Contrasting Rates of Mitochondrial Molecular Evolution in Parasitic Diptera and Hymenoptera

L. R. Castro*, A. D. Austin{dagger} and M. Dowton*{dagger}

*Institute for Conservation Biology, Department of Biology, The University of Wollongong, New South Wales;
{dagger}Centre for Evolutionary Biology and Biodiversity, Department of Environmental Biology The University of Adelaide, Glen Osmond, South Australia


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Conclusions
 Acknowledgements
 References
 
We investigated the putative association between the parasitic lifestyle and an accelerated rate of mt genetic divergence, compositional bias, and gene rearrangement, employing a range of parasitic and nonparasitic Diptera and Hymenoptera. Sequences were obtained for the cox1, cox2, 16S, 28S genes, the regions between the cox2 and atp8 genes, and between the nad3 and nad5 genes. Relative rate tests indicated generally that the parasitic lifestyle was not associated with an increased rate of genetic divergence in the Diptera but reaffirmed that it was in the Hymenoptera. Similarly, a departure from compositional stationarity was not associated with parasitic Diptera but was in parasitic Hymenoptera. Finally, mitochondrial (mt) gene rearrangements were not observed in any of the dipteran species examined. The results indicate that these genetic phenomena are not accelerated in parasitic Diptera compared with nonparasitic Diptera. A possible explanation for the differences in the rate of mt molecular evolution in parasitic Diptera and Hymenoptera is the extraordinary level of radiation that has occurred within the parasitic Hymenoptera but not in any of the dipteran parasitic lineages. If speciation events in the parasitic Hymenoptera are associated with founder events, a faster rate of molecular evolution is expected. Alternatively, biological differences between endoparasitic Hymenoptera and endoparasitic Diptera may also account for the differences observed in molecular evolution.


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Conclusions
 Acknowledgements
 References
 
Since the late 1980s, mitochondrial DNA (mtDNA) analysis has become established as a powerful tool for evolutionary studies of animals. Information on factors that impact on the evolution of mtDNA provides critical background knowledge to improve the level of analytical sophistication in a range of studies, such as phylogeny estimation, population genetics, and mutation research. In particular, three features of the mitochondrial (mt) genome have been the focus of several recent studies: substitutional rate heterogeneity, deviation from compositional stationarity, and variation in mt genome organization.

Compositional Bias
A common feature of nucleotide sequences is compositional bias, the occurrence of the four bases A, G, C, and T in unequal proportions. The degree of compositional bias varies widely among genes and organisms. For example, the G+C-content of third positions of codons from nuclear and mitochondrial protein–encoding genes in bacteria, vertebrates, and insects ranges from 4% to 98% (Bernardi et al. 1985Citation ; Ikemura 1985Citation ; Jukes and Bhushan 1986Citation ; Sueoka 1988Citation ; Liu and Beckenbach 1992Citation ). When taxa under study exhibit a similar pattern and degree of compositional bias, they are said to exhibit stationarity, sometimes referred to as base compositional equilibrium (Saccone, Pesole, and Preparata 1989Citation ). Variation in compositional bias among taxa is known as deviation from stationarity (Collins, Wimberger, and Naylor 1994Citation ).

Substitutional Rate Heterogeneity
Rates of nucleotide substitution are known to vary widely among phylogenetic groups (Wu and Li 1985Citation ; Britten 1986Citation ; Kocher et al. 1989Citation ; Martin, Naylor, and Palumbi 1992Citation ; Adachi, Cao, and Hasegawa 1993Citation ; Martin and Palumbi 1993Citation ; Cantatore et al. 1994Citation ; Martin 1995Citation ; Mindell and Thacker 1996Citation ; Nunn and Stanley 1998Citation ). This has led to the rejection of a universal molecular clock (which implies that for any given macromolecule the rate of evolution is approximately constant over time in all evolutionary lineages [Zuckerkandl and Pauling 1965Citation ]) and has prompted investigators to search for possible causes of rate heterogeneity. Although it is unlikely that rates of molecular evolution are determined by a single factor, several individual factors have been associated with variation in rates of molecular evolution (Britten 1986Citation ; Adachi, Cao, and Hasegawa 1993Citation ; Mindell and Thacker 1996Citation ).

