INTRODUCTION

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The last 20 years or so has seen a growing number of fungal infections coincident with a dramatic increase in the population of severely immunocompromised patients. These infections are due mainly to impairments in host defence mechanisms as a consequence of viral infections, especially the human immunodeficiency virus epidemic, hematological disorders such as different types of leukemia, organ transplants, and more intensive and aggressive medical practices. Many clinical procedures and treatments, such as surgery, the use of catheters, injections, radiation, chemotherapy, antibiotics, and steroids, are risk factors for fungal infections. However, these procedures are necessary, and so the incidence of fungal infections will increase accordingly.

Until only a few years ago, pathogenic fungi were a well-defined group, some of which were limited to geographical regions and were well known by clinicians. However, the situation has changed considerably, and new infectious agents are continually appearing, around 20 species yearly. These new opportunistic pathogens have increased the base of knowledge of medical mycology, and unexpected changes have been seen in the pattern of fungal infections in humans. It is also possible that most of the recently reported taxa have caused infections which previously passed unnoticed due to inadequate diagnostic expertise. This situation and the rapid appearance of such a wide range of new pathogens have created a growing interest in fungal systematics. Fungal taxonomy is a dynamic, progressive discipline that consequently requires changes in nomenclature; these changes are often difficult for clinicians and clinical microbiologists to understand. Another difficulty for microbiologists inexperienced in mycology is that fungi are mostly classified on the basis of their appearance rather than on the nutritional and biochemical differences that are of such importance in bacterial classification. This implies that different concepts have to be applied in fungal taxonomy. Generally, medical mycologists are familiar with only one aspect of pathogenic fungi, i.e., the stage that develops by asexual reproduction. Usually microbiologists ignore or have sparse information about the sexual stages of these organisms. However, the sexual stages are precisely the baseline of fungal taxonomy and nomenclature. It seems evident that in the near future, modern molecular techniques will allow most of the pathogenic and opportunistic fungi to be connected to their corresponding sexual stages and integrated into a more natural taxonomic scheme. The aim of this review is to update our present understanding of the systematics of pathogenic and opportunistic fungi, emphasizing their relationships with the currently accepted taxa of the phyla Ascomycota and Basidiomycota.

The basic rank in taxonomy is the species. However, exactly what different mycologists consider to be a species can vary widely, and there are different approaches for delineating them. Attempts to reach a consensus for a universal definition of species have been unsuccessful, and consequently several very different concepts have been used. However, the genetic basis for some of these concepts is largely unknown. The widespread occurrence of asexual reproduction by asexual propagules (conidia) and of hyphal anastomosing can cause confusion because a mycelium in its natural environment seems to be a single physiological and ecological unit but in reality is a genetic mosaic (74). Therefore, in mycology, the distinction between a population and an individual is not always easy, and this can create confusion in genetic studies (87).

Different concepts have been used by mycologists to define the fungal species. The morphological (phenetic or phenotypic) concept is the classic approach used by mycologists; in this approach, units are defined on the basis of morphological characteristics and ideally by the differences among them. The polythetic concept is based on a combination of characters, although each strain does not have to have the same combination. The ecological concept, which is based on adaptation to particular habitats rather than on reproductive isolation, is often used for plant-pathogenic fungi. The biological concept, which was developed before the advent of modern phylogenetic analysis, emphasizes gene exchange (i.e., sexual and parasexual reproduction) within species and the presence of barriers that prevent the cross-breeding of species (111). A species is considered to be an actual or potential interbreeding population isolated by intrinsic reproductive barriers (13). However, application of the biological-species concept to fungi is complicated by the difficulties in mating and in assessing its outcome (302). Also, whether a cross is considered fertile or sterile depends on the frame of reference. In this sense, published accounts of crosses between different species are often difficult to interpret because authors have failed to specify the type of infertility and its severity (428). The biological-species concept cannot be applied to organisms that do not undergo meiosis (484). It is applied only to sexual fungi, whereas asexual ones need only possess similar characteristics to each other. However in asexual fungi, genetic exchange through somatic hybridization is a theoretical possibility, although it is limited by vegetative incompatibility (87). For asexual dermatophytes, the cohesion-species concept, based on a demographic exchangeability of phenotypes, has been used to explain the proliferation of disjunct phenotypes. The demographic exchangeability would be the ecological analogue of the genetic exchangeability of sexual species (523).

Two recent and important developments have greatly influenced and caused significant changes in the traditional concepts of systematics. These are the phylogenetic approaches and incorporation of molecular biological techniques, particularly the analysis of DNA nucleotide sequences, into modern systematics. Molecular techniques, which were previously used only in research laboratories, are now commonplace. These developments have provided new information that has caused the biological-species concept to come under criticism in favor of the phylogenetic-species concept. This new concept has been found particularly appropriate for fungal groups in which no sexual reproduction has been observed (deuteromycetes). Hence, new concepts, specifically formulated within the field of phylogenetics, are becoming familiar to mycologists. Population studies and molecular data are increasingly showing that many widely used morphospecies actually comprise several biological or phylogenetic species. One of the problems for morphologists involves deciding how many base differences are required for strains to be considered different species. This has been partly solved by the phylogenetic-species concept, especially when based on cladistic analysis of molecular characteristics, which offers consistency in the delineation of species. Cladogram topology indicates the existence of monophyletic groups, which may represent species or supra- or subspecific taxa (302, 484). Peterson and Kurtzman (430) correlated the biological-species concept with the phylogenetic-species concept by comparing the fertility of genetic crosses among heterothallic yeasts. They demonstrated that the D₂ region, a variable region of the 25S rDNA gene, is sufficiently variable to recognize biological species of yeasts and that conspecific species generally show less than 1% nucleotide substitution.

However, the definition of the phylogenetic-species concept is also complex, and several different definitions have been proposed. There appear to be two main approaches. The character-based concept, or diagnostic approach, defines a species as a group of organisms that have common observable attributes or combinations of attributes. In contrast, the history-based concept insists that organisms must be historically related before they can be considered members of any given species (23). Some of the different species concepts currently in use are difficult to distinguish, and so mycologists should be familiar with them and clearly define which they are using to recognize species.

Little is known about evolutionary relationships among fungi. Only recently have some data become available, although they are still sparse. The simple morphology, the lack of a useful fossil record, and fungal diversity have been major impediments to progress in this field (35). Classically, studies on fungal evolution have been based on comparative morphology, cell wall composition (19, 20), cytologic testing (538), ultrastructure (174, 242, 243), cellular metabolism (308, 562), and the fossil record (238). More recently, the advent of cladistic and molecular approaches has changed the existing situation and provided new insights into fungal evolution.

The phylogenetic relationships among higher fungal taxa remains uncertain, mainly because of a lack of sound fossil evidence, and remains a source of much controversy. The proposed phylogenetic relationships among the Animalia, Plantae, and Fungi kingdoms depend on the molecular regions and methods used by different investigators. Phylogenetic analysis has shown that the fungal kingdom is part of the terminal radiation of great eukaryotic groups (Fig. 1) which occurred one billion years ago (499, 540). Surprisingly, although mycology has been classically considered a branch of botany, there is also evidence that the kingdom Fungi is more closely related to Animalia than to Plantae (390). The analysis of amino acid sequences from numerous enzymes indicated that plants, animals, and fungi last had a common ancestor about a billion years ago and that plants diverged first (135). Another former hypothesis, i.e., that fungi are derived from algae, has been definitively abandoned (238).

