The Complete Sequence of the Mitochondrial Genome of the Crustacean Penaeus monodon: Are Malacostracan Crustaceans More Closely Related to Insects than to Branchiopods?

Kate WilsonGo,, Valma Cahill, Elizabeth Ballment and John Benzie

Australian Institute of Marine Science, Townsville, Queensland, Australia


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
The complete sequence of the mitochondrial genome of the giant tiger prawn, Penaeus monodon (Arthropoda, Crustacea, Malacostraca), is presented. The gene content and gene order are identical to those observed in Drosophila yakuba. The overall AT composition is lower than that observed in the known insect mitochondrial genomes, but higher than that observed in the other two crustaceans for which complete mitochondrial sequence is available. Analysis of the effect of nucleotide bias on codon composition across the Arthropoda reveals a trend with the crustaceans represented showing the lowest proportion of AT-rich codons in mitochondrial protein genes. Phylogenetic analysis among arthropods using concatenated protein-coding sequences provides further support for the possibility that Crustacea are paraphyletic. Furthermore, in contrast to data from the nuclear gene EF1{alpha}, the first complete sequence of a malacostracan mitochondrial genome supports the possibility that Malacostraca are more closely related to Insecta than to Branchiopoda.


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
Mitochondrial genome sequence and structure is widely used to provide information on phylogenetic relationships and on the genetic structure of populations and patterns of gene flow. This information may derive from studies of gene order, the sequences of individual genes, restriction fragment length polymorphism (RFLP) analysis of mtDNA, or the sequences of complete genomes.

The giant tiger prawn, Penaeus monodon, is the major species of shrimp farmed in aquaculture worldwide. The Penaeidae fall within the order Decapoda (class Malacostraca), which contains most commercially important crustacean species. Analyses of penaeid mitochondrial genetics have been carried out using RFLP analysis and single-gene sequence analysis (Palumbi and Benzie 1991Citation ; Bouchon, Souty-Grosset, and Raimond 1994Citation ; Klinbunga et al. 1999Citation ). However, there is no complete mitochondrial genome sequence available for any decapod, or even for any malacostracan crustacean. To date, the only complete crustacean mitochondrial genome sequences available are those of two branchiopods: the brine shrimp Artemia franciscana (Branchiopoda, Anostraca) (Valverde et al. 1994Citation ) and the water flea Daphnia pulex (Branchiopoda, Cladocera) (Crease 1999Citation ). Thus, there is a dearth of mitochondrial sequence data available for phylogenetic comparisons within the Crustacea and related groups.

Recently, partial sequence (8,754 bp) was published for the southern pink shrimp, Penaeus notialis (García-Machado et al. 1999Citation ). This sequence encompassed seven protein- coding genes, and the aligned amino acid sequences of this species, Artemia, and four insects were used to make inferences about phylogeny within the Arthropoda. The resulting analysis supported a paraphyletic Crustacea, with Penaeus being more closely related to insects than to the branchiopod crustacean, Artemia. However, this analysis was based on only partial mitochondrial sequence from P. notialis and a single branchiopod, A. franciscana, as the complete mitochondrial sequence of D. pulex was not then available for inclusion in the analysis. Moreover, the mtDNA of Artemia appears to be evolving at an accelerated rate (Crease 1999Citation ; see fig. 5 ), which could affect molecular phylogenetic inference (Hillis, Moritz, and Mable 1996Citation ).



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Fig. 5.—Phylogenetic trees based on analysis of concatenated, aligned protein-coding sequences from 13 mitochondrial coding genes. For all parsimony and distance trees, only nodes with >70% bootstrap support have been retained (Hillis and Bull 1993Citation ). All trees are rooted with L. terristris as outgroup. A, Tree derived from maximum-likelihood analysis; the same tree was obtained using both star decomposition and local rearrangement options. Bootstrap probabilities shown are local bootstrap probabilities for the subsequent node estimated by the RELL method with 1,000 replications (Adachi and Hasegawa 1996Citation b). The total branch length of the tree is 349.73, and the log likelihood of the data given this tree is -48,236.81. B, Tree obtained using parsimony analysis. A single tree of 8,282 steps was found with a consistency index excluding uninformative characters of 0.761, a homoplasy index excluding uninformative characters of 0.239, and a retention index of 0.561. Similar trees were obtained from the neighbor-joining algorithm in the program MEGA using distance matrices generated using either number of differences or p-distances. C, Tree obtained using distance analysis, as implemented in the PHYLIP program. Bootstrap values (100 replicates) are shown. Similar trees were obtained from the neighbor-joining algorithm in the program MEGA using distance matrices generated using either the Poisson correction or the gamma parameter ({gamma} = 1 or {gamma} = 2)

