Institute of Zoology, University of Mainz, Mainz, Germany
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Abstract |
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Introduction |
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The phylogeny of the hemocyanin superfamily, as well as the evolutionary changes associated with the emergence of the different proteins, cannot be understood without considering the phylogeny of the Arthropoda. The origin and the evolutionary relationships of this taxon are one of the most extensively debated issues in evolutionary biology (e.g., Fortey and Thomas 1997
) (fig. 1 ). As already pointed out by Mangum et al. (1985)
, the exclusive, but not universal, appearance of the hemocyanins in the Euarthropoda provides an additional argument in favor of the monophyly of this taxon. In this paper, the evolutionary history of the hemocyanin superfamily is inferred and correlated with the recent advances in the understanding of arthropod phylogeny.
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Materials and Methods |
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Sequence Similarities
Two different Monte Carlo shuffling approaches were applied to estimate the significance of the observed amino acid sequence similarities. The Z score (observed value - mean value/standard deviation) was obtained by invoking the "-ran" option of the GAP program of the GCG package, version 8.0 (Genetics Computer Group, Wisconsin) with 100 shuffles. The Z score gives the significance estimate in terms of standard deviations above the mean. In a second approach, which took into account database sizes, the PRSS3 program (FASTA package; Pearson 2000) was used to estimate probability scores (assuming a gap creation penalty of -12 and a gap length penalty of -2 with 1,000 shuffles).
Molecular Phylogenetic Inference
The program packages PHYLIP, version 3.6 (Felsenstein 1999
), and TREE-PUZZLE, version 4.02 (Strimmer and von Haeseler 1996
), were used for phylogenetic analyses. Distances between pairs of protein sequences were calculated according to the following substitution models: Dayhoff's PAM001 matrix (Dayhoff, Schwartz, and Orcutt 1978
), the JTT model (Jones, Taylor, and Thornton 1992
), and Kimura's (1983)
corrected percent differences. Distances were corrected for gamma distribution of evolutionary rates. Indels between pairs of sequences were regarded as missing data. Nucleotide distances were calculated according to the HKY model (Hasegawa, Kishino, and Yano 1985
). Because saturation of silent sites was assumed, only the first and second codon positions were used for phylogenetic inference. Tree constructions were performed by the neighbor-joining method (Saitou and Nei 1987
). Additional trees were constructed using the maximum-parsimony methods implemented in the PROTPARS and DNAPARS programs of the PHYLIP package. The reliability of the trees was tested by bootstrap analysis (Felsenstein 1985
) with 100 or 500 replications (SEQBOOT program). Maximum-likelihood analyses were performed using TREE-PUZZLE, version 4.02, with 1,000 puzzling steps assuming rate heterogeneity with eight gamma categories.
Calibrations and Time Estimations
The distance matrices were calculated according to the above methods and were imported into the Microsoft EXCEL 97 spread sheet program. A relative-rate test was successively applied to single proteins or groups of proteins according to the topology of the tree (cf. Burmester et al. 1998
). Rate constancy of different subtrees was also evaluated by the likelihood ratio test implemented in TREE-PUZZLE, version 4.02. To estimate the divergence times of the different protein families, the replacement rates were calculated separately for each protein family under the assumptions that (1) the Merestomata and Arachnida separated about 450 MYA (Dunlop and Selden 1997
), (2) the brachyuran and palinuran Decapoda separated about 150 MYA (Briggs, Weedon, and White 1993
), and (3) the Orthoptera separated from the other neopteran insects 320 MYA (Kukulovà-Peck 1991
), and the brachyceran nematoceran Diptera separated about 210 MYA (Fraser et al. 1996
) (fig. 1
). Replacement rates in the text are given as inferred from the PAM distances of the alignment used for phylogenetic inference (see also table 1
). This data set does not include some highly variable regions; thus, the replacement rates of the complete sequences are about 5% higher. The lengths of the branches that lead from the basalmost internal nodes to the node that connects the protein families were extrapolated from the branch-specific rates. These calculations were performed independently for both branches that diverge at this node. The confidence limits were estimated using the observed standard deviation of the inferred replacement rates or based on the maximum-likelihood standard deviations.
