Department of Plant and Microbial Biology, University of California at Berkeley
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Abstract |
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Introduction |
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Here, we extended the previous analyses of C. immitis by including isolates of two of the closest relatives, the nonpathogenic saprophytes Auxarthron zuffianum and Uncinocarpus reesii (Bowman and Taylor 1993
; Pan, Sigler, and Cole 1994
; Bowman, White, and Taylor 1996
). Although these two species have also been collected from the lungs of rodents, they seem to be only transient and apparently harmless inhabitants of animals, and their life cycle does not include the production of spherules or endospores, stages which are presumed adaptations for the infective process (Sigler and Carmichael 1976
). Unlike C. immitis, both A. zuffianum and U. reesii have well-identified sexual stages in their life cycles, and a recombinant population structure is therefore expected. We analyzed fragments of three nuclear genes: (1) CHS1, coding for chitin synthase, which is responsible for the synthesis of chitin, a major component of the fungal cell wall (Pan, Sigler, and Cole 1994
); (2) tcrP, coding for a human T-cell reactive protein of C. immitis (this protein is homologous to 4-hydroxy-phenyl-pyruvate dioxygenase (4-HPPD) proteins and mammalian F antigens; Wyckoff et al. 1995)
; and (3) pyrG, coding for orotidine 5'-monophosphate decarboxylase (OMPD), which catalyses a step in pyrimidine biosynthesis (Radford 1993
).
By combining global samples of the three species, we aimed to root the relationships within C. immitis and determine whether the two taxa previously identified were both monophyletic or one was paraphyletic, still segregating ancestral alleles (Neigel and Avise 1986
; Avise and Ball 1990
). A consistent finding that one taxon was paraphyletic across a number of loci would imply a larger population size in that taxon (Hey and Kliman 1993
). Data on the closest relatives would also help establish whether C. immitis as a whole was monophyletic, which would suggest a single origin of pathogenicity. Previous single-isolate analysis of 11 pathogenic and nonpathogenic relatives of C. immitis has indicated that the capacity to infect humans has arisen multiple times throughout the evolutionary history of the Onygenales (Bowman, White, and Taylor 1996
). Second, we wanted to establish whether the finding of two cryptic species in C. immitis was a feature peculiar to it, perhaps due to its pathogenic nature leading to faster evolution and differentiation into several host niches, or whether cryptic species were a general feature of fungal species, including free-living ones. Finally, we wished to determine if adaptation to a new life style, that of being a pathogen, had led to positive Darwinian selection and increased rates of evolution, both for proteins in general and, more specifically, in gene regions known to produce antigenic responses in humans. For this purpose, we analyzed the numbers of silent and expressed changes within and between reproductively isolated taxa. The ratio Q = (D/P)E/(D/P)S of between-taxa divergence (D) to within-taxon polymorphism (P) for expressed and silent substitutions (E and S, respectively) is a well accepted measure for detecting positive, adaptive evolution, with the neutral expectation being that Q = 1 (McDonald and Kreitman 1991
; Charlesworth 1994
; Hudson 1996
). Positive selection, on the other hand, would lead to an excess of expressed between-taxa divergence (Q > 1) due to rapid fixation of favorable amino acid changes in different taxa, resulting from diversification and adaptation to new environments. A number of surface antigens of parasites or viruses have been shown to be positively selected, evolving rapidly by selective fixation of amino acids thought to be critical in the host-parasite interaction (Sphaer and Mullins 1993
; Endo, Ikeo, and Gojobori 1996
). We therefore expected the dioxygenase fragment containing the T-cell reactive site to be fast-evolving and under positive selection in C. immitis.
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Materials and Methods |
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DNA sequences were aligned manually, and maximum-parsimony genealogies were constructed for each gene separately and for the three genes combined. Observed tree lengths were then compared with the minimum possible lengths in order to identify possible homoplasies within trees and incompatibilities between trees, following the analyses on the C. immitis data (Koufopanou, Burt, and Taylor 1997
). Then, the three species were also analyzed together and gene genealogies were compared to examine the consistency of roots and of branching order of isolates. Finally, all data were analyzed in combination (phylogenetic analyses were performed using PAUP*, version 4.0; Swofford 1999
).
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Results and Discussion |
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In short, three putative reproductively isolated groups in A. zuffianum were indicated by high genetic divergence and shared branches among three gene genealogies: group I, represented here by three isolates, and groups II and III, represented by only one isolate each (AG and UK, respectively). Group III was consistently the most divergent in the species. Mixis, on the other hand was implied by incompatible branches of isolates in group I, in accordance with the presence of a sexual state in the life cycle.