View larger version (23K): [in this window] [in a new window]   FIG. 1.   Phylogenetic tree showing relationships of eukaryotes, based on the nucleotide sequence of 18S DNA. Reprinted from reference 540 with permission of the publisher.

Berbee and Taylor (37), following Hennig's criteria (247), used geological time to calculate the appearance of nonsister taxa in an evolutionary tree. Just as sister lineages are equivalent units because they are equal in age, other lineages should receive equal rank because they diverged at about the same time. On this basis, it has been hypothesized that all the ranks from classes to families have successively appeared between the Cambrian and Tertiary periods.

The number of nucleotide substitutions in DNA sequences is directly proportional to the time passed, and so the number of base changes can be used to estimate the date of evolutionary radiation (238). On this basis, and using reference points such as the appearance of fossilized fungal clamp connections from the fossil record, the absolute timing of the origin of fungal groups has been estimated (36). The three main fungal phyla, Zygomycota, Ascomycota, and Basidiomycota, are thought to have diverged from the Chytridiomycota approximately 550 million years ago. The Ascomycota-Basidiomycota split occurred about 400 million years ago, after plants invaded the land. Many ascomycetes have evolved since the origin of the angiosperms in the last 200 million years. These results, with a few exceptions, are broadly supported by fossil evidence (36).

To be formally recognized by taxonomists, an organism must be described in accordance with internationally accepted rules and given a Latin binomial. The rules that control the bionomenclature are very diverse and depend on the type of organism. Biological nomenclature is regulated by five different codes devoted to plants (197), cultivated plants (64), bacteria (496), viruses (168, 335), and animals (454). However, in an attempt to harmonize the different codes, some efforts are being made to find a unified system (236, 237, 239). Meanwhile, the nomenclature of fungi (including yeasts) is governed by the International Code of Botanical Nomenclature (ICBN) (197) as adopted by each International Botanical Congress. Any proposed changes to the Code are published in Taxon, the official journal of the International Association for Plant Taxonomy, and then debated in the Congress for approval. The Code aims to provide a stable method for naming taxonomic groups, avoiding and rejecting names which may cause error, ambiguity, or confusion. However, strict application of the Code frequently leads to name changes for nomenclatural rather than scientific reasons (235). This causes annoyance among users, who do not usually understand the reasons for the changes. The taxonomy of some pathogenic fungi is particularly unstable and controversial at present. Changes to the names of taxa and their consequent diseases are potentially confusing (340). For example, the name of the fungus Allescheria boydii (so called in the early 1970s) was changed to Petriellidium boydii and then to Pseudallescheria boydii within a very short period. Consequently, infections caused by this organism were referred to as allescheriasis, allescheriosis, petriellidosis, and pseudallescheriosis in the medical literature (403). To help clarify the confusion that changes in fungal names can cause, the Nomenclature Sub-Committee of the International Society for Human and Animal Mycology published its recommendations for mycosis nomenclature (402, 403). According to these recommendations, a disease name should ideally describe a disease, whereas many mycosis names indicate only a causative fungal genus. Disease nomenclature based on the traditional structure "fungus + sis," which can be highly influenced by the taxonomic changes, was discouraged. In addition, the value of names of the "pathology A due to fungus B" construction was emphasized (402), e.g., "subcutaneous infection due to Alternaria longipes."

The dual modality of fungal propagation, i.e., sexual and asexual, has meant that since the last century (463) there has been a dual nomenclature. The fungus, as a whole, comprises a teleomorph (sexual state) and one or more anamorphs (asexual states) (246). The term "holomorph" has been reserved for fungi with teleomorphic sporulation together with all their sporulating or vegetative anamorphs (180). The anamorph and teleomorph generally develop at different times and on different substrates, although in zygomycetes they often occur together. Since each phase has been described in total ignorance of the existence of the other in many cases, the ICBN maintains that it is legal to give them separate binomials. For a long time, the anamorphs that occurred alone have been grouped into anamorph genera because they share some morphological features. These anamorphs have been placed in a separate major high-level taxon called Deuteromycotina or Deuteromycetes. With the advent of molecular approaches in fungal taxonomy, some mycologists have advocated abandoning the dual system of naming because unified classification of all fungi may be possible on the basis of the rDNA sequences of the anamorphs (47, 71, 452). Other authors do not agree with this proposal and have considered it absolutely necessary to conserve deuteromycete taxon names, at least for identification purposes (180, 484). During the Holomorph Conference (453), it was agreed to maintain the term deuteromycetes with a lowercase "d" and not to formally recognize this group of organisms at a particular rank.

Another controversy resulted from the replacement of the terms "anamorph" and "teleomorph" with "mitosporic fungus" and "meiosporic fungus", respectively, in the last edition of Ainsworth and Bisby's Dictionary of the Fungi (238); this source is considered a fundamental framework for fungal terminology and taxonomy. These changes have not been accepted by numerous authors (179, 180, 286), who consider that the anamorph and teleomorph phases of a fungus are determined not simply by the type of cellular processes (meiosis or mitosis) that precede sporulation but also by morphological features. Additionally, it has been argued that the cytological events preceding sporulation have not been investigated in sufficient depth to correlate teleomorph morphology with sexual recombination (179).

These problems particularly involve clinical fungi. The majority of pathogenic fungi are ascomycetes, which only rarely show their teleomorphs in the diagnostic laboratory. In contrast, these fungi frequently develop their anamorphs in routine culture media, and for some species there are at least two anamorphs (synanamorphs). Thus, the application of nomenclatural rules to the complicated life cycle of fungi can create some confusion among the users of fungal systematics. Clinical users have usually had little formal training in the fundamental principles of nomenclature and taxonomy, and they have a limited understanding and appreciation of these concepts. In addition, current reductions in financial support of hospital services means that the rapid identification of pathogenic fungi is an important concern (241). Consequently, a special effort should be made to develop reliable taxonomic systems which are easy to use and do not require complicated and expensive equipment (340).

The correct identification of fungi is of great practical importance not only in the clinical setting but also in plant pathology, biodeterioration, biotechnology, and environmental studies. An enormous number of species of fungi are already known, and so taxonomists are being kept very busy with recognizing and describing new species and grouping taxa. Hence, most species have received only limited study, so that classification has been mainly traditional rather than numerical and has been based on readily observable morphological features. However, some groups of fungi, because of their economical or pathological importance, have been studied more extensively. Other features beside morphology, such as susceptibilities to yeast killer toxins, susceptibilities to chemicals and antifungal drugs, the use of morphograms, molecular techniques, physiological and biochemical tests, secondary metabolites, ubiquinone systems, fatty acid composition, cell wall composition, and protein composition, have been used in classification and also in identification. The increased use and availability of modern techniques have opened up many new areas within systematics and have enabled more traditional ones to be developed further (67). Some of these approaches are detailed below.