 
In this paper, we present the first complete mitochondrial sequence for a malacostracan, P. monodon. We discuss the composition of the P. monodon mitochondrial genome and analyze the effect of nucleotide bias on codon composition across the Arthropoda. Finally, we describe the results of phylogenetic analyses among members of the Arthropoda for which complete mitochondrial sequences are available, paying particular attention to the relationships of the different crustacean groups and Insecta.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
Determination of Sequence
Mitochondrial DNA was extracted from pleopod muscle from a single hatchery-grown P. monodon specimen derived from parents collected near Cairns, North Queensland, Australia, according to Dowling, Moritz, and Palmer (1990)Citation , using 10 mM EDTA in the homogenization buffer. This mtDNA was cut with XbaI to produce three fragments (2.1, 3.8, and 10.0 kb), which were cloned into pUC19. The resulting three plasmids, AIMS-P.mon74, AIMS-P.mon75, and AIMS-P.mon76, were sequenced using fluorescent cycle-sequencing kits from the Applied Biosystems division of Perkin-Elmer. Initial sequencing was carried out using vector primers, and subsequent sequencing was performed by "primer walking"; i.e., primers were synthesized corresponding to previously obtained P. monodon mitochondrial sequence. Sequence was obtained for both strands for the complete genome. To determine the relative orientation of the three clones and to ensure that no sequence was missing due to a failure to clone very small XbaI fragments, primers that faced "out" of the clones were designed and used to amplify junction fragments from intact mitochondrial DNA by the polymerase chain reaction (PCR). In each case, single bands of the predicted size were obtained. These three PCR products were also sequenced using ABI reagents.

Analysis of Codon Bias
Potential amino acid bias due to underlying nucleotide bias was analyzed using the "square plot" analysis of Foster, Jermiin, and Hickey (1997)Citation . In brief, this analyzes the amino acid composition by looking at the proportion of amino acids with codons that have (1) A or T in both the first and the second positions (2) A or T in either the first or the second position, and (3) only G or C in these two positions. The amino acid leucine was excluded from this analysis, as it can be encoded by codons from two different classes (TTR and CTR) and hence can accommodate changes in underlying base composition without a change in encoded amino acids. The data are presented here as a histogram rather than as square plots to show underlying trends across the Arthropoda.

Phylogenetic Analyses
Protein-coding sequences were individually aligned using Pileup (Genetics Computer Group 1994Citation ) with the gap creation penalty set to 5.0 and the gap extension penalty set to 0.3. Sequences were from 10 of the 11 arthropods for which complete mitochondrial genome sequences were available in GenBank: among the insects (Hexapoda), the dipteran flies Drosophila yakuba (X03240, Hexapoda, Insecta, Diptera), Drosophila melanogaster (U37541, Hexapoda, Insecta, Diptera), Ceratitis capitata (CCA242872, Hexapoda, Insecta, Diptera), Anopheles quadrimaculatus (L04272, Hexapoda, Insecta, Diptera), and Anopheles gambiae (L20934, Hexapoda, Insecta, Diptera) and the orthopteran Locusta migratoria (X80245, Hexapoda, Insecta, Orthoptera); the branchiopod crustaceans A. franciscana (X69067, Crustacea, Branchiopoda, Anostraca) and Daphnia pulex (AF117817, Crustacea, Branchiopoda, Cladocera); and from the Chelicerata, the prostriate tick Ixodes hexagonus (AF081828, Chelicerata, Arachnida, Acari) and the metastriate tick Rhipicephalus sanguineus (AF081829, Chelicerata, Arachnida, Acari). The honeybee, Apis mellifera (L06178, Hexapoda, Insecta, Hymenoptera), was not included in the analysis due to the extreme nucleotide bias which confounds phylogenetic inference based on both nucleotide and protein sequence analyses (Galtier and Gouy 1995Citation ; Foster, Jermiin, and Hickey 1997Citation ). The earthworm Lumbricus terrestris (U24570, Annelida, Oligochaeta) was used as an outgroup. Internal sections of nad2 and nad4 were excluded from the analysis due to severe ambiguities in alignment. In addition, all sequences were trimmed at the N- and C-terminal ends to give an exact and unambiguous alignment. The alignments were then concatenated to give a single multiple sequence alignment containing 3,327 characters (2,335 variable and 1,687 parsimony- informative) for phylogenetic analysis. The alignments generated are available from the corresponding author on request.

The aligned sequences were subjected to distance analysis using MEGA 1.01 (Kumar, Tamura, and Nei 1993Citation ) and PHYLIP 3.572c (Felsenstein 1993Citation ), parsimony analysis using PAUP 3.1.1 (Swofford 1993Citation ), and maximum-likelihood analysis using MOLPHY 2.3b (Adachi and Hasegawa 1996bCitation ). Distances between taxa were estimated in the program MEGA using the number of differences, p-distance, the Poisson correction, and gamma probabilities with gamma set to 1 or 2. Trees were then constructed using neighbor joining. Bootstrap probabilities were estimated with 1,000 replications using pairwise deletion for gaps and missing data. Distance analyses were also carried out with the PHYLIP package using the Dayhoff PAM matrix for amino acid substitutions and neighbor-joining tree building. Bootstrap probabilities were estimated in PHYLIP with 100 replicates. For parsimony analysis, the heuristic search option in PAUP 3.1.1 was used. A simple addition sequence was used and tree bisection-reconnection branch swapping was performed. Bootstrap values were estimated using 1,000 replications. For both distance and parsimony analyses, L. terrestris was defined as the outgroup. For maximum-likelihood analysis, the mtREV-24 model of amino acid substitution in mitochondrial protein-coding genes was used (Adachi and Hasegawa 1996aCitation ), and the data were analyzed using both the star decomposition method and local rearrangement options. For the latter, the 200 best trees using stepwise addition were first generated, and these tree topologies were then subjected to local rearrangements to determine the maximum-likelihood tree.


