1 Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, 231 Albert Sabin Way, Cincinnati, OH 45267-0524, USA
2 Department of Internal Medicine, Division of Infectious Diseases, University of Cincinnati College of Medicine, 231 Albert Sabin Way, Cincinnati, OH 45267-0524, USA
Correspondence
Scott P. Keely
scott.keely{at}uc.edu
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the 18SITS15·8SITS2 rDNA sequence reported in this article is AY532651.
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INTRODUCTION |
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P. murina infections in mice serve as the principal model for the study of the immune response. Given its importance as a model of human disease, it is important to clarify the relationship between P. murina and other members of the genus. The goal of the work described herein was to better assess the genetic distinctiveness of P. murina by systematic comparison of its DNA to that of other Pneumocystis species. To enhance the power and accuracy of this comparison, the sequences of the genes encoding the 18S and 5·8S rRNAs were determined along with the internal transcribed spacer (ITS) regions adjacent to these two genes. These and other sequences were used to assess phylogenetic relatedness, concordance of divergence at different loci and time of genealogical divergence. Analyses of the sequence comparisons employed tools that were not available at the time that the other three Pneumocystis species were described. Hence, these studies provide both a foundation for understanding the status of P. murina as a possible species and a more-sophisticated view of the relationships between the previously described species in the genus.
Naming new species can be controversial because there are many ways to conceive of a species (Hey, 2001; Keely et al., 2003b
; Mayden, 1997
). The phylogenetic species concept is well suited to the task of erecting an evolutionarily based taxonomic structure for the genus Pneumocystis. A phylogenetic species is an evolutionary lineage having a unique combination of DNA sequences (Taylor et al., 2000
). Such species can be elucidated by inferences from a bifurcating gene tree, which is usually constructed from a pairwise distance matrix derived from an alignment of orthologous sequences. The distance matrix is produced by determining the best substitution model to fit the data (Posada & Crandall, 1998
). The topology of the tree can be evaluated by a test of reliability, such as the common bootstrap statistic (Felsenstein, 1985
). Additional tests of statistical significance of tree topology have been developed (Strimmer & Rambaut, 2002
). The probability of obtaining a correct species tree can be increased by considering multiple genes at the same time. Analysis of multiple genes also allows application of the genealogical concordance phylogenetic species recognition method, which can detect the occurrence of genetic exchange. If the variation under analysis is occurring within a sexual species, then different genes will tend to produce different trees due to independent assortment of alleles during sexual reproduction (Taylor et al., 2000
). By contrast, the absence of gene flow, which predominates when organisms are different species, will tend to give concordant tree topologies due to the fixation of alleles after genetic isolation.
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METHODS |
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Electrophoretic karyotype analysis.
Pneumocystis chromosomes were prepared for electrophoretic karyotype analysis as described previously (Cushion et al., 1993a, b
). Briefly, Pneumocystis organisms were treated with DNase I (Boehringer Mannheim) at 10 µg ml1 in a solution of 150 mM NaCl/10 mM MgCl2/10 mM Tris at pH 7·2 for 30 min at 37 °C to digest extracellular DNA. To inactivate the DNase, magnesium ions were removed by washing once with 250 mM EDTA and twice with 125 mM EDTA. Organisms were embedded in 0·8 % low-melting-point agarose (Boehringer Mannheim) and treated with 0·25 mg proteinase K ml1 (Boehringer Mannheim) in a solution of 1 % N-lauroylsarcosine (Sigma Chemical)/0·45 M EDTA/0·01 M Tris at 55 °C for 2448 h. Gels for contour clamped homogeneous electrical field (CHEF) electrophoresis contained 1 % FMC SeaKem GTG-agarose (SeaKem) prepared in 0·5xTBE (45 mM Tris/HCl/45 mM boric acid/1·25 mM EDTA) for a total volume of 200 ml and final dimensions of 14x21 cm. Electrophoresis was performed using either a Bio-Rad CHEF DR II or a Bio-Rad CHEF DR III apparatus. Gels were run for 104144 h, at 14 °C, in 0·5xTBE at 3·8 V cm1 with a 50 s initial pulse that was gradually increased to 100 s. Agarose gels were stained with SYBR-Gold (Molecular Probes), illuminated by UV light and photographed. The molecular masses of the chromosomes were determined by linear regression using molecular mass standards.