Some of the hypotheses that explain different molecular rates between taxa are (1) The differences in DNA repair efficiency among lineages, proposed by Britten (1986)Citation —differences in the number of germline DNA replications per year or in the mutation rate itself are possible causes of rate contrasts. (2) The generation time hypothesis (Kohne 1970Citation )—organisms with shorter generation times have a greater number of germ cell divisions per unit time; therefore, they have a higher mutation rate. This holds if the majority of mutations are the result of errors during DNA replication, if the number of germ cell divisions is roughly similar per generation in most organisms, and if the majority of mutations are neutral (Kimura 1979Citation ). (3) The metabolic rate hypothesis (Martin and Palumbi 1993Citation )—increased rates of DNA replication and nucleotide replacement in organisms with higher metabolic rates should lead to higher mutation rates, whereas the increased concentrations of free oxygen radicals in cells with higher metabolic rates should be associated with a higher incidence of DNA damage. (4) The population size hypothesis—populations with small effective sizes will experience faster rates of evolution than populations with larger effective sizes (Ohta 1972Citation , 1992Citation ) because of the increased influence of drift on selection. Smaller population sizes are often associated with longer generation times, thus counteracting the generation time hypothesis.

Gene Rearrangement
It has been proposed that transfer RNA (tRNA) gene rearrangements are ideal phylogenetic markers (Boore et al. 1995Citation ; Kumazawa and Nishida 1995Citation ; Dowton 1999Citation ), representing some of the few retained synapomorphies in the mitochondrial genome useful for retracing ancient evolutionary relationships. Although many organisms have the same arrangement of protein-coding genes and rRNA genes, rearrangements involving tRNA genes are much more common. Among insects, the positions of a few tRNA genes appear to vary, particularly the positions of those near the control region and in the tRNA gene cluster trnA-trnR-trnN-trnS1-trnE-trnF (Shao et al. 2001bCitation ).

Molecular Evolution and Parasitism
A range of studies suggests that an increased rate of mt molecular evolution is coincident with an evolutionary transition to the parasitic lifestyle. For example, Dowton and Austin (1995)Citation found a higher rate of mtDNA sequence divergence in the parasitic wasps compared with nonparasitic wasps. Similar results were found by Hafner et al. (1994)Citation and Page et al. (1998)Citation in parasitic lice and by Nickrent and Starr (1993)Citation in parasitic plants. Possible causes for this correlation are an increased rate of speciation (Page et al. 1998Citation ), increased flux of mutagens (Dowton and Campbell 2001Citation ), or decreased DNA repair efficiency.

On the other hand, there have been few studies examining the association between compositional bias and parasitism. The only studies so far are those of Dowton and Austin (1995, 1997). They found that the A-content was increased in the predominantly parasitic wasps compared with nonparasitic wasps and suggested that this result may be related to the evolution of parasitism. Oxidative damage may be a significant source of mutations in mt genomes. This involves the oxidative or hydrolytic deamination of dC to dU, changing the base pairing from dG to dA during the next round of replication (Wagner, Hu, and Ames 1992Citation ). Consistent with this scenario, Dowton and Austin (1997)Citation found that the A-content of an mt 16S fragment increased in parasitic wasps, at the expense of the G-content.

Some studies also hint that an accelerated rate of gene rearrangement is associated with the parasitic lifestyle. Schistosomes, which are among the most significant parasites of humans, have a unique gene order (Le et al. 2000)Citation . The correlation between frequency of gene rearrangement and parasitism is also supported by the numerous gene rearrangements in the mt genome of the wallaby louse, which is an obligate ectoparasite (Shao, Campbell, and Barker 2001aCitation ). In the Diptera, gene rearrangements have been found only in three species of mosquitoes, which may also be categorized as ectoparasites (Pruess, Zhu, and Powers 1992Citation ; Beard, Hamm, and Collins 1993Citation ). Finally, Dowton and Austin (1999)Citation noticed that all mt gene rearrangements in the Hymenoptera occurred after the parasitic lifestyle was adopted. Primitively nonparasitic Hymenoptera retained the ancestral organization for the insects, whereas many parasitic Hymenoptera had altered genome organizations.