Classification systems of organisms are historically based on observable characteristics. This is the phenotypic approach. The classification and identification of fungi, unlike other important pathogens such as bacteria or viruses, relies mainly on morphological criteria. The fungi of medical importance are microscopic, and the study of their morphology requires the use of the light microscope. The classical light microscopic methods have been enhanced by Nomarski differential interference contrast, fluorescence, cytochemistry, and the development of new staining techniques such as those for ascus apical structures (461). Unfortunately, during infections most pathogenic fungi show only the vegetative phase (absence of sporulation); in host tissue, only hyphal elements or other nonspecific structures are observed. Although the pigmentation and shape of these hyphae and the presence or absence of septa (Fig. 2, structures a, h, and l) can give us an idea of their identity, fungal culture is required for accurate identification. Species-specific antibodies and the use of probes can be very useful in such cases. Although some commercial probes exist, these techniques are not yet available or convenient for routine use in medical mycology. Therefore, the growth of isolates in appropriate culture media, enabling their most characteristic features to be recognized, is still the most common procedure used in practice.

View larger version (30K): [in this window] [in a new window]   FIG. 2.   Diagnostic features of the fungal phyla of clinical relevance. Zygomycota: a, coenocytic hypha; b, zygospore; c, sporangiophore; d, sporangiospores. Basidiomycota: e, basidiomata; f, basidium; g, basidiospores; h, hypha with clamp connections. Ascomycota: i, ascomata; j, ascus; k, ascospores; l, septate hypha. Deuteromycetes: m, pycnidium; n, conidiophore; o, conidiogenous cells; p, conidia. Oomycota: q, zoospore; r, gametangia; s, oospores.

As mentioned above, clinical microbiologists are used to seeing only deuteromycetes (Fig. 2) in cultures of clinical specimens. For the identification and classification of these fungi, the type of conidia (Fig. 2, structure p) and conidiogenesis (the process involved in conidium formation) (100) are considered the most important sets of characteristics to be observed. Cells that produce conidia are conidiogenous cells (Fig. 2, structure o). Often a different structure which bears one or more conidiogenous cells is present; this structure is the conidiophore (Fig. 2, structure n). There are two basic types of conidiogenesis, blastic and thallic (118). In blastic conidiogenesis, there is a small spot on the conidiogenous cell from which the conidia are produced. In thallic conidiogenesis, the entire conidiogenous cell is converted into one or more conidia. Primarily based on ultrastructural observations, Minter et al. (356-358) gave a personal interpretation of the detailed events involved in the formation of conidia and proliferation and regeneration of the cells bearing them. However, the terminology of the conidiogenesis events for the description of the anamorphic species proposed by Minter et al. is not widely followed. Hennebert and Sutton (245) described a set of basic unitary parameters which can aid in the study of the characteristics involved in conidiogenesis, although recognition of some of these parameters is difficult for inexperienced scientists. When sporulation is absent, there are still a number of morphological features to assist in classification. Other structures such as sclerotia, chlamydospores, or the presence of particular hyphal elements, as in anthropophilic dermatophytes, are sometimes very useful in the identification of anamorphs.

In the rare instances that opportunistic fungi develop the teleomorph in vitro (this happens in numerous species of Ascomycota and in a few species of Zygomycota and Basidiomycota), there are many morphological details associated with sexual sporulation which can be extremely useful in their classification. The type of fruiting body (basidioma in basidiomycetes, ascoma in ascomycetes) (Fig. 2, structures e and i) and the type of ascus (a microscopic, unicellular, frequently globose, saccate, or cylindrical structure which often contains eight ascospores produced after meiosis, and is usually developed in the cavity of fruiting bodies) (Fig. 2, structures j and k) are vital for classification. The shape, color, and the presence of an apical opening (ostiole) in the ascomata are important features in the recognition of higher taxa. Variations in ascus structure are currently important in the classification of these fungi, especially at the level of family and above. Ascospores are particularly subject to environmental selection and are consequently of value at lower ranks than are ascus structures. Pigmentation is a metabolic process occurring rather late in ascosporogenesis; as with ascospore septation, it is therefore of most value at lower taxonomic levels.

Electronic microscopy techniques allow the recognition of several details of special taxonomic significance. Cross sections of cell walls observed by transmission electronic microscopy (TEM) reveal significant differences between ascomycetous and basidiomycetous yeasts (296). The value of differences in the ultrastructure of the septum at the base of the ascus and in ascogenous hyphae has become apparent as a result of the studies of Kimbrough (277). In general, the correlation between septal type and family is high in discomycete groups but still requires investigation and analysis in other ascomycetes (277).

The increased availability of scanning electronic microscopy (SEM) has resulted in a number of significant findings and, in some ascomycetes, has greatly facilitated identification at the species level by enabling differences in the surface detail of the ascospores to be clearly visualized (118). Freeze fracturing has revealed fine details of outer wall layers of conidia or ascospores (164, 531).

In recent years, morphological techniques have been influenced by modern procedures, which allow more reliable phenotypic studies to be performed. Numerical taxonomy, effective statistical packages, and the application of computer facilities to the development of identification keys offer some solutions and the possibility of a renaissance of morphological studies. Automated image analyzers, electronic particle sizing, and fractal geometry may have a lot to offer in the analysis of fungal morphology (94, 484).

The phenotypic approach has been largely criticized for its lack of standardized and stable terminology and for its high subjectivity. Moreover, some phenotypic characteristics have been considered to be unstable and dependent on environmental conditions, as with growth in artificial culture. A clear limitation of phenotypic approaches is that they cannot be applied to fungi that do not grow in culture. Consequently, there are many fungi that will remain unclassified as long as taxonomists rely solely on phenotypic characteristics. Another notable problem of classification based on morphological criteria is the above-mentioned dual system of classification, with no consistent correlation between the taxonomies of the ascomycetes and deuteromycetes (245). This is an important difficulty in establishing the taxonomic concept of the fungus as a whole.

Since the distinguishing morphological characteristics of a fungus are frequently too limited to allow its identification, physiological and biochemical techniques are applied, as has been routinely done for the yeasts. However, for poorly differentiated filamentous fungi, these methods are laborious, time-consuming, and somewhat variable and provide insufficient taxonomic resolution. In contrast, molecular methods are universally applicable. Comprehensive and detailed reviews of the use of molecular techniques in fungal systematics have been provided by Bruns et al. (71), Hibbett (249), Kohn (284), Kurtzman (295), Maresca and Kobayashi (325), and Weising et al. (575).

Two important technical advances have stimulated the use of molecular techniques. Firstly, the advent of PCR has allowed the analysis of small numbers of fungal cells or even single spores, dried herbarium material (87), or extinct organisms (192). Second, the selection of universal oligonucleotide primers specific to fungi (244, 474, 559, 582) has provided easy access to nucleotide sequences.

The aim of molecular studies in biodiversity is fourfold: (i) phylogenetic studies, i.e., tracing back the most probable course of evolution and the historic coherence between groups at higher taxonomic ranks; (ii) taxonomic studies, mostly at the level of genera and species; (iii) diagnostic applications, i.e., recognition of defined taxonomic entities; and (iv) epidemiology and population genetics, i.e., monitoring outbreaks of subspecific entities with respect to the analysis of populations and their mode of reproduction. Each of these broad aims and levels of diversity has its own set of optimal techniques. In this review, only phylogenetic and taxonomic studies are discussed.