PCR amplification.
An aliquot of lung homogenate (1 ml) was treated with proteinase K, and genomic DNA was isolated by 2-propanol extraction, as described previously (Keely et al., 2003a). The DNA was dissolved in 0·01 M Tris/0·001 M EDTA, pH 8, and diluted 1 : 20 in sterile water. One microlitre of the DNA dilution was subjected to PCR. Primer sequences are shown in Table 1
. The positions of the primers are shown in Fig. 1
. The gene encoding 18S rRNA was amplified with primers p1 (primer A) (Shah et al., 1996
) and p2 (primer B) (Shah et al., 1996
) without the restriction sites. ITS1, 5·8S rRNA and ITS2 were amplified with primers p3 and p4 based on previous reports (Edman et al., 1988
; Stringer et al., 1989
). PCR was performed under the following conditions: 94 °C hot start for 2 min, 35 cycles of incubation at 94 °C for 20 s, 55 °C for 15 s and 72 °C for 120 s, and 1 cycle of 72 °C extension for 5 min. A portion of the gene encoding the large-subunit mitochondrial rRNA [mtrRNA(LSU)] was amplified with primers PAZ102-H and PAZ102-E (Wakefield et al., 1990
) under the following conditions: 95 °C hot start for 5 min, 30 cycles of incubation at 95 °C for 60 s, 50 °C for 120 s and 72 °C for 60 s, and 1 cycle of 72 °C extension for 5 min. Reaction volumes were 25 µl containing 50 µM each of dATP, dCTP, dGTP, dTTP and 2·5 mM MgCl2 and 100 ng of each primer. Triple Master polymerase (1·5 U) (Eppendorf) and High Fidelity Buffer were utilized for the amplification of 18SITS15·8SITS2. Taq polymerase (1 U) (Promega) was utilized for the amplification of the mtrRNA(LSU) locus.
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Sequences compared in this study.
Portions of the following genes were aligned: manganese-cofactored superoxide dismutase (SODA), accession nos Z79785, AF146751, AF146753, AF146754 and AF146752; dihydropteroate synthase (DHPS), accession nos M86602, U66283, AF139132, AY070270 and AF322064; dihydrofolate reductase (DHFR), accession nos AF322061, AF175561, AF090368, AY017418 and AF186097; mtrRNA(LSU), accession nos U20169, S42926, AF461783 and S42915, and two P. murina sequences, AF257179 and Peters et al. (1994); small-subunit mitochondrial rRNA [mtrRNA(SSU)] (Hunter & Wakefield, 1996
); ITS15·8S rRNAITS2 (Hsueh et al., 2001
; Ortiz-Rivera et al., 1995
; Nimri et al., 2002
), accession nos L27658 and AY328078; 18S rRNA, accession nos X12708 and L27658, Pneumocystis f. sp. mustalae (Shah et al., 1996
), P. jirovecii (Shah et al., 1996
); thymidylate synthase, accession no. S77510, P. jirovecii (Mazars et al., 1995
) and P. murina (Mazars et al., 1995
); 5-enolpyruvylshikimate-3-phosphate synthase (AROM), P. jirovecii (Banerji et al., 1995
), P. murina (Banerji et al., 1995
) and P. carinii (Banerji et al., 1995
); AB000948 (Taphrina carnea), AB000960 (Taphrina virginica), AB000958 (Taphrina robinsoniana), AB000955 (Taphrina nana), D12531 (Taphrina wiesneri), AB000949 (Taphrina communis), AB000959 (Taphrina ulmi), Z75578 [Saccharomyces (Sac.) cerevisiae]; AY046227 (Saccharomyces bayanus); AB040998 (Saccharomyces mikatae); AB040997 (Saccharomyces kudriavzevii); Z75580 (Saccharomyces kluyveri); X97806 (Saccharomyces paradoxus); Z75577 (Saccharomyces castellii). Outgroups and other fungi utilized for the rDNA alignment: Oryza sativa, AF069218; Candida albicans, E15168; Schizosaccharomyces (Sch.) pombe, X58056.
Phylogenetic tree construction.
