*Institute for Conservation Biology, Department of Biology, The University of Wollongong, New South Wales;
Centre for Evolutionary Biology and Biodiversity, Department of Environmental Biology The University of Adelaide, Glen Osmond, South Australia
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Abstract |
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Introduction |
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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 proteinencoding genes in bacteria, vertebrates, and insects ranges from 4% to 98% (Bernardi et al. 1985
; Ikemura 1985
; Jukes and Bhushan 1986
; Sueoka 1988
; Liu and Beckenbach 1992
). 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 1989
). Variation in compositional bias among taxa is known as deviation from stationarity (Collins, Wimberger, and Naylor 1994
).
Substitutional Rate Heterogeneity
Rates of nucleotide substitution are known to vary widely among phylogenetic groups (Wu and Li 1985
; Britten 1986
; Kocher et al. 1989
; Martin, Naylor, and Palumbi 1992
; Adachi, Cao, and Hasegawa 1993
; Martin and Palumbi 1993
; Cantatore et al. 1994
; Martin 1995
; Mindell and Thacker 1996
; Nunn and Stanley 1998
). 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 1965
]) 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 1986
; Adachi, Cao, and Hasegawa 1993
; Mindell and Thacker 1996
).
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)
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 1970
)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 1979
). (3) The metabolic rate hypothesis (Martin and Palumbi 1993
)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 hypothesispopulations with small effective sizes will experience faster rates of evolution than populations with larger effective sizes (Ohta 1972
, 1992
) 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. 1995
; Kumazawa and Nishida 1995
; Dowton 1999
), 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. 2001b
).
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)
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)
and Page et al. (1998)
in parasitic lice and by Nickrent and Starr (1993)
in parasitic plants. Possible causes for this correlation are an increased rate of speciation (Page et al. 1998
), increased flux of mutagens (Dowton and Campbell 2001
), 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 1992
). Consistent with this scenario, Dowton and Austin (1997)
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)
. 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 2001a
). 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 1992
; Beard, Hamm, and Collins 1993
). Finally, Dowton and Austin (1999)
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.
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Materials and Methods |
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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. 1.40
) 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 1994
), as distributed with the BIOEDIT program (Hall 1999
). 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. 2001
) for calculating the nucleotide composition and performing the Tajima's (1993)
relative rate test (RRT). To test for taxon-specific differences in DNA composition, the program STATIO (Rzhetsky and Nei 1995
) was used. With respect to the RRT, as pointed out by Wu and Li (1985)
, 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 1997
). 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)
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)
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 1997
). 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.
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Results and Discussion |
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Nucleotide CompositionDiptera
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 1985
; Beard, Hamm, and Collins 1993
; Crozier and Crozier 1993
; Mitchell, Cockburn, and Seawright 1993
). In Drosophila there is no clear distinction between the nucleotide composition of genes transcribed from the two complementary strands (Clary and Wolstenholme 1985
); 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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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 1967
; Wu and Li 1985
; Tajima 1993
). 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)
, 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 AD). 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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Conclusions |
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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 1972
, 1993
), 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 1997
) and as an explanation for the increased rate of genetic divergence among parasitic lice compared with their hosts (Page et al. 1998
). 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 1992
). 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 1992
). 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).
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Acknowledgements |
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Footnotes |
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Abbreviations: mt, mitochondrial; RRT, relative rate test.
Keywords: compositional bias
gene rearrangement
molecular rates
parasitic Diptera
point mutations
relative rate test
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
.
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