Uncinocarpus reesii
Of 1,273 nucleotides sequenced in the three genes in U. reesii, 27 were polymorphic, a rate of 2.1% (table 2
and fig. 2
). The two introns in dioxygenase were 58 and 60 bp long, with 8.5% of sites being polymorphic. Here, too, 17 of the polymorphic sites were unique to a single isolate (AUS), and if this was removed, polymorphism dropped to 0.8%. Of the 10 isolates, only six had different genotypes. All identical genotypes were reconfirmed by independent DNA extractions, PCR, and DNA sequencing from material newly derived from the UAMH culture collection. Identical genotypes were merged for all of the analyses.
For chitin synthase and dioxygenase, trees have minimal lengths, equal to the number of polymorphic sites in each locus (most-parsimonious tree lengths: CHS1L = 2 steps, 4-HPPDL =17 steps; table 2 ; U. reesii branches in fig. 1 ). For orotidine decarboxylase, there were two equally parsimonious trees, differing in the placement of the AG and AUS isolates, both being two steps longer than the minimum length; this homoplasy was removed when the AUS isolate was excluded, but not when any other U. reesii isolate was excluded, suggestive of multiple hits along branches to this isolate (for OMPD, L = 10 steps; excluding AUS, L = 6 steps = minimum). To find the potentially homoplastic sites, we reincluded the AUS isolate and successively removed sites, first one and then two at a time. Minimal tree length was only obtained when both site 75 and site 240 (see table 2 and fig. 2 ) were excluded from the analysis. As before, the compatibility of the three genealogies was tested by excluding the AUS isolate and combining the data for the three loci. The most-parsimonious tree from the combined data was one step longer than minimum (11 vs. 10 steps), suggesting incompatible genealogies. By successively excluding each of the three loci, we located the incompatibility between the dioxygenase and orotidine decarboxylase loci in branches separating isolates HG, IT1, IT2, IT3, CA1, and AG (i.e., within group II; fig. 1 ). However, the homoplasy in the combined data also was removed when the AUS isolate was reincluded and the two homoplastic sites of the orotidine decarboxylase locus were excluded, implying no additional incompatibility among loci. The branching order in the dioxygenase tree and the extensive intron divergence of the AUS isolate suggest that this is the most widely divergent of the species. Although consistent with the chitin synthase genealogy, this is not consistent with the orotidine decarboxylase one, which shows the AUS isolate together with group II isolates. There is only one common branch in the three genealogies, separating the TX, UT, and CA2 (group I) isolates from the rest (fig. 1 ).
To summarize, in U. reesii the three gene genealogies share a common branch separating group I isolates from the rest, indicating complete sorting of alleles and reproductive isolation between them. Also, there is incompatibility in the placement of group II isolates, implying mixis among these isolates, again as predicted from the life cycle. Finally, the dioxygenase locus alone indicates that the AUS isolate is the most widely divergent of the species, and although this is consistent with the chitin synthase genealogy, it is inconsistent with the orotidine decarboxylase one, which shows the AUS isolate clustering together with group II isolates. The possibility of uncovering conflicting gene genealogies in taxa that were separated a long time in the past but whose speciation has occurred close in time has already been discussed (Avise 1994
). We conclude that there are three potentially noninterbreeding taxa in U. reesii, one of which is represented by a single isolate (AUS).
The inferred phylogenetic history of the three species is depicted in figure 3
. Note that this is a "species" tree rather than a simple gene genealogy, in that it combines information on both divergence and compatibility of three genealogies, and the terminal taxa constitute putatively noninterbreeding units. The tree was rooted with Auxarthron as a monophyletic outgroup, as indicated in 18S rRNA and chitin synthase phylogenies (Pan, Sigler, and Cole 1994
; Bowman, White, and Taylor 1996
). Despite the extensive subdivision at the tips, each of the three named species represents a deep monophyletic clade, indicating early divergence and supporting the current generic classification. The maximum-likelihood analysis (see fig. 3
for parameters) gave no significant deviation from a "molecular clock," implying that the parasitic C. immitis and the nonparasitic U. reesii evolve at similar rates, and providing no indication of accelerated evolution in C. immitis due to adaptation to a novel pathogenic habit. The amount of time the Californian and non-Californian taxa have been reproductively isolated was previously estimated to be 11 Myr based on five gene fragments, assuming a substitution rate at third-base positions of u = 10-9/bp/yr (Koufopanou, Burt, and Taylor 1997, 1998
). For the three fragments analyzed here, this estimate is 14 Myr, and the present tree implies that the UK isolate of Auxarthron may have been isolated from the others for as long as 28 Myr.