Sequences were aligned using DNAMAN software using the default settings. The alignments were optimized by introducing a limited number of gaps, which were not counted in relatedness calculations. In addition, ambiguous regions in the alignment were not scored. The DNA alignments contained 939, 815, 600, 229, 219, 332, 327 and 1800 nt for SODA, DHPS, DHFR, mtrRNA(LSU), mtrRNA(SSU), thymidylate synthase, AROM and 18S rDNA, respectively. For supertree analysis of five Pneumocystis taxa, SODA, DHPS, DHFR, mtrRNA(LSU) and mtrRNA(SSU) sequences were concatenated to form a sequence 2800 nt long.
Relatedness of pairs of aligned sequences for each individual gene and the concatenated sequences were calculated by MEGA 2.1 software utilizing p-distance and pairwise deletion (Kumar et al., 2001). p-Distance is the proportion of nucleotide sites at which the two sequences compared are different. It is obtained by dividing the number of nucleotide differences by the total number of nucleotides compared. Neighbour-joining (NJ) trees were constructed with MEGA 2.1 (Kumar et al., 2001
) and 1000 bootstrap replications were performed for each tree.
The p-distance nucleotide substitution model may not be optimal for phylogenetic analysis of Pneumocystis genes. Thus, optimal models were estimated for SODA, DHPS, DHFR, mtrRNA(LSU), mtrRNA(SSU), 18S rDNA and concatenated alignments by maximum-likelihood analysis utilizing the hierarchical likelihood ratio test implemented in MODELTEST 3.6 (Posada & Crandall, 1998) for PAUP* 4.0b10 (Swofford, 1998
). Each model was utilized to construct a phylogenetic tree by PAUP* 4.0b10 (Swofford, 1998
) and TREEPUZZLE version 5.1 (Strimmer & von Haeseler, 1996
). One-thousand bootstrap replications were performed and the bootstrap values for internal branches were recorded.
Different models and parameters were proposed by the hierarchical likelihood ratio test in MODELTEST (Posada & Crandall, 1998). The Hasegawa, Kishino and Yano (HKY) model (Hasegawa et al., 1985
) was optimal for mtrRNA(LSU), mtrRNA(SSU), DHPS and DHFR. This model assumes that transitions and transversions each have different rates of change and assumes that the base composition frequencies are unequal. The estimated base composition frequencies for mtrRNA(LSU) were: A, 0·3293; C, 0·1111; G, 0·2368; T, 0·3228. In addition, a transition/transversion (Ti/tv) ratio of 1·4392 was estimated. For mtrRNA(SSU), the estimated base composition frequencies were: A, 0·3491; C, 0·1016; G, 0·2109; T, 0·3384; and the Ti/tv ratio was 1·6467. A gamma shape parameter of 0·6877 was estimated to allow for a broad distribution of rates among nucleotide sites (this model is referred to as HKY+G). HKY+G was also optimal for DHPS and DHFR. The estimated base composition frequencies for DHPS were: A, 0·3288; C, 0·1264; G, 0·1914; T, 0·3534; the Ti/tv ratio was 5·5724, and the gamma shape parameter was 0·2445. The estimated base composition frequencies for DHFR were: A, 0·3073; C, 0·1405; G, 0·2146; T, 0·3376; the Ti/tv ratio was 2·0145, and the gamma shape parameter was 0·7177. Interestingly, HKY+G was determined to be optimal for these mitochondrial genes and the DHPS gene from Pneumocystis derived from Old World and New World monkeys (Hugot et al., 2003
).
The general time reversible (GTR) model (Rodriguez et al., 1990) was determined to be optimal for the SODA, 18S rDNA and concatenated alignments. This model is more complex than HKY because it estimates six nucleotide substitution rates (rAC, rAG, rAT, rCG, rCT and rGT) and assumes unequal base composition frequencies. Interestingly, it was shown recently to be the best model for a combined alignment of three Pneumocystis genes derived from Old World and New World monkeys (Hugot et al., 2003
).