    Results
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
Genome Size
Three plasmids were constructed from the three major XbaI fragments of the P. monodon mitochondrial genome. As the total size of these fragments was estimated to be approximately 16 kb, it was assumed that this comprised the majority of the mitochondrial genome. PCR analysis of intact mtDNA using primers that spanned the XbaI sites at the plasmid ends revealed that one XbaI site had been missed in the cloning strategy. This was because there were two XbaI sites which were almost adjacent within a region which was subsequently identified as containing the trnG gene. These sites are separated by only 6 bp and thus add 12 bp to the total sequence length obtained from the plasmids, making a total genome size of 15,984 bp. This sequence has been deposited in GenBank (accession number AF217843).

Gene Content and Order
The gene content was identified primarily by alignment with the known genes from D. yakuba, D. melanogaster, and A. gambiae. In addition, tRNA genes and their anticodons were detected using the program tRNAscan (Lowe and Eddy 1997Citation ). Like all of the other arthropod mtDNAs, the P. monodon mitochondrial genome contains genes for 13 proteins, 22 tRNAs, and two rRNAs. In addition, there is a 991-bp-long A+T-rich region that does not contain any known genes. As the origin of replication of the Drosophila mtDNA maps in the corresponding region in the Drosophila mtDNA genome, this region is presumed to be the equivalent of the vertebrate control region (Wolstenholme 1992Citation ). The order and arrangement of the genes in P. monodon is illustrated in figure 1 . It is identical to that observed in the insects D. melanogaster, D. yakuba, and C. capitata and in the branchiopod D. pulex.



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Fig. 1.—Gene order in the Penaeus monodon mitochondrial genome. tRNA genes are represented by the single-letter code for the cognate amino acid. For Leu and Ser, for which there are two tRNAs, the codon recognized has been specified. The numbers are the numbers of bases between genes, with negative numbers representing probable overlaps between genes (e.g., atp8 and atp6 are separated by -7 bp, ATGTTAA)

 
The genes are densely packed, as in other mitochondrial genomes. Excluding the 991-bp control region, there are a total of only 139 bp found between the different genes in the P. monodon mitochondrial genome. There are three clear cases of overlapping genes, which all occur between adjacent genes encoded on the same strand. There is a 5-bp overlap (TCTAA) between the last codon and the termination codon of cox1 and trnL(taa) and a 7-bp overlap (ATGATAA) between the termination of atp8 and the initiation of atp6 which is seen in most arthropod mitochondrial genomes (the honeybee, A. mellifera, has an even greater overlap, 19 bp); likewise, the end of nad4L and nad4 overlap by 7 bp (ATGTTAA) on the minority coding strand. In addition, there are a further three cases in which an overlap could occur if the complete stop codon present in the mtDNA sequence is used. These are a 2-bp overlap (AA) between the potential termination codon of nad2 (TAA) and the start of trnW, a 1-bp overlap (A) between the termination of nad6 (TAA) and the initiation of cob, and a 2- bp overlap (AG) between the termination of cob (TAG) and the start of trnS(uga). However, it is also possible that RNA processing clips the genes apart upstream of these potential overlaps and that polyadenylation completes the stop codon on the mRNA (Anderson et al. 1981Citation ).

Coding Sequences
The P. monodon mitochondrial genome appears to use the same genetic code as Drosophila. It is certainly the case that UGA specifies an amino acid rather than a stop codon, since otherwise the majority of proteins would show premature termination. It is also reasonable to presume that the amino acids specified by each codon family are the same as in Drosophila, as use of this code gives rise to the expected open reading frames with a high degree of similarity to the same proteins identified from Drosophila and other arthropods. However, this could be formally proven only by purification and sequencing of P. monodon mitochondrial proteins.