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The coalescence time for U. reesii and C. immitis is more than 20 times as long as that of taxa within species complexes (fig. 3 ). Unless some exceptional circumstances have spurred the recent speciation, the long internal branches indicate that taxa at the tips are rather transient over evolutionary time, soon to go extinct and be replaced by others along the same main lineages. In other words, taxa are continuously being generated and going extinct along the named branches, which may represent some basic environmental niche and/or organismal structure or habit, and it is unlikely for more than one taxon from each clade to survive and diverge over long evolutionary periods, a phenomenon recognized by Williams (1992, p. 132) as "normalizing clade selection." Alternatively, the long internal branches may have resulted from poor sampling of the existing fungal diversity, with the implication that more taxa remain to be found. Although U. reesii is still the closest known relative of C. immitis, recently published 18S sequences suggest that Onygena equina and Ascocalvatia alveolata may be closer relatives of C. immitis than A. zuffianum (Landvik, Shailer, and Eriksson 1996
; Sugiyama, Ohara, and Mikawa 1999
). Nevertheless, our data suggest that the discovery of new cryptic subdivisions is likely to be within the existing framework of generic classification. No similar outgroup analysis is provided in the other studies that have discovered cryptic species, so it is difficult to assess the validity of this hypothesis for fungi in general.
In contrast to C. immitis, both A. zuffianum and U. reesii are known to have sexual states, and mating tests could be performed to test the mating compatibilities of strains. No such data exist for A. zuffianum, but limited data on U. reesii (numbers of gymnothecia obtained from a cross; table IV in Sigler and Carmichael 1976
) are consistent with our findings: the most divergent AUS isolate will not mate with either UT, CA1, or HG of groups I and II (but will mate with other strains not included in this study). Isolates IT1 and IT2 from group II will mate readily with CA1 and HG strains from the same group. The IT1 isolate (but not IT2), however, will also mate with the UT strain of group I. As no information is given on the fertility of ascospores in the gymnothecia, it is difficult to evaluate from these data the extent of the correlation between mating incompatibility and genetic divergence of strains. A good correlation has been found between mating incompatibility and sequence divergence at a single locus (nuclear ITS) for isolates from various geographic locations in the colonial green algae Pandorina morum and Gonium pectorale (Coleman, Suarez, and Goff 1994
).
Molecular Evolution
The above analysis identified eight phylogenetically distinct taxa in the three species, three of which are represented by only one isolate. For clarity of presentation, we shall refer to the original species as "species complexes." To examine the evolution of the three loci, we calculated the numbers of silent and expressed nucleotide changes within and between the eight taxa (table 3
). Changes among isolates within taxa were counted as polymorphisms (P), while changes among taxa, i.e., fixed within taxa, were counted as divergences (D). For the three taxa with only one representative, i.e., taxa II and III of A. zuffianum and taxon III of U. reesii, we assumed that all of the observed divergence was fixed within these taxa; in other words, sites that are contributing to the divergence of these isolates will not be found to be polymorphic once more isolates from these taxa have been analyzed. For expressed changes, the minimum number of nucleotide changes needed to convert one amino acid to another was calculated according to the Protpars matrix in PAUP* version 4.0 (Swofford 1999
).
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To test whether the three loci were fixing amino acids in different taxa faster or slower than would be predicted by the amounts of standing polymorphism within the taxa, we calculated for each locus the ratio D/P of divergence over polymorphism for silent and expressed substitutions, respectively, from the pooled polymorphism and within-complex divergence (table 3
). The ratio Q = (D/P)E/(D/P)S is equal to one if loci are evolving according to the neutral hypothesis (McDonald and Kreitman 1991
; Charlesworth 1994
). In all three loci, Q values are <1, indicating negative, purifying selection rather than positive selection, but the deviations from unity are not significant (table 3
). As there is no significant heterogeneity among loci in the divergence-to-polymorphism ratios (G = 0.15 and G = 0.44 for expressed and silent changes, respectively, NS), we were able to combine the data from the three loci, but the deviation was still nonsignificant. The Q ratios remained <1 and nonsignificant if the uncorrected between-complexes divergence values were included in the estimations. As silent sites are likely to be more saturated than replacement sites, correcting the between-complexes divergence values for saturation would tend to decrease the Q values and hence increase the deviation from unity.