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Results and Discussion |
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Evolutionary Relationships Within the Hemocyanin Superfamily
The final alignment of all available sequences from the arthropod hemocyanin superfamily comprises 93 complete amino acid sequences. To minimize the influence of possible alignment errors, long indels and highly heterologous regions were removed. In figure 4 , a 50% majority-rule consensus tree resulting from a neighbor-joining analysis based on the PAM distances is displayed. The general topology of this tree is similar to those previously published (Beintema et al. 1994
; Burmester and Scheller 1996
; Sánchez et al. 1998
) but essentially differs from other recent analyses (Durstewitz and Terwilliger 1997
; Hughes 1999
; Terwilliger, Dangott, and Ryan 1999
). As discussed above, the latter trees are probably biased by the choice of a heterologous (unrelated) or incorrect outgroup. To assess the reliability of the present tree, several additional phylogenetic methods, as well as analysis of the available nucleotide sequences, were applied. In table 2 , the bootstrap confidence values of 10 selected nodes (fig. 4 ) deriving from these approaches are displayed. As pointed out above, in all analyses the monophyly and the general relationships of the major protein families are strongly supported, with the exception of the insect hemocyanin.
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The hemocyanins of the Crustacea and Chelicerata individually form two well-separated clades (fig. 1
and table 1
, nodes C and F). The chelicerate hemocyanin subunits are basal, consistent with the assumption that the Chelicerata is a rather distinct arthropod taxon (Dunlop and Selden 1997
). There is strong statistical support for the hypothesis that crustacean pseudohemocyanins (cryptocyanins) are included within the clade of the true crustacean hemocyanins (Burmester 1999b
; Terwilliger, Dangott, and Ryan 1999
), but their arrangement relative to the known crustacean hemocyanin subunits is not well resolved in the different analyses (table 1
, node G). Most likely they are in sister group position to all known crustacean hemocyanins, although an association with the
-type subunits (see below) cannot be excluded with sufficient confidence. The insect hexamerins are closely related to the crustacean proteins, with the hexamerins forming a well-supported monophylum. Although the statistical (bootstrap) support is not very strong (node H, table 1
), the association of the insect hemocyanin with the hexamerins was supported in all but two analyses, implying an evolution of the hexamerins within the hexapod lineage.
Diversity of PPOs in Holometabolous Insects
Although PPOs have been described by biochemical means in all euarthropodan subphyla, sequences are only available from the malacostracan Crustacea and the holometabolous insects. The clades leading to PPOs of these subphyla are well separated (fig. 4
and table 2
, node B). Although the resolution of the arrangement within the insect PPOs is generally poor, there is consistent support for the existence of three different PPO classes among the insects (adjacent to node B, fig. 4
). While two of them contain PPO sequences only from the Diptera or Lepidoptera, respectively, the third (in which monophyly is, however, supported by only 64%) comprises PPOs from Diptera, Lepidoptera, and Coleoptera. Although up to six different PPO genes have been described in insects, which may exhibit differential expression patterns (Müller et al. 1999
), the physiological relevance of the existence of multiple PPOs is not yet clear. A timescale of PPO evolution cannot be readily inferred because the orthology of the different PPOs is not resolved. It is, nevertheless, noteworthy that the internal arrangement of the single known coleopteran PPO (TmoPPO) within one of the branches (fig. 4
) suggests that different PPO types had already diverged well before the radiation of the Mecopteria (i.e., Lepidoptera, Diptera, Trichoptera, Siphonaptera, and Mecoptera) about 270 MYA (fig. 1 ).
A Timescale for Arthropod Hemocyanin Evolution
While the rates of evolutionary changes are, with a few exceptions among the hexamerins (Burmester et al. 1998
), rather uniform within the different protein families, there is considerable variation between them (fig. 2A
and table 1
). Therefore, no general molecular clock can be employed to infer the divergence times. However, likelihood ratio tests and relative-rate tests suggest that local clocks probably still apply and may be used to infer a timescale of the molecular evolution of the different members of the hemocyanin superfamily, as well as an at least tentative timescale for arthropod phylogeny.