In the present study, we wished to further investigate this apparent association between the parasitic lifestyle and accelerated rates of molecular evolution in mt genomes. We chose the parasitic Diptera as a model group because parasitism has evolved in this order multiple times, permitting multiple tests of this hypothesis. Further, the broad range of parasitic lifestyles displayed by the Diptera provides the potential to discover those aspects of the parasitic lifestyle that contribute to these genetic phenomena. We used molecular sequence data from four mitochondrial genes (cox1, cox2, 16S, nad5) and one nuclear gene (28S) to assess these issues.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Conclusions
 Acknowledgements
 References
 
DNA Extraction
All taxa sampled (see tables 1 and 2 Go ) were collected into 100% ethanol and stored at 4°C before extraction. Flight muscle was dissected from larger flies and the entire thorax used for smaller ones. The head region was excluded because it has been found to contain PCR inhibitors in other insects (Dowton and Austin 1999Citation ). Ethanol was removed by washing three times (30 min each) in 10 mM Tris-HCl (pH 8) containing 100 mM NaCl and 1 mM MgCl2. Tissue was homogenized in 400 µl of 10 mM Tris-HCl (pH 8), 10 mM EDTA, and 1% SDS containing 100 µg of proteinase K (Boehringer Manheim) and incubated overnight at room temperature. DNA was separated from salt-insoluble material by the method of Sunnucks and Hales (1996)Citation . DNA was redissolved in 100 µl of sterile water and stored at 4°C. This DNA solution was used directly in the PCR reaction.


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Table 1 List of Parasitic and Nonparasitic Diptera Sampled

 

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Table 2 List of Hymenoptera Sampled and the Base Composition of the 16S Fragment

 

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Table 2 Continued

 
PCR Amplification
PCR reactions were performed in a total volume of 20 µl. Each PCR reaction contained 10 mM Tris-HCl (pH 9), 50 mM KCl, 0.1% Triton X-100, 1.25–6.25 mM MgCl2, 0.4 µM of each primer, 25 µM of each dNTP (dATP, dCTP, dTTP, dGTP), 0.75 U Taq DNA polymerase (PROMEGA) per reaction, and 0.5 µl of DNA extract. A negative control PCR tube was prepared with the same constituents but lacking DNA. Primer sequences appear in table 3 . Amplifications were performed in a Hybaid Sprint PCR thermocycler using the following program: 5 cycles (30 s at 94°C, 30 s at 45–55°C, and 1 min at 72°C), followed by 30 cycles (30 s at 94°C, 30 s at 55°C, and 1 min at 72°C). In addition, a 5-min extension at 72°C was added at the end of the 35 cycles in order to finish any incomplete amplification. PCR optimization for each template involved the variation of possible primer combinations, MgCl2 concentration, and annealing temperature.


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Table 3 Primer Sequences and Gene/Genome Regions to Which they Anneal

 
Longer amplifications (between the nad3 and nad5 genes) were performed as mentioned previously but using 0.75 U Taq and 2.5 mU Pfu DNA polymerase (Pyrococcus furiosus) and 100 µM of each dNTP per reaction. For these amplifications, we used an Eppendorf Gradient PCR cycler using a long PCR program: an initial denaturation at 92°C for 2 min, followed by 35 cycles (denaturation at 92°C for 10 s, annealing at 45–65°C for 30 s, and extension at 68°C for 2.5 min). Eight different primer combinations were tested, and MgCl2 concentration and annealing temperature were again optimized for each taxon.

In order to remove unincorporated primers and dNTPs before sequencing, double-stranded PCR products were purified using GeneClean Spin Kits (Bio 101) or PEG (polyethylene glycol) precipitation (Maniatis, Sambrook, and Fritsch 1989, p. 1.40Citation ) with some modifications (0.6 volumes of 30% PEG in 1.5 M NaCl was added to each PCR reaction). Cycle sequencing reactions were performed with the ABI Prism Dye Terminator cycle sequencing kit (Perkin-Elmer) with AmpliTaq FS. Longer products were sequenced by walking, designing, and synthesizing additional primers as sequence data accumulated. Both strands of the PCR product were sequenced. Primer sequences were removed from the start and the end of the obtained sequence and sequence ambiguities resolved by comparing the electropherograms using the program SeqEd v. 1.0 (Applied Biosystems, Inc. 1990).