Contrary to expectation, we found no positive selection or increased amino acid polymorphism in the 200-bp-long stretch of the T-cell reactive site of dioxygenase in C. immitis (fig. 2
), implying no strong selection for diversification of the pathogen in this region. Indeed, an alignment of six dioxygenase proteins from a variety of organisms, including the human, mouse, rat, and the protist Tetrahymena, indicates that this stretch is among the most well conserved in the entire molecule (Wyckoff et al. 1995
). Somewhat higher rates of expressed polymorphism were found in serine proteinase and chitinase gene fragments, which are also thought to be involved in the antigenic reaction of the host against this pathogen (Koufopanou, Burt, and Taylor 1997, 1998
). These fragments were not included in this study, as we were not able to amplify them in A. zuffianum or U. reesii using primers designed from the C. immitis sequence. Also surprisingly, there was very low polymorphism in the dioxygenase introns: there were only two variable sites within group I of A. zuffianum, and in U. reesii, there was no intron polymorphism within either of the two taxa (table 3
), although there was considerable intron divergence among taxa in these two species complexes. In C. immitis, only a single variable site was found previously among 317 intron sites from the dioxygenase, serine proteinase and chitinase loci, a rate of 0.3%, compared with 0.7% for third codon sites polymorphic within the two taxa (Koufopanou, Burt, and Taylor 1997, 1998
). Although low in polymorphism, introns of different species complexes are so divergent, with a number of insertions/deletions, that they cannot be aligned unambiguously. Such disparity in the polymorphism and divergence of apparently neutral sites might suggest very quick fixation of mutations arising in these populations.
In short, we did not detect any significant deviation of amino acid divergence from the neutral expectation, as measured by the Q ratio of expressed and silent D/P values, providing no evidence for positive Darwinian selection by rapid fixation of favorable amino acids. Instead, the ratio was less than unity, suggestive of stasis by elimination of mostly deleterious alleles. Slow evolution and amino acid conservation were also indicated by the low values of expressed divergence alone, relative to total divergence. Although powerful in providing an intuitive null hypothesis for the detection of deviations, these tests are weak in that if positive and negative selection are acting on different sites or regions of the same gene, they would tend to cancel each other out, with the outcome possibly appearing as neutral evolution. Recent reviews have indicated that different regions, or even sites, are very likely to be under different selective pressures (Ngai et al. 1993
; Golding and Dean 1998
), thus making it very difficult to detect any particular type of selection when summing across sites. Furthermore, the Q ratio is very sensitive to small changes in the numbers of silent and expressed polymorphisms within populations, which tend to be small, especially when only potentially interbreeding populations are considered. Here, a stretch of 1,155 bp sequenced in a total of 25 isolates from 5 taxa and 3 loci was able to generate only a small number of polymorphic sites and an even smaller number of amino acid polymorphisms. A much larger sample size would thus be required to remove this sensitivity. No extreme heterogeneity was found among regions of the same fragment in our data, including the T-cell reactive site of dioxygenase.
Nevertheless, we did find significant heterogeneity in the rates of evolution of the three loci considered here. From comparisons across species, it is now obvious that proteins evolve at different rates, indicating differences in selective pressures among different molecules. Many proteins from distant organisms are greatly conserved, implying strong negative selection. On the other hand, surface antigens of parasites or viruses tend to be under strong positive selection more frequently than other genes (Endo, Ikeo, and Gojobori 1996
). Fewer studies have scaled the divergence among species to the polymorphism within species (reviewed by Brookfield and Sharp 1994
), and again the results have been mixed, with examples of positive (McDonald and Kreitman 1991
; Eanes, Kirchner, and Yoon 1993
; Kliman and Hey 1993
), neutral (Kliman and Hey 1993
), and negative evolution (Nachman, Boyer, and Aquadro 1994
). The heterogeneity of findings probably reflects a true heterogeneity in the mode of evolution of different genes and populations.
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Conclusions |
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Adaptation to the pathogenic lifestyle in C. immitis does not seem to have been accompanied by accelerated evolution or positive Darwinian selection in the three protein fragments analyzed, one of which included a region known to produce antigenic responses in humans. Instead, our data suggest relative stasis by the action of negative, purifying selection against mostly deleterious amino acid changes.
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Acknowledgements |
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Footnotes |
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1 Present address: Department of Biology, Imperial College at Silwood Park, Ascot, Berks, United Kingdom.
2 Keywords: gene genealogies
concordance
outgroups
cryptic species
molecular evolution
human pathogen
Coccidioides immitis
Auxarthron zuffianum
Uncinocarpus reesii
Ascomycota
Onygenales
3 Address for correspondence and reprints: Vassiliki Koufopanou, Department of Biology, Imperial College at Silwood Park, Ascot, Berks SL5 7PY, United Kingdom. E-mail: v.koufopanou{at}ic.ac.uk
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