One of the groups of genes which is most frequently targeted for phylogenetic studies is the one that codes for rRNA. Introns of several protein-encoding genes, such as the -tubulin (405, 543), actin (101), chitin synthase (59, 526), acetyl coenzyme A synthase (42), glyceraldehyde-3-phosphate dehydrogenase (230), lignin peroxidase (377) or orotidine 5'-monophosphate decarboxylase (443) genes, can also be applied and can provide important information. The main reasons for the popularity of rDNA are that it is a multiple-copy, non-protein-coding gene, whose repeated copies in tandem are homogenized by concerted evolution, and it is therefore almost always treated as a single-locus gene. Furthermore, ribosomes are present in all organisms, with a common evolutionary origin. Parts of the molecule are highly conserved (551, 552) and serve as reference points for evolutionary divergence studies. The conserved regions alternate with variable regions or divergent domains (232). The 5.8S, 18S, and 25S rDNAs are transcribed as a 35S to 40S precursor, along with internal and external transcribed spacers (ITS and ETS). All spacers are spliced out of the transcript. Between each cluster is a nontranscribed or intergenic spacer (NTS or IGS) that serves to separate the repeats from one another on the chromosome. A 5S gene takes a variable position and is transcribed in the opposite direction. The total length of one DNA repeat is between 7.7 and 24 kb (249).

Comparisons of the 18S (also called the small-subunit [SSU]) rRNA sequences have been performed to assess the relationships of the major groups of living organisms (591, 592). For phylogeny of filamentous fungi, the 18S sequence is mostly used completely or in subunits of over 600 bp (70, 225, 233, 411, 502, 525, 586). In the yeasts, the D1 and D2 variable regions of 25S rDNA regions are almost exclusively used (220, 296). This technique is currently being extended to Heterobasidiomycetes (27) and sometimes also to filamentous ascomycetes (199, 329). In only a limited number of fungi have both regions been sequenced. Due to this different choice of target regions, comparison of fungi to all possible relatives is hampered. The 25S variable domains are very informative and allow comparisons from high taxonomic levels down to the species level, although only a limited number of variable positions remain (212). In the 18S gene, the variable domains mostly provide insufficient information for diagnostic purposes (116), and thus large parts of the molecule must be sequenced to obtain the resolution required (225). The ITS regions are much more variable, but sequences can be aligned with confidence only between closely related taxa. These regions are generally used for species differentiation but may also demonstrate patterns of microevolution (196). In contrast, 5.8S rDNA is too small and has the least variability. 5S has been used mainly to infer relationships at the ordinal level (571), where differences could be traced back to the secondary structure of the molecule (48).

The evolutionary distance is generally displayed in the form of trees, and a wide diversity of algorithms are available to construct them. Two basic methods are available: distance matrix methods, resulting in phenograms, and maximum-parsimony methods, resulting in cladograms. Several papers have reported comparisons between the two methods with the same data set (329). If the difference between compared taxa is moderate, the results of the two algorithms are similar, but when dissimilarity values are higher, the number of evolutionary substitutions may be underestimated (509). The statistical significance of the tree found is tested by using resampling algorithms such as the bootstrap method. This subject has been reviewed by Avise (12) and Hillis et al. (250). The tree is usually rooted with an organism at a moderate distance (outgroup) that still can be aligned with confidence. For groups which have unexpected heterogeneity, such as the fission yeasts and the black yeasts, the correct choice of outgroup may be quite important (512).

Among the classical DNA-based methods is the determination of the nuclear DNA (nDNA) guanine-plus-cytosine content. The G+C content of nDNA has been established for many fungi, primarily yeasts. A difference of 2% in the G+C content has been considered to indicate that two strains should be assigned to different species (295). In some insufficiently resolved fungal groups, a difference of 8% has been allowed within species (50). In more precise recent studies with ecologically defined taxa, this difference was reduced to 1% (210). The G+C content is determined by using the T_m from the S-shaped melting curve of the DNA. Occasionally the shape of the curve is deviant; by determination of the first derivative, this could be traced back to the presence of DNAs with different melting velocities. Guého et al. (214) found that these differences may be characteristic of subspecific entities.

The identity or nonidentity of closely related strains can be determined by DNA-DNA hybridization, estimating the velocity of heteroduplex formation compared to the standard kinetics of the individual strains. A relative hybridization value of over 80% is generally regarded as indicating membership in the same species, whereas values of less than 20% are proof of nonidentity (558). Intermediate values have increasingly been found recently, and these probably indicate subspecific entities (495).

In recent years, the methods most widely used for taxonomy at the species level have been sequencing and electrophoretic methods. Many authors have sequenced closely related species to investigate the relationship of the taxa. Such studies have been carried out with larger genera such as Penicillium (310, 311), Fusarium (429), and Trichoderma (292). Teleomorph and anamorph variation is not always congruent (292, 451). The speed of evolution seems heterogeneous and is characterized by different rates of variation between groups (127). In general, about 2% intraspecific variability is maintained within species (195). Occasionally the use of ITS is problematic due to the occurrence of two different types in a single organism (405).

Among the electrophoretic methods, restriction fragment length polymorphism (RFLP) is particularly significant for taxonomy. This technique involves digesting DNA samples with a panel of restriction enzymes. The patterns can be tabulated and compared (546), or phenetic trees can be constructed (73, 142). The first RFLP technique widely used in taxonomy compared patterns of mitochondrial DNA (mtDNA). However, some authors have sequenced the mtSSU rDNA instead (568, 597). mtDNA is generally indicative of differences somewhat below the species level (268), but in groups where microspecies are currently distinguished, such as in the dermatophytes, the differences seem to correspond to teleomorph species (360). RFLP-based typing methods have been used to reveal anamorph-teleomorph connections (185). Most commonly, the RFLP of PCR-amplified rDNA is used. This technique is also known as amplified rDNA restriction analysis (554) and provides a quick insight into relationships between moderately distant fungi (116). Therefore, homogeneity of ITS profiles corresponds well to final ITS sequencing diversity (547). The method is primarily confirmatory; i.e., new strains are quickly assigned to sequenced strains with the use of restriction maps (559). This strategy was used, for example, by Yan et al. (598) to study Phialophora. Amplified rDNA restriction analysis is particularly useful as an inexpensive and simple alternative to SSU rDNA sequencing when broad relationships have already been determined. However, the frequent occurrence of introns in SSU rDNA (183) may hamper quick comparison of strains.

Random primed methods (226) are particularly useful to determine relationships below the level of species, but depending on the length of the primers and the recognized taxonomic diversity of the group under study, the method may help to discriminate species. For such aims, a comparison of several unlinked molecular methods is overdue (545). A popular technique is random amplified polymorphic DNA (RAPD) with 10-mer primers. However, this method is gradually being abandoned because of poor reproducibility. Microsatellites are a special class of tandem repeats, which have a base motif of up to 10 bp that is frequently repeated (up to about 100 times); they are found in many genomic loci with an almost ubiquitous distribution (536). A general profile comparison of microsatellites enables species recognition (195, 389). In addition, due to the high level of polymorphism, individual bands can be informative for the characterization of strains (163, 314, 482).