Ancient Divergence of the Chelicerate Hemocyanin Subunits
The hemocyanins of the Chelicerata are very often multimers of hexamers that are formed by different subunits that occupy distinct positions in the native oligomer (Markl and Decker 1992
). For example, the 24mer (4 x 6) hemocyanin of the tarantula E. californicum is composed of seven different subunits (EcaHcAEcaHcG in fig. 4
). In addition, the sequences of two hemocyanin subunits from the Xiphosura L. polyphemus and T. tridentatus (LpoHcA and TtrHcII) and one from the scorpion A. australis (AauHc6) are known. While the chelicerate hemocyanin subunits form a well-supported common clade, the phylogenetic resolution of the relationships between them is poor. This indicates a lack of phylogenetic signal that may be interpreted as the result of a rapid diversification from a single primordial chelicerate hemocyanin. However, the association of EcaHcB and EcaHcC is strongly supported (99% bootstrap value in neighbor-joining analysis). These subunits are located in the interhexamer contact zones and play an important role in the formation of the tarantula 4 x 6 hemocyanin. Thus, the ancestor of the tarantula hemocyanin was likely a hexamer composed of a-, b/c-, d-, e-, f-, and g-type subunits, with each occupying a distinct position in the hemocyanin structure and the arachnidan 4 x 6 hemocyanin evolving after gene duplication and formation of distinct b and c subunits.
There is also good statistical (99%) and immunological evidence (Kempter et al. 1985
) for a close relationship of subunit A of E. californicum (EcaHcA) and subunit II of the horseshoe crab Limulus (LpoHcII). Xiphosura (L. polyphemus) and Arachnida (E. californicum) diverged about 450 MYA (Dunlop and Selden 1997
). Assuming this date and the orthology of LpoHcII and EcaHcA, a substitution rate of 5.82 ± 0.31 x 10-10 per site per year was inferred (table 1
). Then, the earliest time of divergence of the chelicerate hemocyanin subunits took place about 564 ± 23 MYA, probably in the stemline of the Chelicerata. The date of the EcaHcB/EcaHcC split was estimated to have occurred about 418 ± 18 MYA. Immunological data of the arachnidan hemocyanins suggest that these subunit types split before Araneae and Scorpiones diverged (Kempter et al. 1985
), probably in the Silurian period (Dunlop and Selden 1997
). Therefore, the estimated time is consistent with the available fossil record and indicates that the cheliceratan hemocyanins evolved in an at least approximate clocklike manner. The other E. californicum subunit types diversified about 476 ± 65 MYA. Therefore, the 24mer hemocyanin built by seven different subunits appears to be the original quaternary structure of the arachnidan hemocyanins, and other subunit compositions of hemocyanins in several nonorthognathan Araneae should be interpreted as secondary rearrangements (cf. Markl 1986
).
Hemocyanin Subunit Diversity in the Crustacea
Hemocyanins have been found in most Malacostraca (fig. 1
) but are apparently absent in other crustacean taxa (Mangum 1983
), with the possible exception of the Remipedia (Yager 1991
). Nevertheless, their universal appearance in the other arthropod subphyla indicates that a respiratory hemocyanin was present within the crustacean stemline. Markl (1986)
noted three different hemocyanin subunit types in the higher Decapoda. The seven known crustacean subunits form two distinct clades, representing the
(HamHcA, PinHcA, PinHcB, PvuHc) and
(PinHcC, PvaHc, CamHc6) types (fig. 4
), while ß-type sequences are still unknown. According to the fossil record, the brachyuran and palinuran Decapoda diverged at the end of the Jurassic period, about 150 MYA, while the split of the astacura and palinuran Decapoda was about 180 MYA (Briggs, Weedon, and White 1993
). The rate of amino acid substitution was calculated to be 1.29 x 10-9 per site per year, about two times as fast as that of the chelicerate hemocyanins. This calculation indicates a divergence of the
and ß subunit types 200 ± 13 MYA. This suggests the radiation of the known crustacean hemocyanin subunits in the stemline of the Decapoda, which is in agreement with the immunological data (Markl 1986
). The pseudohemocyanins (cryptocyanins) are included in the clade of the crustacean hemocyanins (Burmester 1999b
; Terwilliger, Dangott, and Ryan 1999
). Consistent with their proposed role as storage proteins, the pseudohemocyanins have a rather high replacement rate of 1.97 x 10-9 per site per year. According to the molecular-clock calculations, crustacean hemocyanins and pseudohemocyanins diverged about 215 MYA (table 3 ).
