the srrna, trna Pro, and trna Val genes since these have also been inverted, a characteristic for which intramitochondrial recombination may have been responsible (Lunt and Hyman, 1997; Dowton and Campbell, 2001). Intramitochondrial recombination specifically involves the breaking and re-joining of DNA double strands, thus facilitating gene rearrangement and gene inversions. Since the trna Pro gene is not juxtaposed to either srrna or trna Val in the Table 2 Genomic characteristics of decapod crustacean mtdnas Species h-strand 13 Protein-coding lrrna gene srrna gene 22 trna genes Putative control region No. of amino acid 1. C. destructor 15,895 62.4 3705 60.0 1302 67.9 917 68.3 1436 70.7 977 65.8 2. Pen. monodon 15,984 70.6 3716 69.3 1365 74.9 852 71.6 1494 68.0 991 81.5 3. Pan. japonicus 15,717 64.5 3715 62.6 1355 69.2 855 67.1 1484 68.9 786 70.6 4. Portunus trituberculatus 16,026 70.2 3715 68.8 1332 73.8 840 70.1 1468 72.0 1104 76.3 5. Pag. longicarpus a 3698 69.6 1303 77.1 789 77.2 1458 74.1 1 5 GenBank accession numbers: AY383557, NC_002184, NC_004251, NC_005037, and NC_003058, respectively. Chi-square tests indicated that the A + T composition of C. destructor differed significantly from Pen. monodon, Pan. japonicus, and Por. trituberculatus ( p < 0.001). a Incomplete mtdna sequence (Hickerson and Cunningham, 2000).

A.D. Miller et al. / Gene 331 (2004) 65 72 69 putative ancestral gene order, and the srrna and trna Val genes have retained the same order (although inverted), independent inversions and translocations probably occurred. 3.3. Protein-coding genes Translation initiation and termination codons of the 13 protein-coding genes in C. destructor are summarized in Fig. 3. Putative secondary structures for the 22 trna genes of the C. destructor (Decapoda: Parastacidae) mitochondrial genome. Watson-Crick and GT bonds are denoted by and +, respectively.

70 A.D. Miller et al. / Gene 331 (2004) 65 72 Table 1. Ten protein-coding genes share ATG initiation codons, while the COI, ND3, and ND6 genes have ACG, ATC, and AAT codons, respectively. Open-reading frames of the protein-coding genes were terminated with TAA or TAG codons in the majority, while the remaining genes had incomplete termination codons, either TA (COI) or T(ND3 and Cyt b). Incomplete termination codons are quite common among animal mitochondrial genes, with the production of the TAA termini being created via post-transcriptional polyadenylation (Ojala et al., 1981). There were two reading frame overlaps on the same strand; ATP6 and ATP8 shared seven nucleotides, as did ND4 and ND4L. Overlap at these gene boundaries and of this length is quite common amongst other crustaceans (Crease, 1999; Hickerson and Cunningham, 2000; Machida et al., 2002; Yamauchi et al., 2002, 2003). A/T base compositional bias was present in the 1st and 3rd codon positions (Table 3). This bias is comparable to that reported for other crustaceans, although the 3rd codon bias for other arthropods has been reported to be much more exaggerated (Crease, 1999). Bias to cytosine on the a-strand was found to be greater than that found on the h-strand, and, concomitantly, the guanine composition was greater on the h-strand in comparison with the a- strand (Table 3). This has been reported for other arthropod taxa, however, the process responsible remains unknown (Yamauchi et al., 2003). 3.4. Transfer RNA genes Twenty-one trna genes were identified on the basis of their respective anticodons and secondary structures (Fig. 3). Gene sizes and anticodon nucleotides were congruent with those described for other crustacean species. The D- arm was absent from the trna Ser(UCU) gene secondary structure, however, this feature has been commonly observed in metazoans (Wolstenholme, 1992). The 22nd transfer RNA gene (trna Val ) could not be confidently identified since the DNA sequence does not form a conventional clove-leaf structure in this mt genome (Fig. 3). Further, the anticodon AAC displayed by the putative C. destructor trna Val is not typical for crustaceans, which typically possess a TAC anticodon. This anticodon discrepancy corresponds to the third wobble position. In addition, the putative trna Val gene displays significant mispairings at the AA- and T-arm stems, and the D-arm appears to be absent, although the latter has been observed in another arthropod for this gene (Shao and Barker, 2003). It is possible that the trna Val gene is completely absent from the C. destructor mt genome, however, this has not been observed in any other arthropod. Due to the fact the putative trna Val intervenes two translocated gene clusters (Fig. 2) and has been possibly inverted and translocated itself, the unconventional clover-leaf structure and mispairings maybe residual artefacts of gene rearrangement processes. 3.5. Ribosomal RNA genes BLAST searches indicate that the lrrna gene intervenes trna Pro and the control region, while the srrna gene intervenes trna Leu(CUN) and trna Ile with both rrnas being encoded by the h-strand. The rrna gene boundaries were estimated via nucleotide sequence alignments with Pen. monodon and Panulirus japonicus (Gen- Bank accession number NC_004251). The arrangement of the rrna genes in C. destructor is atypical of arthropods sequenced so far. The rrna genes of the chelicerate Varroa destructor have also been separated, although these are encoded by opposite strands (Evans and Lopez, 2002). Also, the rrna genes of the insect Thrips imaginis have been reported to have undergone translocation and both are encoded on the a-strand (Shao and Barker, 2003). The rrna genes are arranged close together in all other arthropods, usually separated only by a single transfer RNA gene, and both encoded on the h-strand. While there is very little known about the transcription of rrna genes in arthropods, this mechanism has been researched more thoroughly in mammals, especially Homo sapiens (Montoya et al., 1982; Clayton, 1984; Taanman, 1999). We can assume that since the rrna genes in arthropods, except C. destructor, V. destructor, and T. imaginis, are arranged in a similar way to H. sapiens, then the mechanisms of transcription may be comparable or even identical. The proximity of the rrna genes to the transcription promoter site (within the control region) ensures that the rrna genes are expressed at much higher rate than other mt genes. However, in C. destructor the rrna genes are separated and the srrna is now located 5202 bp upstream of the control region. It has been suggested that under such circumstances two sets of promoter and termination elements may exist (Shao and Barker, 2003). However, a comprehensive investigation is required in order to elucidate the mechanism and relative rates of rrna gene transcription in C. destructor. 4. Conclusion The complete mitochondrial DNA sequence was determined for the Australian freshwater crayfish C. destructor (Decapoda: Parastacidae). The 15,895-bp genome is circular and has the same gene composition as other metazoans. However, the gene order is atypical of the putative arthropod ancestral gene arrangement and all other arthropod genomes sequenced to date. Eleven genes appear to have been translocated, three of which have also undergone inversions. Both duplication/random loss and intramitochondrial recombination may be responsible for these rearrangements. We are currently in the process of screening various species of freshwater crayfish and marine clawed lobsters

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