The location and putative initiation and termination codons of the protein-coding sequences are shown in table 1 . Twelve out of the 13 protein coding genes appear to initiate with the codon ATN. However, as is the case with several other arthropod species, the initiation codon of cox1 is unclear. In P. monodon, it appears to start with ACG based on alignment with other species. In Drosophila, Locusta, and Daphnia, there is a tetranucleotide sequence (ATAA or ATTA) immediately preceding the apparent first codon, and it has been suggested that this tetranucleotide could serve as the initiation codon (Clary and Wolstenholme 1983Citation ; de Bruijn 1983Citation ). However, no such sequence is present in P. monodon, and we are not aware of any model indicating how a tetranucleotide might function in this capacity. The absence of such a tetranucleotide has also been observed in other arthropods, e.g., A. gambiae (Beard, Mills, and Collins 1993Citation ), leading to the conclusion that each of the disparate array of first codons in aligned cox1 genes most likely does serve as the initiation codon. An alternative model to explain these atypical start codons is that they are converted to more conventional start codons in the mRNA; for example, in the tomato, the cox1 gene starts with an ACG codon, but this is converted to an AUG in the mRNA by RNA editing (Kadowaki et al. 1995Citation ). The presumed initiation context of cox1 genes from arthropods, for which there is complete mitochondrial sequence, plus that of the penaeid shrimp P. notialis, for which there is partial mitochondrial sequence (GenBank accession numbers X84350X84357), is shown in figure 2 .


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Table 1 Protein-Coding Genes in the Penaeus monodon Mitochondrial Genome

 


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Fig. 2.—Initiation context for cox1 genes from a variety of arthropods. The first five to seven codons are shown in uppercase letters. Where the first codon is not a typical start codon for arthropod mitochondrial DNA (ATN), the upstream context is shown in lowercase letters. In the case of the drosophilid flies, the preceding tetranucleotide is postulated to act as the initiation "codon." However, while this theory might also be applicable to the locust and to Daphnia, the upstream sequence for the Anopheles species, Ceratitis capitata, and Penaeus monodon do not conform to this pattern. The presumed initial amino acids are shown on the right-hand side of the figure

 
Nine of 13 genes have a complete termination codon, either TAA (eight genes) or TAG (cytochrome b only). The other genes all have incomplete termination codons, T, or possibly TA for nad5 (table 1 ). These are believed to be converted to complete TAA codons by the addition of A residues during RNA processing (Anderson et al. 1981Citation ).

tRNAs
The predicted structures of the P. monodon tRNAs are shown in figure 3 . All tRNAs have the typical cloverleaf structures of other mitochondrial tRNAs except for trnS(ucg), which recognizes the codon AGN. This latter tRNA lacks the dihydrouridine loop, as observed for this tRNA in all other arthropod mitochondrial genomes (Wolstenholme 1992Citation ).



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Fig. 3.—The inferred secondary structure of the 22 tRNAs in the Penaeus monodon mitochondrial genome. The tRNAs for Leu and Ser are identified in the figure by the codons recognized, rather than by the anticodon present in the tRNA itself

 
The anticodons are identical to those observed in D. yakuba, except for the trnK anticodon, which is UUU in P. monodon, in contrast to CUU in D. yakuba. The trnK anticodon is also UUU in the related shrimp P. notialis and in the honeybee A. mellifera (Crozier and Crozier 1993Citation ), but it is CUU in all other arthropods known to date. In fact, UUU is the anticodon which would be predicted for lysine because, in most mtDNAs, two-codon families which end in a purine have a U in the wobble position of the anticodon. In the case of D. yakuba and other arthropods, the CUU anticodon for lysine (codons AAA and AAG) requires C-A pairing (Clary and Wolstenholme 1985Citation ).

Base Composition
The A+T content of the P. monodon genome is 70.6% (A, 5,636; C, 2,669; G, 2,028; T, 5,651 on the strand that contains the majority of open reading frames). The 991-bp control region has an AT composition of 81.5%. These values are lower than those for the published insect genomes (75.3%–84.9% for total A+T content and 86.0%–96.0% for the AT-rich control region), but higher than those for the crustaceans A. franciscana (64.5% total AT content, 68.0% AT in the control region) and D. pulex (62.3% total AT, 67.1% AT in the control region). Among the chelicerates, the horseshoe crab is 67.7% A+T in the 8,459 bp sequenced (Staton, Daehler, and Brown 1997Citation ), and the ticks Ixodes and Rhipicephalus are 72.6% and 77.9% AT-rich, respectively.

There are a total of 3,716 codons in all 13 protein-coding genes, excluding stop codons. The overall AT composition of protein-coding regions is 69.3% but at third positions it is 83.7%. When the nucleotide composition of the third positions of fourfold-degenerate codons is examined, it is 84.9% AT (see table 2 ). This contrasts with much lower values of 68.7% in Artemia and 62.9% in Daphnia. The corresponding value for the partial sequence available for P. notialis (for the protein- coding genes atp6, atp8, cox1, cox2, cox3, nad2, nad3, and nad5) is 74.6%. Table 2 illustrates that these values for the two penaeid shrimp, representing the class Malacostraca, are intermediate between those for the branchiopod crustaceans and the insects. The percentage of A+T in the third positions of fourfold-degenerate codons for the two tick species is similar to that for the penaeids.