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Conclusions |
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Acknowledgements |
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Footnotes |
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1 Keywords: Chelicerata
Crustacea
hemocyanin
hexamerin
Hexapoda
phenoloxidase
2 Address for correspondence and reprints: Thorsten Burmester, Institute of Zoology, University of Mainz, D-55099 Mainz, Germany. E-mail: burmeste{at}mail.uni-mainz.de
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literature cited |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Averof, M., and M. Akam. 1995. Insect-crustacean relationships: insights from comparative developmental and molecular studies. Philos. Trans. R. Soc. Lond. B Biol. Sci. 347:293303.[ISI]
Beintema, J. J., W. T. Stam, B. Hazes, and M. P. Smidt. 1994. Evolution of arthropod hemocyanins and insect storage proteins (hexamerins). Mol. Biol. Evol. 11:493503.[Abstract]
Boore, J. L., D. V. Lavrov, and W. M. Brown. 1998. Gene translocation links insects and crustaceans. Nature 392:667668.
Börner, C. 1909. Neue Homologien zwischen Crustaceen und Hexapoden. Zool. Anz. 34:100124.
Briggs, D. E. G., M. J. Weedon, and M. A. White. 1993. Arthropoda (Crustacea excluding Ostracoda). Pp. 321342 in M. J. Benton, ed. The fossil record 2. Chapman and Hall, London.
Brusca, R. C., and G. J. Brusca. 1990. Invertebrates. Sinauer Associates, Sunderland, MA.
Burmester, T. 1999a. Evolution and function of the insect hexamerins. Eur. J. Entomol. 96:213225.
. 1999b. Identification, molecular cloning and phylogenetic analysis of a non-respiratory pseudo-hemocyanin of Homarus americanus. J. Biol. Chem. 274:1321713222.
Burmester, T., H. C. Massey Jr., S. O. Zakharkin, and H. Bene. 1998. The evolution of hexamerins and the phylogeny of insects. J. Mol. Evol. 47:93108.[ISI][Medline]
Burmester, T., and K. Scheller. 1996. Common origin of arthropod tyrosinase, arthropod hemocyanin, insect hexamerin, and dipteran arylphorin receptor. J. Mol. Evol. 42:713728.[ISI][Medline]
Crampton, G. C. 1922. A comparison of the first maxillae of apterygotan insects and crustacea from the standpoint of phylogeny. Proc. Entomol. Soc. Wash. 24:6582.
Dayhoff, M. O., R. M. Schwartz, and B. C. Orcutt. 1978. A model of evolutionary change in proteins. Pp. 345352 in M. O. Dayhoff, ed. Atlas of protein sequence structure. Vol. 5, Suppl. 3. National Biomedical Research Foundation, Washington, D.C.
Dohle, W. 1997. Myriapod-insect relationships as opposed to an insect-crustacean sister group relationship. Pp. 305315 in R. A. Fortey and R. H. Thomas, eds. Arthropod relationships. Systematic Association Special Volume Series 55. Chapman and Hall, London.
Dunlop, J. A., and P. A. Selden 1997. The early history and phylogeny of the chelicerates. Pp. 221236 in R. A. Fortey and R. H. Thomas, eds. Arthropod relationships. Systematic Association Special Volume Series 55. Chapman and Hall, London.
Durstewitz, G., and N. B. Terwilliger. 1997. cDNA cloning of a developmentally regulated hemocyanin subunit in the crustacean Cancer magister and phylogenetic analysis of the hemocyanin gene family. Mol. Biol. Evol. 14:266276.[Abstract]
Felsenstein, J. 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39:783791.
. 1999. PHYLIP (phylogeny inference package). Version 3.6alpha. Distributed by the author, Department of Genetics, University of Washington, Seattle.
Fortey, R. A., and R. H. Thomas. 1997. Arthropod relationships. Systematic Association Special Volume Series 55. Chapman and Hall, London.
Fraser, N. C., D. A. Grimaldi, P. E. Olsen, and B. Axsmith. 1996. A Triassic Lagerstätte from eastern North America. Nature 380:615619.
Friedrich, M., and D. Tautz. 1995. Ribosomal DNA phylogeny of the major extant arthropod classes and the evolution of myriapods. Nature 376:165167.