Alignment
The edited sequences generated for the cox1, cox2, 16S, and 28S genes, together with nucleotide sequences of other nonparasitic Diptera obtained from GenBank were separately aligned using CLUSTAL V (Thompson, Higgins, and Gibson 1994Citation ), as distributed with the BIOEDIT program (Hall 1999Citation ). These alignments were subsequently manually adjusted by removal of length polymorphic regions. After the exclusion of regions of questionable alignment, the data set comprised 251 characters for 16S, 232 for 28S, 282 for cox1, 288 for cox2 and 174 for nad5. Nucleotide and amino acid alignments are available as supplementary material from the MBE web page (www.molbiolevol.org).

Nucleotide Composition and Relative Rate Tests
Unequivocally alignable positions were imported into MEGA version 2.1 (Kumar et al. 2001Citation ) for calculating the nucleotide composition and performing the Tajima's (1993)Citation relative rate test (RRT). To test for taxon-specific differences in DNA composition, the program STATIO (Rzhetsky and Nei 1995Citation ) was used. With respect to the RRT, as pointed out by Wu and Li (1985)Citation , the choice of the reference out-group taxon is critical to minimize the error in the estimations of substitutions per site. Because the vast majority of parasitic Diptera belong to the suborder Brachycera, we chose representatives from the only other dipteran suborder (the Nematocera) as the out-group taxon, specifically Aedes (for which all of the investigated gene fragments are reported in GenBank) and a chironomid (for which we independently generated gene sequence information). However, for the nuclear gene comparisons, previous studies indicate that the Culicomorpha have an accelerated substitution rate compared with other Diptera (Friedrich and Tautz 1997Citation ). For this reason we chose a representative from the Nematocera outside of the Culicomorpha (Psychodomorpha: Psycodidae: Nemapalpus flavus) as an out-group for the nuclear gene comparisons, to maximize the discriminating power of the test. Nemapalpus flavus sequence was obtained from GenBank.

Tajima's (1993)Citation test considers three sequences, 1, 2, and 3, with sequence 3 as the out-group. This test examines E(nijk) = E(njik) where i, j, and k represent different nucleotides in sequences 1, 2, and 3, respectively. Under the molecular clock hypothesis, E(nijk) = E(njik), irrespective of the substitution model and whether or not the substitution rate varies with the site. If the hypothesis is rejected, then the molecular clock hypothesis can be rejected for this set of sequences. RRTs were performed using the data obtained for the mitochondrial 16S, cox1, cox2, and nad5 genes, and for 28S (D2) nuclear region. Some RRTs and compositional stationarity tests were performed using the hymenopteran data (16S gene) from Dowton and Austin (2001)Citation to establish comparisons.

Identification of tRNAs
The region between the cox2 and atp8 genes normally contains two tRNA genes, whereas that between the nad3 and nad5 genes normally contains six tRNA genes. Sequences generated from these regions were submitted for tRNA gene search using the program tRNA-Scan SE (v. 1.1, http://genome.wustl.edu/eddy/tRNAscan-SE; Lowe and Eddy 1997Citation ). The parameters for the tRNA scan program were set to search for mitochondrial-chloroplast DNA as the source and using the invertebrate mitochondrial genetic code. When long tracts of noncoding sequence were apparent, the cove cutoff score was reduced to 10 and the search repeated. Nucleotides at the boundaries of tRNA genes of the sequence were translated to amino acid sequences by the program Translation Machine (www.2ebi.ac.uk/translate/) using invertebrate-mitochondrial translation tables.


    Results and Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Conclusions
 Acknowledgements
 References
 
The purpose of the present study was to determine whether the parasitic lifestyle is associated with mutation rate heterogeneity, departure from compositional stationarity, and an accelerated rate of mt gene rearrangement among the Diptera. To this end, sequence data were generated from four mitochondrial (16S, cox1, cox2, and nad5) and one nuclear (28S) gene fragments from 11 to 12 dipteran taxa.

Nucleotide Composition—Diptera
Base composition was heavily biased toward adenine and thiamine in all taxa sampled and in both nuclear and mitochondrial genes (table 4 ). In particular, the bias was most pronounced at the third codon position of protein-coding genes. These results are similar to those reported for other insect species (Clary and Wolstenholme 1985Citation ; Beard, Hamm, and Collins 1993Citation ; Crozier and Crozier 1993Citation ; Mitchell, Cockburn, and Seawright 1993Citation ). In Drosophila there is no clear distinction between the nucleotide composition of genes transcribed from the two complementary strands (Clary and Wolstenholme 1985Citation ); however, we found a slightly higher AT-content for the 16S and nad5 genes (minor mt strand) compared with the cox1 and cox2 genes (major sense strand) (table 4 ).