For the SODA locus, the GTR model with a proportion of invariable sites fitted the data the best (referred to as GTR+I). The proportion of invariable sites was 0·4094; the estimated base composition frequencies were: A, 0·3470; C, 0·0957; G, 0·1455; T, 0·4118; and the rate matrix was: A-C, 1·0000; A-G, 7·7290; A-T, 1·8147; C-G, 1·8147; C-T, 7·7290; G-T, 1·0000. GTR, with a gamma shape parameter and a proportion of invariable sites (referred to as GTR+G+I), was optimal for the 18S rDNA sequence alignment. The estimated base composition frequencies were: A, 0·2591; C, 0·2073; G, 0·2622; T, 0·2714; the gamma shape parameter was 0·6220, the proportion of invariable sites was 0·3987, and the substitution rate matrix was: A-C, 1·0000; A-G, 2·6109; A-T, 1·0000; C-G, 1·0000; C-T, 4·5713; G-T, 1·0000. Similarly, the GTR model of substitution with a gamma shape parameter (GTR+G) was optimal for the concatenated sequence alignment. Estimated base composition frequencies were: A, 0·3141; C, 0·1409; G, 0·1942; T, 0·3508. The gamma shape parameter was 0·7925 and the rate matrix was: A-C, 1·0000; A-G, 5·9787; A-T, 1·6157; C-G, 1·6157; C-T, 5·9787; G-T, 1·0000.
Tree concordance analysis.
COMPONENT software (version 2.0, R. D. M. Page; http://taxonomy.zoology.gla.ac.uk/rod/cpw.html) was utilized to determine the total possible topologies for the set of taxa. The tree topology and branch lengths that have the greatest likelihood of generating the data were determined (Strimmer & von Haeseler, 1996). Gene tree topologies were evaluated by the one-sided and two-sided KishinoHasegawa methods implemented in TREEPUZZLE version 5.1 (Strimmer & von Haeseler, 1996
). Log-likelihood, likelihood ratios and standard errors (SE) were calculated for each gene alignment and set of trees. Confidence sets were defined by the upper and lower bounds of an approximate 95 % confidence interval (1·96xSE and +1·96xSE).
Phylodating speciation.
18S rRNA gene NJ trees were constructed from distance matrices produced by GTR+G+I (see above for parameter values) and Kimura's two-parameter (K2P) (Kimura, 1980) nucleotide substitution models. K2P is a special case of GTR: it assumes transitions have one rate and transversions have another; it also assumes nucleotide base frequencies are equal (i.e.
A=
C=
G=
T) (Kimura, 1980
). NJ trees were calibrated with an evolutionary rate of 1·26x1010 substitutions per site per lineage per year, which is based on the fungal fossil record (Berbee & Taylor, 2001
). Times of divergence were determined for each node, as described previously (Nei et al., 2001
). ITS15·8S rRNAITS2 divergence times were estimated as described previously (Kasuga et al., 2002
). Poisson correction distances were determined for DHFR as described previously (Nei et al., 2001
). Poisson correction corrects for multiple hits and assumes (1) equality of substitution rates among sites and (2) equal amino acid frequencies. These distances were utilized to construct a NJ tree, which was linearized and calibrated using 140 million years as the time of divergence of C. albicans and Sac. cerevisiae (Berbee & Taylor, 2001
).
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RESULTS AND DISCUSSION |
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P. murina isolated from SCID and wild mice are genetically similar
It was shown previously that multilocus enzyme electrophoresis (MLEE) is useful for the investigation of species-level genetic diversity of Pneumocystis organisms (Mazars et al., 1997). In this study, MLEE analysis was performed for Pneumocystis organisms isolated from 22 weaning rabbits, 30 rats and 17 mice [13 BALB/c/U42 (BU) hybrid white mice and four outbred U42 white mice]. It was shown that three distinct populations occurred in rats, but only one was found in mice and one in rabbits (Mazars et al., 1997
). This suggested that organisms isolated from mice and rabbits have very little or no genetic diversity.
To further investigate the genetic diversity in mice, we amplified and sequenced a portion of the mtrRNA(LSU) locus from a wild mouse putatively infected with P. murina. A comparison of 210 nt of this sequence (wild-type) and the homologous region from a SCID mouse (Peters et al., 1994) indicated that there was one G
A substitution and four one-nucleotide indels between them. A second comparison between the wild-type sequence and another P. murina sequence (GenBank accession no. AF257179) showed that there were one T
C substitution, two G
A substitutions and three one-nucleotide indels. Thus, the wild-type sequence was >98 % identical to those from two laboratory mice. This high level of similarity is indicative of strain-level but not species-level variation (Stringer, 1996
). MLEE and sequence analysis taken together suggest that one species propagates in mice. However, these data do not exclude the possibility of discovering additional Pneumocystis species in mice.
