Department of Zoology, Duke University
Abstract
The entire mitochondrial gene order of the crustacean Pagurus longicarpus was determined by sequencing all but approximately 300 bp of the mitochondrial genome. We report the first major gene rearrangements found in the clade including Crustacea and Insecta. At least eight mitochondrial gene rearrangements have dramatically altered the gene order of the hermit crab P. longicarpus relative to the putatively ancestral crustacean gene order. These include two rearrangements of protein-coding genes, the first reported for any nonchelicerate arthropod. Codon usage and amino acid sequences do not deviate substantially from those reported for other crustaceans. Investigating the phylogenetic distribution of these eight rearrangements will add additional characters to help resolve decapod phylogeny.
Introduction
The metazoan mitochondrial genome is a circular, double-stranded DNA molecule that is highly variable in DNA sequence but conservative in gene content (Wolstenholme 1992
). Mitochondrial gene order has been found to vary across the Metazoa, generating interest in using mitochondrial DNA (mtDNA) gene order for phylogenetic inference (Boore, Lavrov, and Brown 1998
). Although the apparent rarity of gene rearrangements provides few characters for phylogenetic reconstruction (Curole and Kocher 1999
), useful characters will accumulate as more mitochondrial genomes are sequenced. Of the nine arthropods whose complete mitochondrial gene order has been determined, gene rearrangements have been restricted to tRNA genes, with the exception of protein-coding gene movements in two lineages of ticks. The two complete crustacean mitochondrial genomes sequenced to date have revealed no major deviation from the gene order found in Drosophila yakuba, aside from two tRNA gene movements in Artemia franciscana.
The phylogenetic position of crustaceans within the arthropods is not agreed upon, and the monophyletic status of the Crustacea has even been called into question (Ballard et al. 1992
; Friedrich and Tautz 1995
; Regier and Shultz 1997
). Here, we describe the mitochondrial gene order of the hermit crab Pagurus longicarpus and report the first major gene rearrangements in any nonchelicerate arthropod. Comparisons with partial crustacean genomes suggest that the rearrangements in the hermit crab P. longicarpus appear to have arisen within the Decapoda. Because they are restricted to the Decapoda, these rearrangements will not help resolve the issue of crustacean monophyly. On the other hand, exploring their phylogenetic distribution may provide additional characters to help discern relationships among decapod groups.
Materials and Methods
DNA was extracted from a live P. longicarpus individual obtained from Beaufort, N.C., using the Genome kit (BIO 101) according to the manufacturer's instructions. With the exception of approximately 300 bp of an 850-bp AT-rich region, 15,630 bp of the mtDNA molecule was sequenced directly from a combination of overlapping 0.52.0-kb PCR products and a long PCR 7.5-kb product cloned into TOPO XL vectors (Invitrogen). The overlapping 0.52.0-kb fragments were PCR-amplified in 50 mM Tris HCl (pH 9.0), 20 mM ammonium sulfate, 0.005% BSA, and 2.5 mM MgCl2, with 200 µM of each dNTP, 0.5 mM of each primer, and 1.25 U Amplitaq Gold polymerase (Perkin Elmer) in a total volume of 50 µl.
The 7.5-kb fragment was initially amplified using the Boehringer Mannheim Expand Long Template PCR system with conditions specified for system 3. PCR products were inserted into the pCR®XL plasmid vector using the Invitrogen TOPO XL cloning kit. Plasmid DNA was purified from each clone using the QIAGEN plasmid miniprep kit. The 7.5-kb fragment was sequenced with M13 and T7 reverse primers and then subsequently with newly designed internal primers. As a check against PCR error, three different clones were sequenced, and a two-out-of-three rule was used to choose between base ambiguities. The 0.52.0-kb fragments were cycle-sequenced with the PCR amplification primers and internal primers when necessary. All cycle-sequencing was performed with ABI PRISM BigDye terminator chemistry and analyzed on ABI 373 or 377 automated sequencers (ABI/Perkin-Elmer).
Mitochondrial protein and RNA gene sequences were initially identified using BLAST searches. Confirmation of gene sequences was then done by alignment with Drosophila yakuba mitochondrial sequences using CLUSTAL W, version 1.4. The amino acid sequences of the protein-coding genes were inferred from the Drosophila genetic code. A preliminary screen for tRNA genes was done using tRNAscan-SE 1.1 (Lowe and Eddy 1997
). The default search mode was used (cove cutoff score = 15), specifying mitochondrial/chloroplast DNA as the source and using the invertebrate mitochondrial genetic code for tRNA structure prediction. tRNA secondary structures were detected in this manner for 20 out of the 22 tRNA's, the two remaining tRNA's were detected by inspecting noncoding sequences for tRNA-like secondary structures by eye.
Contiguous sequences were obtained for the entire mitochondrial genome with the exception of approximately 300 bp within the AT-rich noncoding region. This sequence is deposited in GenBank under accession number AF150756.