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Table 2 Base Composition at the Third Codon Positions of Fourfold-Degenerate Codons

 
The codon usage in P. monodon is shown in table 3 . A feature of most arthropod genomes sequenced to date is that the bias toward the nucleotides A and T also leads to a bias in the amino acids used. This can be analyzed by comparing the proportions of amino acids with A or T versus C or G at the first and second codon positions. Penaeus monodon appears to share the bias of all arthropod genomes toward amino acids encoded by AT-rich codons (fig. 4 ). In P. monodon, the bias toward AT-rich codons appears to be very similar to that observed in Artemia and Daphnia and is slightly less than that observed in the insects. The gradation in the proportion of codons with A or T in both of the first two positions which is observed in the different taxonomic groups indicates a lack of evolutionary stationarity; i.e., there is heterogeneity in the evolutionary process leading to differing average codon and, hence, amino acid compositions.


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Table 3 Codon Usage in 13 Protein-Coding Genes of Penaeus monodon

 


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Fig. 4.—Amino acid content of protein-coding genes in arthropod mitochondrial genomes is affected by codon bias. The histogram represents the proportion of amino acids for codons that have (1) A or T in both the first and the second positions (AT/AT), (2) A or T in either the first or the second position (AT/CG or CG/AT), or (3) only G or C in these two positions (CG/CG). Leucine and termination codons are excluded. All arthropod taxa for which complete mitochondrial genome sequences are available are included

 
Phylogenetic Analysis
The P. monodon mitochondrial sequence represents the 12th complete arthropod mitochondrial genome sequenced to date; there are now complete mitochondrial sequences available for seven insects, two chelicerates, and three crustaceans. Hence, it was of interest to see whether mitochondrial sequence comparisons could yield any evidence on phylogenetic relationships between the different subphyla represented, particularly on crustacean/insect relationships. As the phylogenetic comparisons being made are between widely divergent phylogenetic groups, most sequence signal will be obscured by saturation. This was indeed found to be the case when nucleotide sequences of individual genes (small- and large-subunit rRNA genes and cox1) were used in phylogenetic analyses: the phylogenetic trees produced from these data were largely unresolved (nodes supported by bootstrap values of <70% were considered insufficiently supported; Hillis and Bull 1993Citation ). However, by aligning the amino acid sequences of all mitochondrially encoded proteins and using the concatenated set of aligned sequences in phylogenetic analysis, more robust phylogenetic trees were obtained.

The phylogenetic analysis used data from 11 of the 12 complete arthropod mitochondrial genomes available (10 from the database plus P. monodon). Sequence from the honeybee A. mellifera was omitted as the extreme AT bias in this species makes phylogenetic analyses even of protein-coding regions unreliable (Foster, Jermiin, and Hickey 1997Citation ), as evidenced by the fact that A. mellifera clustered together with the ticks with a bootstrap value of 99–100 when included in a similar phylogenetic analysis (Black and Roehrdanz 1998Citation ). The earthworm L. terrestris (Boore and Brown 1995Citation ) was used as the outgroup.

Three different tree structures resulted from application of different phylogenetic algorithms (fig. 5AC ). In all cases, the two chelicerate species form a clade which is quite distinct from a Hexapoda/Crustacea clade. In the first tree structure (fig. 5A ), Artemia and Daphnia form a clade separate from Hexapoda (represented by the class Insecta) and P. monodon; in the second (fig. 5B ), all Crustacea and Hexapoda form a single clade, with Penaeus being the crustacean most closely related to the Hexapoda and Artemia being the most distantly related. In the third tree, Penaeus and Hexapoda form one clade, and the relationship of Daphnia and Artemia to each other and to this clade remains unresolved. The most striking feature held in common by all three trees is the paraphyly of the Crustacea, with Penaeus being more closely related to the insects than to the branchiopods. This relationship of Penaeus to Insecta is supported by bootstrap values of >90% with all tree-building methods.


    Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
The length of the P. monodon mitochondrial genome falls within the range observed for most animal mitochondrial genomes, including those of the other arthropods for which complete sequences are available: the insects D. yakuba (16,019 bp) (Clary and Wolstenholme 1985Citation ), D. melanogaster (19,517 bp) (Lewis, Farr, and Kaguni 1995Citation ), A. gambiae (15,363 bp) (Beard, Mills, and Collins 1993Citation ), A. quadrimaculatus (15,455 bp) (Cockburn, Mitchell, and Seawright 1990Citation ), C. capitata (15,980 bp) (Spanos et al. 2000), A. mellifera (16,343 bp) (Crozier and Crozier 1993Citation ), and L. migratoria (15,722 bp) (Flook, Rowell, and Gellissen 1995Citation ), the crustaceans A. franciscana (15,822 bp) (Valverde et al. 1994Citation ) and D. pulex (15,333 bp) (Crease 1999Citation ), and the chelicerates I. hexagonus (14,539 bp) and R. sanguineus (14,710 bp) (Black and Roehrdanz 1998Citation ).