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Table 4 Base Compositions in Dipteran Taxa Sampled

 
To test for taxon-specific differences in DNA composition, the program STATIO (Rzhetsky and Nei 1995Citation ) was used because it accounts for correlated changes caused by phylogeny. We found the null hypothesis—DNA composition stationarity—rejected for all genes (16S, 28S, cox1, cox2, and nad5) when all taxa were included (parasitic Diptera, nonparasitic Diptera, and the out-group which includes the nonparasitic Aedes [for mt genes] or Nemapalpus [for the nuclear gene] and Nematocera: Chironomus) (table 5 , part A). To test whether parasitic Diptera were causing the departure from stationarity, we performed the same test including (1) just the nonparasitic taxa (table 5 , part B), and (2) just the parasitic taxa (table 5 , part C). In all cases (except for the cox2 gene in nonparasitic Diptera), the hypothesis of stationarity was rejected. Overall, we found that the dipteran mt genes depart from compositional stationarity but that the departure was not particularly associated with the parasitic taxa. In their study using dipteran 28S and 18S sequences, Friedrich and Tautz (1997)Citation found that the Culicidae caused a departure from compositional stationarity. For this reason, we repeated the test without the Culicomorpha: Aedes for the mt genes and without Chironomus (also from the Culicomorpha) for both nuclear and mt genes. Again, for genes 16S, cox1, and nad5, compositional stationarity is rejected. For 28S the hypothesis was never rejected when Chironomus was excluded from the analysis (table 5 , parts D and E). The departure from stationarity when using the 28S gene seems to be entirely caused by the taxon Culicomorpha.


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Table 5 Test for DNA Composition Stationarity

 
Nucleotide Composition—Hymenoptera
The composition stationarity test using the hymenopteran data was designed in a similar way, using just the 16S gene. A first test was performed using parasitic wasps (various Apocrita), together with the nonparasitic out-group (Symphyta: Hartigia) and the primitively ectoparasitic ingroup (Symphyta: Orussus). In this case, the hypothesis of composition stationarity was rejected (P = 0.000). When the test was performed in each group separately, we found that there was no deviation from stationarity in the Symphyta (P = 0.3503) but that the hypothesis of composition stationary was still rejected in the Apocrita (P = 0.003). Because previous studies showed that the Braconidae has a particularly high A-content (Dowton and Austin 1997Citation ); we repeated this test for the Apocrita after removing the braconid. In this case, stationarity was not rejected (P = 0.4369) (table 5 , part F). These stationarity tests confirmed that compositional bias increased in the lineage leading to the Apocrita, coincident with the adoption of the parasitic lifestyle, and that a secondary increase in compositional bias occurred in the lineage leading to the Braconidae.

Relative Rate Tests
There are several approaches for calculating molecular rates. The most common method used to test for rate variation is the RRT (Sarich and Wilson 1967Citation ; Wu and Li 1985Citation ; Tajima 1993Citation ). With this test, we can determine if rates of molecular evolution are the same for two different taxa by comparing how divergent they are from a known out-group. In order to establish whether parasitism is associated with increased rates of point mutation in the Diptera, we performed several RRTs according to Tajima (1993)Citation , comparing one parasitic brachyceran taxon with one nonparasitic brachyceran taxon using a nonparasitic nematoceran as the out-group. In order to assess how sensitive the results of these tests would be to the choice of an out-group and a nonparasitic ingroup, we performed the RRT with either of the two out-groups (Aedes or Chironomus for mt genes and Nemapalpus or Chironomus for the nuclear gene) and two ingroups (Ceratis or Drosophila).

When performing the RRT, we observed differences in branch lengths when using the different out-groups, with Aedes and Nemapalpus generally having a short branch lengths and Chironomus a very long branch length. Nevertheless, even when Aedes or Nemapalpus were used as the out-groups (i.e., when differences are more likely to be detected), very few RRTs indicated significantly different rates. For example, although we detected some differences for the 16S gene (table 6 , part A), these differences were not restricted to parasitic groups. For all other genes, using either Aedes, Nemapalpus, or Chironomus as out-groups, only isolated taxa showed significant rate heterogeneity, but generally inconsistently so (table 6 , parts A–D). A notable exception to this was Ogcodes, which had increased molecular rates compared with the nonparasitic ingroup in all comparisons (table 6 ).