Analysis of the 18S rRNA locus of P. murina
The 18S rRNA gene of P. murina contained 2179 nt and a composition of 27·1 % A, 28 % T, 19·3 % C and 25·6 % G. These percentages are the same as those in the three other Pneumocystis species. The region spanning nucleotides 17742165 is occupied by a 391 bp group I intron. The composition of the intron is slightly different from the coding region: 29·9 % A, 27·0 % T, 17·6 % C and 25·4 % G. The intron is located at the same site as it is in P. carinii (Liu & Leibowitz, 1993). It is 89·4 and 78·8 % identical to the introns in P. carinii (Liu & Leibowitz, 1993
) and ferret Pneumocystis, respectively. Interestingly, the intron is not present in the 18S rRNA gene of P. wakefieldiae and P. jirovecii (Cushion et al., 1993b
; Liu et al., 1992
; Ortiz-Rivera et al., 1995
). The variability with respect to the presence of this intron shows that P. murina is different from P. wakefieldiae and P. jirovecii. However, the intron information is difficult to use as an index of degree of divergence because the frequencies of intron insertion and deletion are not known.
Table 2 shows that the coding region of the P. murina 18S rRNA gene was most similar to 18S rRNA genes in P. carinii (16 nucleotide substitutions between the sequences) and P. wakefieldiae (22 substitutions). By contrast, the mean number of nucleotide substitutions for all Pneumocystis was 38±4·33 SE (range, 1651). Thus, the P. murina 18S rRNA gene is most closely related to its orthologues in the two Pneumocystis species found in rats. At 38, the average number of nucleotide substitutions for all Pneumocystis is substantially greater than the mean for Taphrina (23·6±2·98 nucleotide substitutions per gene, range 1038) and several times greater than Saccharomyces (7·2±1·55 nucleotide substitutions per gene, range 018). Similarly, the number of substitutions between the three Pneumocystis types found in rodents is comparable or greater to that exhibited by half of the Taphrina species and most of the Saccharomyces species.
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To explore the reproducibility of these relationships, additional phylogenetic trees were constructed. An NJ tree was derived from the entire 18S rRNA locus sequence and consisted of the same five Pneumocystis taxa shown in Fig. 3(A) and many additional Ascomycota representatives, such as 15 euascomycete species (e.g. Neurospora crassa) and four hemiascomycete species (e.g. C. albicans and Sac. cerevisiae), as described in Berbee & Taylor (2001)
. Another NJ tree was derived from a portion of the thymidylate synthase gene and included the five Pneumocystis taxa shown in Fig. 3(A)
, as well as C. albicans, Sac. cerevisiae and three protozoa (Keely et al., 1994
; Cushion et al., 2004
). As in Fig. 3(A)
, similar branch patterns and statistical support were seen for Pneumocystis in these two trees (trees not shown). These relationships were confirmed by maximum-likelihood analysis of 56 nucleotide substitution models (see Methods). Thus, all trees suggested that the Murinae clade is monophyletic.
Concordance of gene genealogies
A phylogenetic species is an evolutionary lineage that has a unique combination of DNA orthologue sequences (Taylor et al., 2000). The evolutionary history of phylogenetic species can be depicted by a bifurcating gene tree. The branches can be evaluated by a test of reliability, such as the common bootstrap statistic, and robustness can be assessed by comparing the concordance of topologies of multiple gene trees.
NJ trees were constructed from SODA, DHPS, DHFR, mtrRNA(LSU) and mtrRNA(SSU) gene sequences and compared to 18S rDNA. As expected, P. murina and P. carinii clustered together and 100 % bootstrap support was observed in all trees. This level of significance was also observed for the cluster of P. jirovecii and non-human primate-derived Pneumocystis (Demanche et al., 2001; Denis et al., 2000
; Guillot et al., 2001
; Hugot et al., 2003
). To further increase the signal-to-noise ratio, a supertree was constructed from a concatenated gene alignment. All of the branches had 100 % bootstrap support (Fig. 4
).