The gene order in the P. monodon mitochondrial genome is identical to that of the three dipteran species D. yakuba, D. melanogaster, and C. capitata, as well as to the branchiopod D. pulex. All other arthropod mtDNAs for which complete mitochondrial sequences are available show distinct differences in the placement of certain genes. The two Anopheles species differ in the placement of two tRNAs, Locusta in the placement of one tRNA, Apis in the placement of eight tRNAs, Artemia in the placement of the block of trnI to trnQ, and Ixodes in the placement of one tRNA, and Rhipicephalus shows major rearrangements. In the latter species, the block of genes from nad1 to trnQ appears to have transposed, and there are two further tRNA rearrangements.

Boore et al. (1995)Citation , and Boore and Brown (1998)Citation , and Boore, Lavrov, and Brown (1998)Citation argue that rearrangements of metazoan mtDNA are highly unlikely to be duplicated by convergences and hence can be used to infer phylogenetic lineages. These authors therefore conclude that the Crustacea and Insecta form a sister group, with the Chelicerata and Myriapoda occupying separate arthropod lineages (Boore, Lavrov, and Brown 1998Citation ). The arrangement of genes in the P. monodon genome is in accord with this hypothesis. This arrangement is identical to that observed in the crustacean D. pulex and appears to be also conserved in the partially characterized genomes of the malacostracan crustaceans Homarus americanus (Boore et al. 1995Citation ) and P. notialis (García-Machado et al. 1999Citation ). Taken together, these data provide further support for the proposed relationship between Crustacea and Insecta (Boore, Lavrov, and Brown 1998Citation ), and the conclusion that the drosophilid mitochondrial gene order represents the ancestral crustacean/insect mitochondrial gene order (Crease 1999Citation ).

Phylogenetic relationships among the major arthropod groups were also investigated by sequence comparisons using concatenated protein-coding sequences. The resulting trees all gave strong support for several conclusions regarding arthropod phylogeny, although it should be noted that these trees are necessarily based on the restricted set of arthropod taxa for which complete mitochondrial sequences are currently available. In particular, the available sequences only represent three of the four extant arthropod subphyla, with no complete mitochondrial sequence from the Myriapoda being available in the database.

The first conclusion is that the Chelicerata form a separate clade from Crustacea/Hexapoda. This appears to be widely supported by molecular evidence on gene sequence (Friedrich and Tautz 1995Citation ; Black and Roehrdanz 1998Citation ), on mitochondrial gene order (Boore, Lavrov, and Brown 1998Citation ), and on the molecular basis of development (Averof and Akam 1995Citation ). It is also a view held by most morphological taxonomists (e.g., Snodgrass 1938Citation ; Kukalová-Peck 1992Citation ; Wheeler, Cartwright, and Hayashi 1993Citation ), although there have been proposals that have placed crustaceans closer to chelicerates (Schram and Emerson 1991Citation ; Briggs, Fortey, and Willis 1992Citation ).

The second conclusion is that Crustacea are a paraphyletic group. This contradicts many phylogenetic trees proposed on the basis of morphological data (Snodgrass 1938Citation ; Wheeler, Cartwright, and Hayashi 1993Citation ). However, the concept of crustacean paraphyly has been proposed previously based on both morphological and molecular characters (Briggs and Fortey 1989Citation ; Averof and Akam 1995Citation ), although the details of the paraphyly may vary (see discussion below). The conclusion that Crustacea is paraphyletic with respect to Hexapoda was also drawn by García-Machado et al. (1999)Citation in their analysis of partial mitochondrial sequence of P. notialis.

The third conclusion relates to the relationship of the two classes of crustaceans that are now represented by complete mitochondrial genome sequences, the Malacostraca and the Branchiopoda, to each other and to the Hexapoda, represented here by the class Insecta. A corollary of several theories supporting the idea that the Crustacea are paraphyletic is that Hexapoda derive from a crustacean ancestor. If this is the case, then it is an inescapable conclusion that some Crustacea will be more closely related to Hexapoda than to other crustacean groups. This theory is consistent with the trees generated in the present analysis, which indicate that P. monodon is related to the Hexapoda (Insecta), with bootstrap support ranging from 92% to 100%. The exact relationship of the two branchiopods both to each other and to the Penaeus/insect clade is unclear, but in all cases our analysis indicates that the branchiopods are less closely related to insects than is the malacostracan P. monodon. This result was also obtained in the analysis of García-Machado et al. (1999)Citation , although their analysis included only one branchiopod, A. franciscana, which appears to be subject to accelerated evolution (Crease 1999Citation ; see long branch lengths for Artemia in fig. 5 ).

This conclusion on the relative relationships of Malacostraca/Branchiopoda/Insecta contradicts the results of two other molecular studies that included representatives of both crustacean classes. In the first, a combined analysis of the 18S and 28S nuclear ribosomal RNA genes gave strong support for a single crustacean/hexapod clade and weak support for a possible closer relationship of the branchiopod Artemia salina to Hexapoda than the malacostracan Procambarus clarkii (Friedrich and Tautz 1995Citation ). In a more wide-ranging analysis using protein-coding sequence from the nuclear gene encoding EF1{alpha}, Regier and Schultz (1997, 1998)Citation propose that Crustacea is polyphyletic, with Branchiopoda forming a single clade with Hexapoda and Chelicerata being more closely related to this clade than Malacostraca.