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Table 6 Relative Rate Test Calculations Using Different Outgroups and Different Ingroups for Each of the Five Genes Studied

 
The same trends were observed when using the tephritid as the nonparasitic ingroup, although fewer differences were detected in the 16S gene, and more differences were detected in the nad5 gene. Again, these differences were not restricted to parasitic groups (table 6 , parts C and D). Finally, to establish that the trends reported previously for the parasitic Hymenoptera (Dowton and Austin 1995Citation ) would be detected using the same tests as applied here, we repeated these tests using hymenopteran sequences (fig. 1 ). This demonstrated that, in contrast to that observed within the Diptera, the rates of molecular evolution are heterogeneous when comparing parasitic with nonparasitic wasps in both nuclear and mitochondrial genes.



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Fig. 1.—RRT (Tajima 1993Citation ) comparing (A) parasitic and nonparasitic Hymenoptera and (B) parasitic and nonparasitic Diptera

 
Gene Arrangement
Sequence data were obtained from the junction of the cox2 and atp8 genes (which normally contain two tRNA genes for lysine and aspartate) of nine dipteran taxa and from the junction of the nad3 and nad5 genes (which normally contain six tRNA genes for alanine, arginine, aspargine, serine, glutamic acid, and phenylalanine) of 11 dipteran taxa. In each case, the 5' region of the nucleotide and protein sequenced products showed homology to previously sequenced cox2 and nad5 genes, respectively (compared with Drosophila, Locusta, and Apis) (see Supplementary Material). The atp8 primer anneals immediately 5' to the start codon, and all sequences contained a start codon (ATN: Crozier and Crozier 1993Citation ) immediately 3' to the primer sequence. The identified tRNA genes in each case could be folded into cloverleaf structures and exhibited conserved features (fig. 2 Go ). These properties collectively suggest that the sequenced fragments represent bona fide mitochondrial gene fragments, rather than nuclear pseudogenes.



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Fig. 2.—Inferred secondary structure of the two tRNAs situated between the cox2 and atp8 genes and of the six tRNAs between the nad3 and nad5 genes for one of the parasitic flies sampled (Comptosia). tRNAs are labeled with the abbreviations of their corresponding amino acids. Nucleotide sequences are from 5' to 3' as indicated for the first tRNA. Arms of tRNAs (clockwise from top) are the amino acid acceptor (AA) arm, the T {Psi} C (T) arm, the anticodon (AC) arm, and the dihydrourine (DHU or D) arm

 


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Fig. 2 (Continued)

 
All taxa sampled in this study had the same organization of tRNA genes compared with Drosophila yakuba (see fig. 3 Go for an example), which is considered to have the ancestral organization for the Diptera (Flook, Rowell, and Gellissen 1995Citation ). Thus, no differences between the tRNA organization of the parasitic and nonparasitic flies were found (table 7 ). In the Hymenoptera, both these gene junctions show elevated rates of gene rearrangement (Dowton and Austin 1999Citation ; L. R. Castro, A. D. Austin, and M. Dowton, unpublished data) in parasitic wasps but not in nonparasitic wasps.



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Fig. 3.—Sequence of the nad3-ARNSEF-nad5 region for one of the Diptera sampled (Chironomus). The direction of transcription of the gene is shown by arrows; tRNAs are boxed. Corresponding anticodons are indicated by double line boxes. Stop codons are indicated by asterisks

 


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Fig. 3 (Continued)

 

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Table 7 Characteristics of the Junctions Between the cox2 and atp8 Genes and of the Junctions Between nad3 and nad5 Genes for Various Dipteran Taxa

 
Interestingly, Chironomus had a different genome organization between the nad3 and nad5 genes compared with that of two other nematocerans, Aedes and Anopheles. These mosquitoes have a derived RANSEF organization (bold letters denote that the gene is encoded on the minor sense strand) (HsuChen and Dubin 1984Citation ; Beard, Hamm, and Collins 1993Citation ; Mitchell, Cockburn, and Seawright 1993Citation ), whereas Chironomus had the ancestral organization for the Diptera (ARNSEF). Pruess, Zhu, and Powers (1992)Citation also found the ancestral organization (ARNSEF) for the Diptera: Simuliidae (also a member of the Chironomoidea). Our results suggest that the rearrangement observed in the two mosquitoes may be a synapomorphy within the Culicidae or the Culicoidea.