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These data showed that all of the loci analysed are diverged from each other to the same extent. Such concordance of gene trees is indicative of genetic isolation, which leads to the formation of species.
Phylodating P. murina
The rRNA locus has been used to estimate the time that various fungi diverged from each other. This locus is particularly useful for this purpose because it contains regions that evolve at different rates, thereby allowing comparisons above and below the genus level. For example, there is a 10- to 20-fold difference in the rate of nucleotide substitution between ITSs and the 18S rRNA locus (Kasuga et al., 2002). In order to determine the divergence times for P. murina, the branch lengths in Fig. 3(A)
were re-estimated under the assumption of a molecular clock (Takezaki et al., 1995
). This re-estimate produced a linearized tree, shown in Fig. 3(B)
. The molecular clock appeared to have no significant effect on the topology of the tree. The linearized tree was calibrated to time by using a rate of 1·26x1010 substitutions per site per lineage per year, which has been proposed to be the rate for fungi (Berbee & Taylor, 2001
). Times of divergence were determined for each node utilizing the MEGA program, as described previously (Nei et al., 2001
). As an internal control, the divergence time was calculated for C. albicans/Sac. cerevisiae to be 142 million years, which agrees with a previous estimate (Berbee & Taylor, 2001
).
As shown in Fig. 3(B), Pneumocystis appears to have diversified approximately 100 million years ago. P. murina appears to have split from P. carinii about 39 million years ago. The deeper position of the P. wakefieldiae branch suggests that it may have evolved millions of years prior to this time. To test these estimates, an NJ tree of DHFR was constructed and linearized utilizing a calibration point of 140 million years for C. albicans/Sac. cerevisiae (tree not shown). As observed for the 18S rRNA gene, P. murina split from P. carinii approximately 33 million years ago.
The evolution of P. murina may be beyond the resolution of individual genes such as the 18S rRNA gene because they are slow-evolving (Philippe et al., 1994). To circumvent this possibility, additional approaches were utilized to calculate divergence times. One way is to compare loci that are known to undergo rapid evolution. Fungal ITSs evolve faster than adjacent rRNA genes and are useful for determining divergence dates below the genus level (Kasuga et al., 2002
). To this end, the ITS15·8S rRNAITS2 (ITS5·8S) region of P. murina was analysed by aligning it individually to ITS5·8S of P. wakefieldiae, P. carinii, P. jirovecii and macaque Pneumocystis. The Kimura two-parameter (K2P) corrected pairwise distance between P. murina and P. carinii was 0·20. This value was also seen for P. wakefieldiae, but greater K2P values were observed for P. jirovecii (0·33) and macaque Pneumocystis (0·36). These K2P values are larger than those of several cross-genera fungal pairs: Histoplasma capsulatum and Blastomyces dermatitidis, Arthroderma incurvatum and Trichophyton rubrum, Eremascus albus and Ascosphaera apis (Kasuga et al., 2002
). We next estimated the divergence time for P. murina utilizing the ITS5·8S as described by Kasuga et al. (2002). Let T=K/2r, where T is the divergence time, K is the K2P corrected distance since divergence and r is the evolutionary rate of ITS5·8S. The mean evolutionary rate for several sister fungi is 1·4±1·3x109 substitutions per site per lineage per year (Kasuga et al., 2002
). Therefore, using this rate, P. murina split from P. carinii approximately 71 million years ago. The reason this estimate is twice that of the 18S rDNA locus is unclear, but it is known that fungal ITS evolutionary rates can vary several fold (Kasuga et al., 2002
). Rate variability is also seen in animals (3x109 to 8x109) and plants (1·7x109 to 8x109) (Depres et al., 1992
; Richardson et al., 2001
). Since the rate (r) of Pneumocystis ITS5·8S is unknown, it was estimated with the equation r=K/2T, where T is the divergence time inferred from DHFR and rRNA NJ trees. The mean rate is 2·8x109, which shows that the ITS5·8S locus evolved 20 times faster than the 18S rRNA gene. Using this rate, P. murina evolved about 36 million years ago, which is consistent with the evolution of its host (Nei et al., 2001
).