The reasons for the disparity in phylogenetic trees obtained with the analysis of different molecules are unclear. One possibility is that the heterogeneity in the evolutionary processes occurring in the different arthropod lineages leads to a misleading result. This was certainly the case in the results of Black and Roehrdanz (1998)Citation , who carried out a similar analysis using concatenated protein sequences which placed A. mellifera in the same clade as the hard ticks with 99% bootstrap support. The reason for the latter result is the extreme AT bias in the Apis mitochondrial genome, which has a significant influence on the resulting amino acid composition of the encoded proteins (see fig. 4 ). However, as figure 4 illustrates, although this bias is evident in the P. monodon mitochondrial genome, it is not as extreme as in the case of Apis or, indeed, any of the other insects. In fact, the degree to which amino acid composition is affected by codon bias appears to be no different for P. monodon than for A. franciscana, and only slightly higher than for D. pulex. Hence, it is unlikely to affect the relative positions of these three species on a phylogenetic tree with respect to each other or to Insecta. In addition, the proposed phylogenetic tree structure is also strongly supported by maximum-likelihood analysis. This method, although still susceptible to producing erroneous phylogenetic trees in cases of extreme bias in nucleotide or amino acid composition, is far less sensitive to small compositional differences than are distance and parsimony analyses (Galtier and Gouy 1995Citation ).

A more likely explanation for the different trees is that each of these molecular phylogenies is based on analysis of only one or two genes or, in the case of the present paper, a set of genetically linked genes. There is always a danger that such analyses may give misleading results due to factors such as insufficient informative characters or convergent evolution.

A more far-fetched, but not impossible, scenario is that some horizontal transfer of either nuclear or mitochondrial sequences has occurred from insects to crustaceans or vice versa. As increasing amounts of data emerge from complete genome sequences, evidence suggests that the evolutionary origins of different groups of genes may differ. For example, Rivera et al. (1998)Citation recently demonstrated that some groups of genes in eukaryotes are derived from archaebacteria, whereas other groups appear to derive primarily from proteobacteria, leading to the conclusion that there may have been considerable horizontal gene transfer in the evolutionary process.

If the phylogeny suggested by this analysis of mitochondrial sequences is correct, this would have strong implications for the polarity of evolution of morphological characters used in the taxonomy of Crustacea and Hexapoda. In particular, it would imply that characters which are used to distinguish crustaceans from hexapods have been secondarily lost in hexapods, rather than being autapomorphies of Crustacea. However, as the Crustacea are notoriously diverse in body form (Schram 1986Citation ) and the characters that serve to uniquely define them are few, it is possible to develop plausible scenarios by which this may have happened. In fact, evolutionary scenarios which would conform with the proposed phylogeny have been already developed on both morphological and molecular-developmental grounds (Averof and Akam 1995Citation ; Telford and Thomas 1995Citation ).

For example, one character which has been used to define Crustacea is the structure of the head. It has been described as having five segments, two preoral segments bearing the first and second antennae and three postoral segments bearing, respectively, the mandibles and the first and second maxillae (Cisne 1982Citation ; Schram 1986Citation ). Insects, in contrast, have been described as having six segments in the head, three preoral and three postoral (Lawrence, Nielsen, and Mackerras 1991Citation ). However, the nature of the most anterior portion of the crustacean head was never clear (see Averof and Akam 1995Citation ), and recent work indicates that both crustaceans and insects have six cephalic segments, as revealed by looking at the expression of genes that specify segmental identity (Popadic et al. 1998Citation ; Scholtz 1998Citation ). Another commonly cited character defining Crustacea is the presence of a second antenna on the second appendage-bearing segment of the head. However, it is easy to envisage secondary loss of such a structure in Insecta, and, indeed, some insects develop small appendages on this segment during the embryonic stage which could be vestiges on an earlier adult appendage (Tamarelle 1984Citation ).

A third set of characters often deemed to be unique to Crustacea relate to embryology and larval development, namely, the nature of the embryonic fate map and the formation of a nauplius larva (larvae with three sets of appendages used for propulsion, corresponding to the antennules, antennae, and mandibles) or passage through an egg-nauplius phase (Anderson 1982Citation ; Schram 1986Citation ). However, more recent studies have contested Anderson's (1982)Citation claim that crustacean egg cleavage is spiral (Hertzler and Clark 1992Citation ), and the wisdom of using a character as variable as fate maps in phylogenetic analysis has also been questioned (Averof and Akam 1995Citation ). Moreover, as larval development has clearly undergone considerable diversification within Hexapoda, ranging from ametabolous insects that show no marked metamorphosis at all, to hemimetabolous insects with a gradual larval-to-adult transition, to the holometabolous insects which show complete metamorphosis from larval to adult forms (Chapman 1991Citation ), it is certainly possible to envisage that the nauplius larval stage has been secondarily lost.