Karyotyping, i.e., the migration of chromosomes under the influence of an electric field, has been commonly used for a long time in plant taxonomy and also in mammal and bacterial taxonomy. However, some problems have complicated the use of this technique in fungal taxonomy (e.g., the smallness of the fungal chromosomes and the difficulty of observing condensed chromosomes during meiosis) (284). Techniques such as electron microscopy, pulsed-field gel electrophoresis (354, 413, 493), and its recent modification, contour-clamped homogeneous electric field electrophoresis (535) can contribute to the precise determination of karyotypes. The high intraspecific variability of the electrophoretic karyotypes of fungi makes this technique especially applicable for studies of populations in pathogenic yeasts (133, 279, 318, 421, 427) and filamentous fungi (68, 535).

Physiological and biochemical techniques. Because numerous fungi grow relatively rapidly in pure culture, it is possible to use physiological and biochemical techniques to identify and classify them (65, 424, 426). These techniques have been successfully used in the study of black yeasts (115, 117, 120, 128, 600). The different ranges of growth temperature have been used as a complementary tool in the identification both of asexual (339) and sexual (353) fungi. Growth rates on defined media under controlled conditions are also valuable in studies of complex genera such as Penicillium (431). Commercially available kits such as the API system have also been used to identify filamentous fungi (155, 450, 573). Paterson and Bridge (426) have published a compilation of the physiological techniques used in the identification of filamentous fungi. They list a variety of biochemical methods which range from simple agar-based tests to more sophisticated chromatographic and electrophoretic methods (426).

Secondary metabolites. Secondary metabolites are compounds neither essential for growth nor key intermediates of the organism's basic metabolism but presumably playing some other role in the life of fungi. They are usually found as a mixture of closely related molecules with a peculiar and rare chemical structure (238). The most common are steroids, terpenes, alkaloids, cyclopeptides, and coumarins, and some of these are mycotoxins. The advent of thin-layer chromatography, especially the simple technique of directly spotting thin-layer chromatography plates with small samples of culture cut from petri dishes, has made it possible to qualitatively assess secondary metabolites much faster than by conventional extraction, purification, and concentration techniques. This improvement has resulted in huge amounts of new secondary-metabolite data, which is now being incorporated in databases (67, 424). The pattern of secondary-metabolite production has for a long time been of great use in the identification and classification of lichens. This pattern has been used less in the taxonomy of ascomycetes and basidiomycetes, although it is well known that these organisms produce a vast array of such compounds (87). The use of this method in fungal taxonomy has been questioned because the production of these compounds can be affected by environmental conditions and the detection procedure presents some difficulties. However, its potential in ascomycete systematics is well illustrated by the chemotaxonomic studies performed in Eurotiales (170-172) and in Xylariales (581). In these orders, individual species can often be recognized on the basis of particular metabolite profiles. Integrated approaches involving morphology, physiology, and secondary metabolites have been used in several attempts to clarify the systematics of some fungi (65, 66, 361, 366, 371).

Ubiquinone systems. Besides the use of secondary metabolites in taxonomy, some use has also been made of other compounds which play an essential role in metabolism and which are primary rather than secondary metabolites, e.g., ubiquinones (coenzyme Q) (87). These compounds are important carriers in the electron transport chain of respiratory systems. The number of isoprene units attached to the quinone nucleus varies, and such differences in ubiquinone structure are excellent indicators in the classification of genera and subgeneric taxa in bacteria and yeasts. Although less common, these techniques are also being used in the taxonomy of black yeasts (596) and filamentous fungi (595). The results provided by these techniques sometimes correlate with those provided by molecular techniques, although conclusions based purely on ubiquinone systems are debatable and, depending on the method used, can provide different sets of data (466).

Fatty acid composition. Cellular fatty acid composition is routinely determined in bacterial systematics (370, 557). Both the type of fatty acid present and its relative concentration are useful characteristics for separating taxa. Until recently, these techniques were only rarely used in fungal taxonomy. Although fewer different fatty acids are produced by fungi than by bacteria (306), these analyses are increasingly used for differentiating fungi (6, 10, 49, 69, 510). Pyrolysis gas chromatography, pyrolysis mass spectrometry, gas chromatography, and partition aqueous polymer two-phase systems are among the numerous methods used to determine these compounds (49). Recently, gas chromatography, combined with methods of multivariate statistical analysis, has successfully been used to study the fatty acids of numerous and varied filamentous fungi, including oomycetes, zygomycetes, basidiomycetes, and even sterile mycelia (49, 510). These techniques have also proved to be useful at intraspecific level (510). However, several technical aspects of the procedure used must be highly controlled to minimize sources of variation, which can influence the results enormously. The culture conditions and temperature are among the most important factors to be standardized (510).

Cell wall composition. Numerous studies have shown differences in the structure and composition of the cell wall of fungi which have been used in the definition of high fungal taxa. For example, cellulose is a particular component of the alkali-insoluble fraction of oomycetes, while ascomycetes and basidiomycetes contain both chitin and glucan in these fractions. In contrast, the zygomycetes have chitosan and polyglucuronic acid. Significant variations in the sugar composition of cell walls have been observed in the different genera of dermatophytes (200b, 396, 397). The walls of yeast cells that have been subjected to hydrolysis yield substantial amounts of glucose and mannose, but species differ in the presence or absence of smaller amounts of other polysaccharides (fucose, galactose, rhamnose, and xylose). This feature has also been used in classification of fungi (87). Bartnicki-Garcia (20) has reviewed the biochemical and physiological characteristics of the cell wall components which have been used to delimit high taxa.

Protein composition. Isoenzyme patterns produced by electrophoretic techniques (zymograms) have determined generic relationships and differentiated species (87, 349, 375). Apart from electrophoresis, immunological techniques and protein sequencing also have suitable resolution for interspecific characterization (173, 284, 334). Isozyme analysis is considered to be an economical and practical technique for screening large populations. What is more, the characteristics determined by this technique are generally accepted to be of independent genetic origin (284). Allozyme (allelic isozyme) data are commonly used in phylogenetic studies (284).

Before DNA-sequencing methods became available, it was practically impossible to infer a reliable evolutionary tree containing all forms of life. Whittaker, in the context of Five Kingdoms (animals, plants, fungi, protists, and monera) (326), summarized evolutionary thought (583). The four multicellular forms of these traditional taxonomic kingdoms were considered to be eukaryotes, and only the monera were identified with the preceding prokaryotes. These two categories of organisms, defined mainly by the presence or absence of nuclear membranes, are considered to be independent and coherently related groups. For a long time, fungi were regarded as a single kingdom belonging to the aforementioned five-kingdom scheme. However, the organisms usually considered to be fungi are very complex and diverse; they include multicellular and filamentous absorptive forms and unicellular assimilative forms, among others, and can reproduce by different types of propagules or even by fission. This general concept cannot be based on a single phylogenetic line but, rather, on a way of life shared by organisms of different evolutionary backgrounds. This has recently been confirmed by ultrastructural, biochemical, and especially molecular biological studies. Hence, there has been controversy about the higher fungal taxa; i.e., some authors consider that fungi should be assigned to two kingdoms, Protoctista and Eumycota (274), and others consider that they should be assigned to three, Chromista, Fungi, and Protozoa (238) (Table 1). When it is useful to refer to fungi in this polyphyletic sense, the name can be used nonitalicized and noncapitalized to differentiate them from the kingdom Fungi.