Results and Discussion
The Mitochondrial Genome of the Hermit Crab P. longicarpus
Aside from approximately 300 bp within the AT-rich region that proved difficult to sequence, we obtained the entire mitochondrial genome of P. longicarpus. Our sequences include both rRNA genes, all 22 tRNA genes, and all 13 protein-coding genes commonly found in the mitochondria of metazoan animals (Wolstenholme 1992
). All orientations and gene sequence junctions are shown in figure 1
. Each of the 22 putative tRNA sequences can be folded into cloverleaf structures similar to those found in other metazoan animals (fig. 2
). The weakest inferred structures are tRNASer(AGN) and tRNAVal, with six base mispairings each (fig. 2
). Although technical difficulties (most likely secondary structure) prevented sequencing of the entire AT-rich region (estimated to be approximately 850 bp in size), we obtained the sequence of its flanking regions (38 bp 5' and 500 bp 3'). Before discussing the gene rearrangements, we will discuss characteristics of the mitochondrial genome.
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Translation Initiation and Termination Signals
All termini of the 13 protein-coding genes are shown in figure 1 and summarized in table 1
. As with the Drosophila genetic code, ATT, ATA, and ATG initiate translation in 12 out of 13 of the protein-coding genes (table 1
). For the initiation of COI in P. longicarpus, we propose a novel 4-bp initiation codon (ATCA) based on the locations of COI initiation in D. yakuba and D. pulex (Clary and Wolstenholme 1985;
Crease 1999
) and the fact that there are no other initiation codons near the beginning of COI in P. longicarpus. A 4-bp initiation codon (ATTA) has also been proposed for the COI of Daphnia pulex (Van Raay and Crease 1994
). For 11 of the 13 protein-coding genes, the 3' sequence regions revealed a termination codon of TAA, while COI used TAG. For ND4, we could find no complete termination codon. Following Crease (1999)
, we inferred an incomplete termination codon beginning with the base T. This association between incomplete termination codons and tRNA genes is common in metazoan mtDNA (Wolstenholme 1992
). One possible explanation is that complete termination codons are formed by polyadenylation of transcripts, as has been observed for some human mtDNA genes (Ojala, Montoya, and Attardi 1981
).
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tRNAs and Codon Usage
Twenty tRNAs have anticodons identical to those of D. yakuba and D. pulex, while two differ: tRNALys (TTT instead of CTT) and tRNASer(AGN) (TCT instead of GCT). This usage of TTT for tRNALys is consistent with most metazoan mtDNA systems. As with tRNALys, the difference for SerineSer(AGN) corresponds to the third base in the wobble position. The presence of these two anticodons in P. longicarpus could be due to codon usage. According to the codon usage inferred for the 13 P. longicarpus amino acid sequences, tRNALys is coded by AAA (rather than AAG) in 85.0% of the codons, while tRNASer is coded by AGA 52.0% of the time. This codon usage makes the anticodon of tRNALys more efficient than the anticodon found in D. yakuba (table 2
).
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Phylogenetic Utility of Mitochondrial Gene Rearrangements in the Decapoda
To demonstrate a mitochondrial gene rearrangement, we must know the original gene order. Inferring the primitive gene order in crustaceans is relatively straightforward, since several insects (e.g., D. yakuba) have a gene order identical to that of one of the two previously reported crustacean genomes (D. pulex; Crease 1999
). The other previously reported crustacean gene order (the brine shrimp A. franciscana; Valverde et al. 1994
) shows two tRNA rearrangements that are unique relative to all other arthropods. This suggests that the A. franciscana tRNA rearrangements are likely autapomorphies with respect to the taxa sampled to date.
In contrast, we found a total of eight rearrangements in the decapod hermit crab P. longicarpus relative to the putatively primitive D. pulex/D. yakuba gene order (fig. 3
). Two of these include the first reported rearrangements of arthropod protein-coding genes outside of the Chelicerata (Black and Roehrdanz 1998
; Campbell and Barker 1998
, 1999). These two rearrangements involve NADH2 and NADH3 (regions 2 and 3 in fig. 3
). Only one rearrangement in P. longicarpus involved an inversion of the direction of transcription (tRNAVal, fig. 3
).
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With eight rearrangements, it may be possible to demonstrate the intermediate gene orders that led to the arrangement found in P. longicarpus. Using this information in conjunction with other molecular data will help to evaluate the probabilities of homology and convergence in gene order. An investigation of the phylogenetic distribution of these rearrangements, together with a multigene sequencing effort, is currently in progress.
Acknowledgements
We must thank K. Tieu, who first discovered that there is something unusual about the hermit crab mitochondrial genome. We also thank C. Morrison for help in the cloning procedure and D. Snyder for running the ABI 373 and 377 automated sequencers (ABI/Perkin-Elmer). This work was supported by NSF grant DEB-9615461 to C.W.C.
Footnotes
Ross Crozier, Reviewing Editor
1 Keywords: crustacean mitochondrial DNA
gene order
gene rearrangements
hermit crab
Pagurus longicarpus.
2 Address for correspondence and reprints: Cliff Cunningham, Department of Zoology, Duke University, Durham, North Carolina 27708-0325. E-mail: cliff{at}duke.edu
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