Another approach to estimate the divergence dates of Pneumocystis species is to examine the evolution of their host mammals. This entails the hypothesis that Pneumocystis species co-evolved with their hosts. Two recent evolutionary studies of Pneumocystis derived from different mammalian orders support this view (Demanche et al., 2001; Hugot et al., 2003
). According to fossil records, rats and mice diverged from each other about 14 million years ago. However, since fossil dates provide only a minimum estimate of divergence dates between species, they may not be useful for confirming molecular dates. Molecular data suggest an older split of 3040 million years ago (Nei et al., 2001
). This estimate is remarkably consistent with the rRNA and DHFR values, suggesting that P. murina has been living in its host as a separate species for 3040 million years. These data suggest that either P. murina is a species or the rate of evolution between 18S rRNA in these fungal genera is different. It is important to note that Pneumocystis has only one gene encoding rRNA (Giuntoli et al., 1994
). By contrast, nearly all known fungi have hundreds of rRNA-encoding genes (Bollon, 1982
). The relationship of gene number and rate of evolution is not clear, but it is possible that one gene will evolve more rapidly than 100 of them.
Conclusion
To summarize, the divergence of the 18S rRNA gene of P. murina is greater than it is among species in other fungal genera that evolved tens of millions of years ago. Divergence is not limited to the 18S rRNA gene, but occurs throughout the genome of P. murina. Molecular dating studies confirmed that P. murina is very old, at least as old as its mammalian host, the mouse. Gene trees are concordant, which is consistent with a long period of genetic isolation of P. murina. Finally, there is every indication that few Pneumocystis species share the same habitat, since they are host-restricted (Durand-Joly et al., 2002; Gigliotti et al., 1993
). Thus, even if they could exchange genes, multiple factors prevent this from happening, thereby allowing speciation to occur. Thus, the picture is clear; the genetic divergence and age of P. murina show that it is a phylogenetic species. Therefore, a new name, Pneumocystis murina, is proposed to reflect this new knowledge.
Recognizing species is not a simple matter. However, once sufficient sequence data are available, there are many tools that provide investigators with the means to determine the statistical significance of observed sequence divergence among members of the genus Pneumocystis. The work described above showed that the amount of sequence information needed to obtain statistically significant results exceeds that recommended previously in published guidelines. However, the following steps will provide ample support of proposed new species names. In addition to the International Code of Botanical Nomenclature requirements described elsewhere (Stringer et al., 2001), researchers should submit at least four sequences, one mitochondrial and three nuclear (and all other gene sequences), to GenBank. They should also analyse genes that have been analysed in other species in the genus. This approach will allow further gene tree concordance analysis. The length of the sequence alignments and extent of genetic variation in them should be sufficient to perform these tests. DNA and any other genetic reagents such as DNA clone libraries should be submitted to public culture collections or similar repositories.
Description of Pneumocystis murina sp. nov. Keely, Fischer, Cushion & Stringer
Pneumocystis murina (mu.ri'na. L. adj. murina murine, of the mouse, after the host in which the organism is found, Mus musculus).
Formerly known as Pneumocystis carinii f. sp. muris (Anonymous, 1994).
Non-filamentous yeast-like organisms (trophic forms) resident in the pulmonary alveoli of Mus musculus. Extracellular and adhere to Type I pneumocytes of the alveolar lumen with clusters of admixed presumptive developmental stages extending into the alveolar lumen. The vegetative cells (trophic forms), measuring 15 µm, are uninucleate, of irregular shape, thin-walled and composed of two plasma membranes. Asci (cysts), measuring 58 µm, are thick-walled, globose, with two plasma membranes and contain eight round to ovoid ascospores, each 12 µm; when empty, they appear falciform or irregular. P. murina is morphologically indistinguishable at the light microscopic level from Pneumocystis species that reside in other mammalian lungs, but ultrastructural studies show the filopodia of P. murina to be thinner and more abundant than those of Pneumocystis from rabbits (Dei-Cas et al., 1994; Nielsen et al., 1998
).