Analysis of the SSU rDNA sequence shows that fungal organisms belong to four monophyletic groups: Acrasiomycota, Myxomycota, Oomycota, and Fungi (71). It also shows that the first two groups diverged separately prior to the terminal radiation of eukaryotes (231, 499, 540), which is consistent with morphology and function, because these two groups include slime molds, which are at present in the kingdom Protozoa. Similarly, phylogenetic analysis shows that Oomycota grouped with different types of algae (41, 167, 540), which is also consistent with their morphology and structure. At present, Oomycota are included in the kingdom Chromista (238). The rank "kingdom" has also been questioned, arguing that this term has no molecular definition (415). In addition, it has been argued that if animals, plants, and fungi are considered to be taxonomic kingdoms, we must recognize at least a dozen other eukaryotic groups as kingdoms. These groups have at least as much independent evolutionary history as that which separates the three traditional kingdoms (415). Above the rank of kingdom, the now-recognized three primary lines of evolutionary descent termed "urkingdoms" or "domains," i.e., Eucarya (eukaryotes), Bacteria (initially called eubacteria), and Archaea (initially called archeobacteria), have been proposed (591, 592). In this context, the term "kingdom" indicates the main lines of radiation in the particular domain.

The kingdom Fungi is organized into phyla and then into classes and orders. However, some authors prefer to avoid particular categories above orders. Mycologists previously used the term "division" instead of "phylum" because it is more inherent to botany, but in the last 5 years the term "phylum," used mainly by zoologists, has been used as an alternative and has practically replaced "division." The four phyla presently accepted in Fungi are Chytridiomycota, Zygomycota, Ascomycota, and Basidiomycota (Table 1). However, the inclusion of Chytridiomycota in the Fungi has been controversial because these organisms possess flagella. On this basis, they have been included in the Protoctista (326), but comparison of cell wall polysaccharides (19) and lysine synthesis (562) linked them firmly with the other classical phyla of Fungi. Analysis of 18S rDNA sequences also showed that chytridiomycetes cluster with representatives of Ascomycota and Basidiomycota (61) in a separate clade from ciliate protists. Subsequent analysis of many fungal 18S rDNA sequences (70) showed that Chytridiomycota and Zygomycota are difficult to separate and are basal to the Ascomycota and Basidiomycota. Although Chytridiomycota form the basal branch in the kingdom Fungi, flagella may have been lost more than once during the evolution of the ancestors through to Zygomycota, Ascomycota, and Basidiomycota. It has been demonstrated that among the nonflagellated fungi, the Zygomycota diverged first, and the branch defining the Ascomycota and Basidiomycota as terminal sister groups is strongly supported (33, 70) (Fig. 1).

There are thought to be so many species of fungi that insects are the only group with more varieties. Currently, approximately 1.4 million living species of microorganisms, fungi, plants, and animals have been recorded (198, 478). Most of these species are insects (950,000) and plants (250,000). On the basis of the current rate of discovery, it has been estimated that the number of undiscovered and undescribed species ranges from 1 million to more than 10 million (478). Fungi by far outnumber bacteria and viruses. In 1995, approximately 70,000 fungal species were accepted (236) compared to only 3,100 known bacteria species and 5,000 viruses (478). However, the number of fungal species so far discovered is probably only a small proportion of those that actually exist, since few habitats and regions have been intensively studied. Many extreme environments are still unexplored or not adequately explored. It has been conservatively argued that the total number of fungal species in the world must be at least 1 to 1.5 million (234, 478), meaning that we have so far recognized only about 5 to 7% of the world's mycobiota. Other authors indicate that less than 20% of the terrestrial and marine fungi have been discovered (283). Since all proposed new species are catalogued in the Index of Fungi (edited twice yearly by the International Mycological Institute, Egham, United Kingdom), we can calculate that 800 to 1,500 new species are described annually. It is worth mentioning that some of the recently described new species have been found only in humans; these include Dissitimurus exedrus, Calyptrozyma arxii, Hormographiella spp., Onychocola canadensis, Ramichloridium mackenziei, Polycytella hominis, Pyrenochaeta unguis-hominis, Phoma cruris-hominis, Phoma oculo-hominis, Botryomyces caespitosus, and some dermatophytes.

The huge number of fungi constitutes a potentially serious problem to public health. An increasing number of fungi that are normally harmless to the immunocompetent host are able to invade the neutropenic host and cause severe infections.

There are three main groups of pathogenic fungi which are quite different from one another. Firstly, dermatophytes are a group of obligate parasites which attack human skin, nails, and hair. Secondly, dimorphic saprobes are a group of normally soil-borne fungi which have developed a different morphology in order to adapt to the hostile environment of the human body. These organisms can cause disease in healthy people. The third group, which is the most numerous, consists of opportunistic saprobes, which can attack humans whose immune systems are deficient in some way or artificially suppressed (274). The pathogenic and opportunistic fungi are distributed among three phyla of the kingdom Fungi (Fig. 2), i.e., Zygomycota, Ascomycota, and Basidiomycota, although the phylum Chromista (Oomycota and Hyphochytriomycota) also contains two human pathogens and several animal pathogens. Some of the most relevant taxonomic developments concerning the members of medical interest belonging to such phyla are addressed below.

The phylum Oomycota comprises approximately 700 species. Cellulose and -glucan are the main components of their cell walls. Some of them are virulent plant pathogens, while others are aquatic species and fish pathogens. Only a few members of this phylum, particularly species of Phythophthora and Pythium, have been the focus of molecular studies aimed at species level and below (24, 71, 328). Only the species Pythium insidiosum, belonging to the order Peronosporales, is an opportunistic pathogen in warm-blooded animals (118). It only rarely causes infection in humans (561, 572). Its life cycle was described by Mendoza et al. (346). An immunodiffusion test, which can be an important aid for its diagnosis, was developed by Pracharktam et al. (439). Rhinosporidium seeberi, which belongs to the phylum Hyphochytriomycota, causes rhinosporidiosis in humans and is characterized by the formation of cysts in the subepithelial tissue of nasal mucosa. These infections occur in India and South America. It is not a well-known fungus because it cannot be grown on routine laboratory media.

The Zygomycota are a group of lower fungi whose thalli are generally nonseptate (coenocytic) (Fig. 3, structures l and m). After the fusion of isogamic sex organs (gametangia) (Fig. 3, structure c), they produce a single, dark, thick-walled, often ornamented sexual spore, called zygospore (Fig. 3, structure b). In host tissue, they are recognized by their broad, aseptate, hyaline, randomly branched hyphal elements. Zygomycota are divided into two classes (Table 1) and 11 orders (238). Fungi of clinical interest are found in only two of them: the small group of Entomophthorales, which have forcibly ejected spores, and the Mucorales, whose spores arise by cleavage of the sporangial plasma and are passively liberated (404, 602). In culture, these fungi show typical characteristics described below.

View larger version (48K): [in this window] [in a new window]   FIG. 3.   Typical characteristics of zygomycetes. a, mycelial colony; b, zygospore; c, gametangium; d, sporangium; e, sporangiole; f, merosporangia; g, columella; h, sporangiospores; i, merospores; j, stolon; k, rhizoids; l, coenocytic hypha; m, section of a hypha; n, sporangiophore.