García-Machado, E., M. Pempera, N. Dennebouy, M. Oliva-Suarez, J. C. Mounolou, and M. Monnerot. 1999. Mitochondrial genes collectively suggest the paraphyly of Crustacea with respect to Insecta. J. Mol. Evol. 49:142149.[ISI][Medline]
Gaykema, W. P. J., W. G. J. Hol, J. M. Vereifken, N. M. Socter, H. J. Bak, and J. J. Beintema. 1984. 3.2 Å structure of the copper-containing, oxygen-carrying Panulirus interruptus hemocyanin. Nature 309:2329.
Hasegawa, M., H. Kishino, and K. Yano. 1985. Dating of the human-ape splitting by a molecular clock of mitochondrial DNA. J. Mol. Evol. 22:160174.[ISI][Medline]
Hazes, B., K. A. Magnus, C. Bonaventura, J. Bonaventura, Z. Dauter, K. H. Kalk, and W. G. J. Hol. 1993. Crystal structure of deoxygenated Limulus polyphemus subunit II hemocyanin at 2.18 Å resolution: clues for a mechanism for allosteric regulation. Protein Sci. 2:597619.
Hughes, A. L. 1999. Evolution of the arthropod prophenoloxidase/hexamerin protein family. Immunogenetics 49:106114.
Jaenicke, E., H. Decker, W. Gebauer, J. Markl, and T. Burmester. 1999. Identification, structure and properties of hemocyanins from diplopod Myriapoda. J. Biol. Chem. 274:2907129074.
Jones, D. T., W. R. Taylor, and J. M. Thornton. 1992. The rapid generation of mutation data matrices from protein sequences. CABIOS 8:275282.
Kawabata, T., Y. Yasuhara, M. Ochiai, S. Matsuura, and M. Ashida. 1995. Molecular cloning of insect pro-phenoloxidase: a copper-containing protein homologous to arthropod hemocyanin. Proc. Natl. Acad. Sci. USA 92:77747778.
Kempter, B., J. Markl, M. Brenowitz, C. Bonaventura, and J. Bonaventura. 1985. Immunological correspondence between arthropod hemocyanin subunits. II. Xiphosuran (Limulus) and spider (Eurypelma, Cupiennius) hemocyanin. Biol. Chem. Hoppe Seyler 366:7786.
Kimura, M. 1983. The neutral theory of molecular evolution. Cambridge University Press, Cambridge, England.
Kukulovà-Peck, J. 1991. Fossil history and evolution of hexapod structures. Pp. 141179 in I. D. Naumann, ed. The insects of Australia. Melbourne University Press, Melbourne, Australia.
Linzen, B., N. M. Soeter, A. F. Riggs et al. (14 co-authors). 1985. The structure of arthropod hemocyanins. Science 229:519524.
Mangum, C. P. 1983. Oxygen transport in the blood. Pp. 373429 in D. E. Bliss and L. H. Mantel, eds. Biology of Crustacea. Vol. 5. Academic Press, New York.
Mangum, C. P., J. L. Scott, R. E. L. Black, K. I. Miller, and K. E. van Holde. 1985. Centipedal hemocyanins: its structure and implication for arthropod phylogeny. Proc. Natl. Acad. Sci. USA 82:37213725.
Markl, J. 1986. Evolution and function of structurally diverse subunits in the respiratory protein hemocyanin from arthropods. Biol. Bull. 171:90115.[ISI]
Markl, J., and H. Decker. 1992. Molecular structure of the arthropod hemocyanins. Adv. Comp. Environ. Physiol. 13:325376.
Miller, K. I., M. E. Cuff, W. F. Lang, P. Varga-Weisz, K. G. Field, and K. E. van Holde. 1998. Sequence of the Octopus dofleini hemocyanin subunit: structural and evolutionary implications. J. Mol. Biol. 278:827842.[ISI][Medline]
Müller, H. M., G. Dimopoulos, C. Blass, and F. C. Kafatos. 1999. A hemocyte-like cell line established from the malaria vector Anopheles gambiae expresses six prophenoloxidase genes. J. Biol. Chem. 274:1172711735.
Nellaiappan, K., and M. Sugumaran. 1996. On the presence of prophenoloxidase in the hemolymph of the horseshoe crab, Limulus. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 113:163168.
Nicholas, K. B., and H. B. Nicholas Jr. 1997. GeneDoc: analysis and visualization of genetic variation. http://www.cris.com/Ketchup/genedoc.shtml.