Other features which in the past have been used to argue that Crustacea and Hexapoda occupy distinct evolutionary lineages, namely, the origin of the mandibles (gnathobasic vs. whole-limb) and the nature of the limbs (uniramous vs. bi- or polyramous) have already effectively been discounted as being only apparent, rather than real, differences by a number of authors (Kukalová-Peck 1992Citation ; Averof and Akam 1995Citation ; Telford and Thomas 1995Citation ; Scholtz, Mittmann, and Gerberding 1998Citation ).

The proposed phylogeny also has implications for the relative affinities of different crustacean classes to each other and to Insecta. The divergence between the branchiopod clade and the malacostracan/insect clade is consistent with the fossil record. Although their exact affinities may be disputed, fossils with substantial similarities to both Branchiopoda and Malacostraca are known from the Cambrian (Dahl 1983Citation ; Briggs 1983Citation ), and these classes were certainly established as independent lineages well before the appearance of the first fossil insects in the Devonian (Kukalová-Peck 1991Citation ). Moreover, there are certain features held in common between Malacostracan crustaceans and insects that are not shared with "simpler crustaceans," most notably the detailed structures of the compound eye and of the segmental nervous systems (Averof and Akam 1995Citation ; Telford and Thomas 1995Citation ; Osorio, Averof, and Bacon 1995Citation ). The former has been alluded to as "one of the most disconcerting problems of arthropod phylogeny" by Tiegs and Manton (1958)Citation , who argued that Arthropoda were polyphyletic and hence that the malacostracan and insect eyes had evolved by convergence.

A question that the present paper cannot address is the relative position of the myriapods on the proposed phylogenetic tree, as no complete myriapod mitochondrial gene sequences were publicly available at the time of writing. Traditionally, it has been assumed that Hexapoda evolved from a myriapodlike ancestor and that Crustacea formed a sister group to this "Atelocerate clade" (Kristensen 1991Citation ). However, almost all molecular analyses indicate that Crustacea, rather than Myriapoda, are sister to Insecta (Wheeler, Cartwright, and Hayashi 1993Citation ; Friedrich and Tautz 1995Citation ; Boore, Lavrov, and Brown 1998Citation ), and arguments have also been put forward on morphological grounds, taking into account key features of Myriapoda, Hexapoda, and Crustacea, that make this scenario plausible (Averof and Akam 1995Citation ; Osorio, Averof, and Bacon 1995Citation ; Telford and Thomas 1995Citation ).

One conclusion that is clear from the present paper and the recent completion of the D. pulex mitochondrial genome sequence (Crease 1999Citation ) is that while identity of mitochondrial gene order may be a strong indicator of phylogenetic relatedness due to the low probability of convergence (Boore and Brown 1998Citation ), differences in mitochondrial gene order do not necessarily reflect major phylogenetic differences. This is illustrated in the Arthropoda by the fact that two widely divergent crustaceans and three dipteran flies all share the same mitochondrial gene order, while those of other dipterans and hexapods differ in the placement of one or more tRNAs. Most strikingly, analysis of the mitochondrial genome of two hard ticks, which lie within the same chelicerate family, has revealed major mitochondrial rearrangements in four out of five subfamilies within that group (Black and Roehrdanz 1998Citation ; Campbell and Barker 1998Citation ). Thus, the various tRNA rearrangements present in the mitochondrial genomes of Anopheles, Locusta, Apis, and Artemia all appear to be characters that were derived after the initial radiation of hexapods and crustaceans.

In summary, the complete sequence of the malacostracan crustacean P. monodon gives further weight to the conclusion that the mtDNA gene order observed in D. yakuba is the ancestral crustacean/hexapod arrangement. Phylogenetic analysis comparing available arthropod mitochondrial genomes indicates a sister group relationship between Malacostraca and Insecta and hence throws further fat on the controversial fire of arthropod phylogeny. However, a more in-depth understanding of arthropod phylogeny will require the completion both of the sequence of a greater variety of arthropod mitochondrial genomes, most notably with the inclusion of some myriapod genomes, as well as a better understanding of the processes of mitochondrial evolution.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
The authors thank Lars Jermiin for his assistance with the analysis of codon bias in arthropod genomes and with the maximum- likelihood analysis, and Gerhard Scholtz for discussions of crustacean phylogeny. We also thank two anonymous reviewers for their helpful comments on the manuscript. This is contribution number 994 of the Australian Institute of Marine Science.


    Footnotes
 
B. Franz Lang, Reviewing Editor

1 Keywords: Malacostraca complete mitochondrial genome sequence arthropod phylogeny crustacean/insect relationships penaeid shrimp Back

2 Address for correspondence and reprints: Kate Wilson, Australian Institute of Marine Science, PMB 3, Townsville MC, Queensland 4810, Australia. E-mail: k.wilson{at}aims.gov.au Back


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Accepted for publication February 1, 2000.