The order Mucorales is the most clinically important. Its members are widely distributed and are found in food, soil, and air. Several species are thermophilic. Eleven genera, containing 22 species of medical interest, have been described and illustrated by de Hoog and Guarro (118). The most frequent, significant mucoralean genera in the clinical laboratory are Rhizopus and Absidia. Mucor is abundant as a contaminant but is very rarely an etiologic agent. However, in recent years, some rare genera have been associated with human infections, e.g., Cokeromyces (273), Saksenaea (330), Apophysomyces (140), and Chlamydoabsidia (79). The clinical picture is rather similar throughout the whole group. Most rhinocerebral mycoses are found in patients with diabetes mellitus, although other types of infection (e.g., gastrointestinal, pulmonary, cutaneous and systemic) are also frequently reported. Disseminated and severe infections are encountered mainly in patients with immunological disorders (156, 304, 481).

Recognition of a clinical isolate which belongs to mucoralean fungus is easy and can be done by observing only the macroscopic features. Lax, woolly colonies which spread over the whole petri dish in a few days and which have small dots (sporangia) (Fig. 3, structures a and d) on the periphery are sufficient to identify them. This identification can be microscopically confirmed by the presence of nonseptate, wide, hyaline hyphae with the typical fertile structures of mucorales. Although serological tests have been developed (129, 267), the identification of the species is rather more complicated. The procedure is both time-consuming and labor-intensive and usually requires special media, some of which are not commercially available. Some species have considerable morphological variation, and in these cases it is important to culture the fungi under optimal conditions to determine the variability of characteristics (29, 480, 481, 578). The species is identified almost exclusively by careful microscopic observation and measurements of several morphological characteristics which are typical of asexual reproduction. This process takes place by means of sporangiospores (Fig. 3, structure h) produced in sporangia (multispored) (structure d), in sporangioles (with one or very few spores) (structure e), or in merosporangia (spores in rows) (structure f). A sporangium generally has a central columella (structure g), which may extend and be visible below the sporangium as a swelling known as the apophysis. Other structures such as zygospores, chlamydospores, or a combination of them are produced only in some organisms. The value of zygospore morphology is limited because the sexual spore has never been reported for many taxa (29). However, the production of zygospores was considered to be very useful in clinical zygomycete taxonomy, especially for the identification of rare, heterothallic zygomycetes (578). An important limitation to this technique is the need for tester strains to try to stimulate zygospore production (578). Several species creep over the agar surface with stolons (Fig. 3, structure j), and the substrate can be penetrated by means of rhizoids (structure k). These structures also have a certain diagnostic value. Fatty acid composition has been investigated in Mortierella (6). A variety of biochemical and physiological tests (carbon and nitrogen assimilation, fermentation, requirement for thiamine, maximum temperature of growth, etc.) are also used by different authors (481, 578). Both SEM and TEM are important aids for the detailed observation of numerous structures and allow the morphology of surface ornamentations to be discerned. They can also resolve aspects of the ontogeny of sporangiospores (29, 404).

Benny (29) suggested exploring some traditional characteristics by phylogenetic studies. He considered it useful to investigate possible differences in sporangiospore ontogeny and distribution of chemical compounds and to search for unknown zygospores. It seems logical to expect that molecular studies will provide more consistent data. Molecular studies have only very rarely been performed in the Mucorales. Radford (443) compared sequences of the orotidine 5'-monophosphate decarboxylase gene and showed that Mucorales was a sister group of basidiomycetes. As a result of a preliminary cladistic analysis based on morphology and SSU rDNA sequences (407), Benny (29) pointed out that many characteristics used to define mucoralean families probably do not indicate relationships but are still useful for identification. The lack of agreement between molecular data and morphological observations could indicate that many of the characteristics which are considered advanced in the Mucorales have arisen several times in the order (29).

In the order Entomophthorales, there are only three species occasionally pathogenic to humans: Conidiobolus incongruus (261, 569), Basidiobolus ranarum (112, 276), and Delacroixia coronata (Conidiobolus coronatus) (385). These species are characterized by forcibly discharged spores borne on tubular sporophores. There are very few molecular studies of the taxonomy of this order (383).

Ascomycota is the largest phylum of Fungi. It comprises almost 50% of all known fungal species and approximately 80% of the pathogenic and opportunistic species. As a reference framework for the systematic of ascomycetes, the University of Umea and the International Mycological Institute publish jointly the periodically revised Outline of the Ascomycetes, which is developed through the journal Systema Ascomycetum. It is published about every 4 years and is based on data provided by numerous specialists (153). Proposals for changes at the generic level and above are compiled in twice-yearly issues of that journal. The classification scheme included in these outlines is followed by the majority of mycologists involved in ascomycetes systematics.

The basic characteristic which differentiates ascomycetes from other fungi is the presence of asci (Fig. 4, structures e to g) inside the ascomata (structures c and d). However, even in the absence of these important diagnostic characteristics, the ascomycetes can be recognized by their bilayered hyphal walls with a thin electron-dense outer layer and a relatively electron-transparent inner layer (238). The arrangement of the asci has played a major role in supraordinal systematics, and for a long time the ascomycetes have been grouped into six classes, i.e., Hemiascomycetes, Plectomycetes, Pyrenomycetes, Discomycetes, Laboulbeniomycetes, and Loculoascomycetes (372). The Plectomycetes are characterized by the presence of closed, more or less spherical fruiting bodies (cleisthothecia) (Fig. 4, structure d), with an irregular distribution of the asci in the cavity, and the ascospores are released after the disintegration of the thin walls of the asci. The dimorphic pathogens (Histoplasma, Coccidioides, Emmonsia, etc.) are included in this group, as are the teleomorphs of the dermatophytes and of the frequent and cosmopolitan Penicillium and Aspergillus among others. The Pyrenomycetes have pyriform (flask-shaped) fruiting bodies (perithecia) (Fig. 4, structure c) with usually saccate or cylindrical asci. The ascospores are forcibly extruded from the ascus. The teleomorphs of several opportunistic fungi are also included in this class. The class Loculoascomycetes comprises species with bilayered asci, sometimes enclosed in stromatic ascomata. The sexual states of numerous pigmented (dematiaceous) pathogenic fungi are included in this latter class. The class Hemiascomycetes comprises the yeasts. In the remaining two classes there are no fungi of medical interest. An alternative, long-accepted classification scheme placed the ascomycetes into the classes Hymenoascomycetes and Loculoascomycetes, depending on whether the ascus wall was single (Fig. 4, structure f) or bilayered (structure g), respectively (17, 18, 317). Several other taxonomic schemes have been established over the years and have been accepted to greater or lesser extents (238).

View larger version (49K): [in this window] [in a new window]   FIG. 4.   Typical characteristics of ascomycetes. a, mycelial colony; b, yeast colonies; c, perithecium; d, cleistothecium; e, ascus; f, unitunicate asci; g, bitunicate ascus; h to m, anamorph types; n, conidiophore; o, conidiogenous cell; p, hyphae; q, detail of a uniporate septum; r, detail of a multiporate septum.