    Conclusions
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Conclusions
 Acknowledgements
 References
 
We found that a departure from compositional stationarity, rate heterogeneity, and genome organization was not generally associated with a range of parasitic Diptera, when compared with nonparasitic Diptera. This contrasts strongly with trends observed within the Hymenoptera, in which each of these genetic phenomena is accelerated in parasitoids compared with nonparasitoids. Our sampling strategy purposefully targeted dipteran parasitoids from a broad cross section of superfamilies to test the notion that parasitism is generally associated with these genetic phenomena. Clearly, it is not.

However, a collateral aim of this study was to examine whether particular variants of the parasitic lifestyle were associated with an increased rate of molecular evolution. This study suggests that ectoparasitism is not one such variant because a range of ectoparasitoids did not display this property. Of the endoparasitic lineages sampled (Acroceridae, Nemestrinidae, and Tachinidae), the acrocerids were the only family to display a significantly elevated rate of genetic divergence. Further, the extent of this increased rate was much less in the acrocerid than generally observed in parasitic Hymenoptera (compare fig. 1A with 1B ).

On the basis of our results, the most likely explanation for the increased rate of molecular evolution in the Hymenoptera is their increased rate of speciation compared with the dipteran parasitic lineages. According to the Nearly Neutral Theory (Ohta 1972Citation , 1993Citation ), if speciation is associated with founder events from populations of small effective size, faster rates of molecular evolution are expected in the relatively speciose Hymenoptera. This explanation predicts that both mitochondrial and nuclear genes will exhibit an elevated rate of divergence in the parasitic Hymenoptera (but not in the parasitic Diptera), a prediction supported by our 28S data. The effective population size has been pointed out as one of the two scenarios that could produce correlated rates across genes from different genomes (Eyre-Walker and Gaut 1997Citation ) and as an explanation for the increased rate of genetic divergence among parasitic lice compared with their hosts (Page et al. 1998Citation ). However, there are also specific differences between hymenopteran and dipteran endoparasitoid biologies (see subsequently) that may also account for the observed differences in molecular rates between these two groups.

Most endoparasitic Diptera avoid the encapsulatory defense response of the host by having a tracheal system in contact with the outside air (Eggleton and Belshaw 1992Citation ). In contrast, most endoparasitoid Hymenoptera live free within the host hemocoel (the larvae have a closed spiracular system which is fluid-filled and becomes air-filled in later instars, but it does not generally open until just before emergence from the host). In the Hymenoptera, the encapsulation response of the host is usually suppressed biochemically (Eggleton and Belshaw 1992Citation ). These differences make it difficult to speculate as to whether it is the increased radiation or aspects specific to hymenopteran endoparasitoid biology that lead to an increase in the rate of molecular evolution. It would be particularly interesting to examine the rates of molecular evolution in representatives of the dipteran pipunculids or rhinophorids because these have more similar endoparasitoid biologies compared with Hymenoptera (their larvae are directly exposed to the host hemocoel).


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Conclusions
 Acknowledgements
 References
 
Thanks to Judy Bellati, Dave Britton, Kris French, John Jennings, Chris Lambkin, and David Yeates for the provision and identification of taxa. Comments from Phillip England and James Bower greatly improved an earlier draft. This work was supported by grants from the Australian Research Council and the Sir Mark Mitchell Foundation. The sequences reported in this paper are deposited in GenBank (accession numbers AF456846AF456879, AF465476AF465483, and AF484024AF484044).


    Footnotes
 
Ross Crozier, Reviewing Editor

Abbreviations: mt, mitochondrial; RRT, relative rate test. Back

Keywords: compositional bias gene rearrangement molecular rates parasitic Diptera point mutations relative rate test Back

Address for correspondence and reprints: L. R. Castro, Institute for Conservation Biology, Department of Biology, The University of Wollongong, Wollongong 2522, New South Wales, Australia. lydaraquelcastro{at}hotmail.com . Back


    References
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Conclusions
 Acknowledgements
 References
 

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Accepted for publication February 25, 2002.





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