P. murina is very different at the DNA sequence level from other Pneumocystis species. P. murina 18S rRNA gene sequences are most similar to those from P. carinii and P. wakefieldiae, with a 0·9 and 1·1 % divergence versus a 2·5 and 1·8 % divergence with the same sequences from P. jirovecii and Pneumocystis from ferret. DNA sequences from regions in the genes of the P. murina SODA diverged from those of P. carinii by 16 % and P. jirovecii by 27 %; DHPS by 6 and 15 %; DHFR by 17 and 31 %; mtrRNA(LSU) by 8 and 20 %; mtrRNA(SSU) by 10 and 18 %; thymidylate synthase by 6 and 21 %; ITS regions within the nuclear rRNA locus by 28 and 45 %; and in the pentavalent AROM gene, by 7 and 17 %, respectively. P. murina and P. carinii have an intron in the 3' region of the 18S rRNA gene while P. wakefieldiae lacks this intron (Edman et al., 1988; Liu & Leibowitz, 1993
).
The type strain is ATCC PRA-111T (=CBS 114898T). Extracted from lungs of 6- to 8-week-old SCID mice (C3SnSmn.CB17-PrkdcSCID/J, The Jackson Laboratory, Bar Harbor, Maine). Cryopreserved samples are stored at the Cincinnati Veterans Affairs Medical Center, Cincinnati, OH.
Latin diagnosis of Pneumocystis murina sp. nov. Keely, Fischer, Cushion et Stringer
Non filiosae similes fermento formae (formae trophicae) quae inhabitant in alveolis pulmoneis Mus musculus. Extracellulares et haestae in Typi Primi pneumocytis alveolaris luminis cum coryumbis admixtarum progredientum formarum extendentium in alveolae lumen. Cellae holitariae (formae trophicae), 15 µm, sunt uninucleatae, irregulares, tenuitunicatae, consistae de duabus membranis plasmaticis. Asci (cysti), 58 µm, sunt crassitunicati, globosi, cum duabus membranis plasmaticis, et continent octo rotundos ad ovatiles ascospores, quisque 12 µm; vacui, apparent falciformi aut irregulares. P. murina est morphologiciter inspectandus, facili microscopio uso, a Pneumocystis qui in pulmonibus mammaliarum habitant, sed studia ultrastructuralia demonstrant filopodia P. murina esse tenuioria et abundantiora quam filopodia Pneumocystis e leporibus (Dei-Cas et al., 1994; Nielsen et al., 1998
).
P. murina est dissimilior ordine DNA quam aliae Pneumocystis speciae. P. murina 18S rRNA ordines genium erant simillimae illis a P. carinii et P. wakefieldiae, cum 0·9 % et 1·1 % differentia versus 2·5 % et 1·8 % differentia cum eodem ordine a P. jirovecii et Pneumocystis ferretiae. DNA ordines a regionibus in genibus P. murina dismutasi manganesi-cofactorati superoxidi erant dissimiles ab illis P. carinii a 16 % et P. jirovecii a 27 %; dihydropteroati synthasi a 6 et 15 %; dihydrofolati reductasi a 17 et 31 %; mitochondrialis magni subunitatis RNA a 8 et 20 %; mitochondrialis parvi subunitatis ribosomalis RNA a 10 et 18 %; thymidylati synthasi a 6 et 21 %; internae transscriptae seperantes regiones intra nuclearem ribosomalem RNA locum a 28 et 45 %; et in pentavalenti AROM geni, a 7 et 11 %, proprie. P. murina et P. carinii habere intronum in 3' regione 18S rRNA genis, sed P. wakefieldiae caret hunc intronum (Edman et al., 1988; Liu & Leibowitz, 1993
).
TYPUS: Consociatae Civitates Americae, Cincinnatensis, OH. Extractae e pulmonibus sexocto hebdomades SCID mures (C3SnSmn.CB17-PrkdcSCID/J, Officina Iacsoni, Vectis Portus, Maine). Curae compagium tradidae sunt ut exempla cryoservati Collectioni Americano Culturarum Typarum (numerus accessioniz ATCC PRA-111T). Exempla cryoservata servata sunt ad Medium Medicum Rerum Veteranorum Cincinnatensis, Cincinnatensis, OH.
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ACKNOWLEDGEMENTS |
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Received 18 November 2003;
revised 18 February 2004;
accepted 23 February 2004.
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