The traditional systems of ascomycete classification have been much criticized due to their artificial nature. It has been repeatedly argued that the supposed similarities among these major groups may not reflect homology. Another much questioned aspect has been the confusing terminology. One example is the term "plectomycete," which has been defined as a closed ascoma (162) and also as scattered asci within the ascomal cavity (317). Another problem inherent in traditional classifications is the convergence of fruiting bodies (77, 320). It has been demonstrated that species which normally produce flask-shaped fruiting bodies can be induced to form closed fruiting bodies under certain environmental conditions (563). Some species, such as the recently reported opportunistic fungi, Microascus spp., have both the fruiting-body type of the Pyrenomycetes and the ascus arrangement characteristic of the Plectomycetes. For these reasons, it has been considered that there are too few stable morphological features which are useful for inferring relationships among higher taxa. Therefore, many mycologists currently deemphasize supraordinal rankings and the relationships that they define (150) while others support the use of these supraordinal rankings as reflecting natural groupings.

Molecular data are adding a new dimension to the understanding of the relationships among the different groups of ascomycetes. One of the first phylogenetic trees to depict the evolutionary history of ascomycetes was published by Berbee and Taylor (35), who studied morphological convergence in true fungi. They recognized three main groups of ascomycetes: (i) the basal ascomycetes, which included the Schizosaccharomycetales (fission yeasts) and Pneumocystis; (ii) the true yeasts, i.e., filamentous or unicellular ascomycetes without fruiting bodies; and (iii) the filamentous ascomycetes with fruiting bodies. Numerous authors subsequently provided sequences of many other fungi which have been useful for filling some of the gaps in the evolutionary history of ascomycetes. Soon it may be possible to build a phylogenetic tree for all ascomycetes. Meanwhile, it is evident that some traditional classes such as the Pyrenomycetes (Hypocreales, Microascales, Diaporthales, and Sordariales) and the Plectomycetes (Eurotiales and Onygenales) are also represented by well-supported monophyletic clades. However, the monophyly of the classes Hymenoascomycetes and Loculoascomycetes was rejected (501, 505). There are examples of perithecial and cleistothecial ascomycetes that do not group with these well-supported clades. Despite this, we consider that some of these old class names are useful, at least until sequences of more ascomycetes become available and the boundaries within these groups are defined. Therefore, in this article, and for the sake of simplicity, the taxa are arranged into five morphological groups, i.e., basal ascomycetes, unitunicate Pyrenomycetes, bitunicate Pyrenomycetes, Plectomycetes, and budding yeasts. The relationships among these groups are inferred from the analysis of 18S rDNA and suggest an early radiation of Schizosaccharomycetales and Pneumocystis carinii, which represent a basal branch in the Ascomycota (basal ascomycetes). This was followed shortly by a bifurcation leading to budding yeasts on one branch and filamentous fungi with fruiting bodies on the other. Among the latter, there is another radiation comprising the unitunicate Pyrenomycetes followed by the Loculoascomycetes (bitunicate Pyrenomycetes) and Plectomycetes (Fig. 5). However, the delimitations between these two groups are not well defined.

View larger version (22K): [in this window] [in a new window]   FIG. 5.   Diagnostic unrooted phylogenetic tree showing the relative positions of fungal clades of clinical importance by 18S rRNA sequences.

Eriksson (152) recently also used these groups to demonstrate the value of the signature sequences in the taxonomy of ascomycetes. Signatures are short sequences in moderately conserved areas of DNA, RNA, or protein. They are characteristic of taxa of various ranks and give phylogenetic information that is not provided by other methods. Studying the terminal part of stem-loop E23-1 in the V4 region of 18S rRNA according to the terminology of Neefs et al. (381), Eriksson found that in most cases it consisted of a short helix of 3 or 4 bp and an end-loop of usually 6 bases. Above the helix, there is almost always a bulge of a single C, and below is a bulge of usually 3 bases. Supposed signatures, end-loops, and a larger bulge in this helix were discussed by Eriksson for different groups of ascomycetes and, for comparison, for some basidiomycetes. He found that when numerous taxa of the Plectomycetes and Pyrenomycetes were examined, they practically all had the loop CTCACC, which was not found in any other group of either ascomycetes or basidiomycetes, and they all had a helix of 3 bp. Other noteworthy findings were found in the signature sequences of the other groups examined such as the budding yeasts. For example, a higher A+T content was found in these sequences than in other ascomycetes and basidiomycetes. Surprisingly, a great variation was found in stem-loop E23-1 of the basidiomycetes compared with ascomycetes.

At present, 46 orders are accepted in Ascomycota (238) but only 9 of them include fungi of clinical interest (see Tables 2 to 8).

Pneumocystis carinii is regarded as the only member of the order Pneumocystidales (238), although immunological and molecular data (14, 143, 187, 309, 514, 568), and host species specificity (5, 176, 188, 352) support the existence of different species or varieties (76). The organism has been isolated from a wide range of unrelated mammalian hosts, including humans. It has been known since the beginning of this century (92) and for nearly 70 years has been reported only as the agent of mild infections, against which residual antibodies are developed. It also causes occasional epidemic pneumonia under conditions of overcrowding and malnutrition (76). During the last 15 years, the organism has emerged as one of the most common pulmonary infections in AIDS patients, and its presence has become one of the first indications of the disease. This organism cannot be cultivated in routine laboratory media (108). In vivo, in alveolar tissue, it is characterized by the absence of hyphal elements. The vegetative cells are thin-walled, irregularly shaped, uninucleate, divided by fission and transformed into a thick-walled cyst-like structure (asci), with up to eight internal cells (ascospores) which are at first globose but become falcate. The crescent-shaped cysts are thought to liberate these endospores (238). The detection of P. carinii is enhanced by cellofluor staining (21) or by immunofluorescent staining with monoclonal antibodies (386). PCR-mediated detection has been developed on the basis of thymidylate synthase sequences (412).

For a long time, P. carinii was considered a Protozoan, but morphological studies have demonstrated its fungal affinities (556) and molecular studies have confirmed this (144, 515, 574). First, analysis of the mitochondrial 24S rDNA gene has shown a close relationship between P. carinii and the simple-septate, red basidiomycete yeasts (568). Morphological characteristics such as the absence of laminated cell walls (599) typical of basidiomycetes and the presence of a double-membrane which delimits each developing intracystic body (ascospore) (333), as well as molecular data (540), apparently confirm that P. carinii belongs to the Ascomycota. On this basis, a change in the terminology seems reasonable for descriptions of P. carinii infections. The terms "parasite," "intracystic bodies," "trophozoites," and "infestation" (commonly reserved for parasites) should be abandoned and a terminology which is applicable to fungi should be adopted (483).

The wall structure of the asci determines two basic kinds of Pyrenomycetes: those with a single wall are called unitunicate (Fig. 4, structure f), and those with a bilayered wall are bitunicate (structure g). The unitunicate ascus sometimes has an operculum (a small lid), which opens to liberate the ascospores when the ascus is mature. These asci are called unitunicate-operculate. No pathogenic fungi belong to this group. Other asci have no operculum but have an apical pore and/or a ring-like structure at the tip, acting as a valve, or sphincter, through which the ascospores are violently discharged and are dispersed by the air. These forcibly ejaculated ascospores can reach long distances. Such asci are called unitunicate-inoperculate (274) and are present, for example, in Neurospora (see Table 3). There are still other perithecial fungi with unitunicate asci which have no obvious mechanism for ascospore release. In this case, the ascospores are released into the cavity of the ascomata, when the ascus wall disintegrates. These ascospores are never forcibly