Pearson, W. R. 2000. Flexible sequence similarity searching with the FASTA3 program package. Methods Mol. Biol. 132:185219.[Medline]
Regier, J. C., and J. W. Shultz. 1997. Molecular phylogeny of the major arthropod groups indicates polyphyly of the Crustaceans and a new hypothesis for the origin of the arthropods. Mol. Biol. Evol. 14:902913.[Abstract]
Saitou, N., and M. Nei. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4:14061425.
Sánchez, D., M. D. Ganfornina, G. Gutiérrez, and M. J. Bastiani. 1998. Molecular characterization and phylogenetic relationship of a protein with oxygen-binding capabilities in the grasshopper embryo. A hemocyanin in insects? Mol. Biol. Evol. 15:415426.[Abstract]
Shultz, J. W., and J. C. Regier. 2000. Phylogenetic analysis of arthropods using two nuclear protein-encoding genes supports a crustacean + hexapod clade. Proc. R. Soc. Lond. B Biol. Sci. 267:10111019.[ISI][Medline]
Söderhäll, K., and L. Cerenius. 1998. Role of the prophenoloxidase-activating system in invertebrate immunity. Curr. Opin. Immunol. 10:2328.[ISI][Medline]
Spears, T., and L. G. Abele. 1997. Crustacean phylogeny inferred from 18S rDNA. Pp. 169187 in R. A. Fortey and R. H. Thomas, eds. Arthropod relationships. Systematic Association Special Volume Series 55. Chapman and Hall, London.
Strimmer, K., and A. von Haeseler. 1996. Quartet puzzling: a quartet maximum likelihood method for reconstructing tree topologies. Mol. Biol. Evol. 13:964969.
Swofford, D. L., G. J. Olsen, P. J. Waddell, and D. M. Hillis. 1996. Phylogenetic inference. Pp. 407514 in D. M. Hillis, C. Moritz, and B. K. Mable, eds. Molecular systematics. Sinauer, Sunderland, Mass.
Telfer, W. H., and J. G. Kunkel. 1991. The function and evolution of insect storage hexamers. Annu. Rev. Entomol. 36:205228.[ISI][Medline]
Terwilliger, N. B., L. J. Dangott, and M. C. Ryan. 1999. Cryptocyanin, a crustacean molting protein, and evolutionary links to arthropod hemocyanin and insect hexamerins. Proc. Natl. Acad. Sci. USA 96:20132018.
Thompson, J. D., T. J. Gibson, F. Plewniak, F. Jeanmougin, and D. G. Higgins. 1997. The CLUSTAL X interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25:48764882.
Turbeville, J. M., D. M. Pfeifer, K. G. Field, and R. A. Raff. 1991. The phylogenetic status of arthropods, as inferred from 18S rRNA sequences. Mol. Biol. Evol. 8:669686.[Abstract]
van Holde, K. E., and K. I. Miller. 1995. Hemocyanins. Adv. Protein Chem. 47:181.[ISI][Medline]
Volbeda, A., and W. J. G. Hol. 1989. Crystal structure of hexameric hemocyanin from Panulirus interruptus refined at 3.2 Å resolution. J. Mol. Biol. 209:249279.[ISI][Medline]
Wheeler, W. C. 1990. Nucleic acid sequence phylogeny and random outgroups. Cladistics 6:363368.
Wilson, K., V. Cahill, E. Ballment, and J. Benzie. 2000. The complete sequence of the mitochondrial genome of the crustacean Penaeus monodon: are malacostracan crustaceans more closely related to insects than to branchiopods? Mol. Biol. Evol. 17:863874.
Xylander, W. 1996. The phenoloxidase from the hemolymph of Diplopoda. Pp. 411420 in J. J. Goeffroy, J.-P. Mauriès, and M. Nguyen Duy-Jacquemin, eds. Acta Myriapodologica. Vol. 169. Mémoires du Muséum national d'Histoire naturelle, Paris.
Yager, J. 1991. The Remipedia (Crustacea): recent investigations of their biology and phylogeny. Verh. Dtsch. Zool. Ges. 84:261269.
Zrzav, J., V. Hyp
a, and M. Vlá
ková. 1997. Arthropod phylogeny: taxonomic congruence, total evidence and conditional combination approaches to morphological and molecular data sets. Pp. 97107 in R. A. Fortey and R. H. Thomas, eds. Arthropod relationships. Systematic Association Special Volume Series 55. Chapman and Hall, London.