C. P. Kurtzman's Evolving Concepts of Species, Genus and Higher Categories

FEMS Yeast Research, 18, 2018, foy103

doi: 10.1093/femsyr/foy103 Advance Access Publication Date: 20 September 2018 Minireview

MINIREVIEW

C. P. Kurtzman’s evolving concepts of species, genus Downloaded from https://academic.oup.com/femsyr/article/18/8/foy103/5104380 by guest on 29 September 2021 and higher categories Marc-Andre´ Lachance

Department of Biology, University of Western Ontario, London, ON N6A 5B7, Canada

Corresponding author: Tel. 1-519-661-3752; E-mail: [email protected] One sentence summary: Cletus P. Kurtzman revolutionised yeast systematics; this is an overview of the evolution of his thoughts on yeast species, genera and higher categories. Editor: Teun Boekhout

ABSTRACT Cletus P. Kurtzman transformed the way yeast systematists practice their trade and how they perceive the yeast species. He redefined many genera of ascomycetous yeasts and provided a sound basis upon which to base higher taxonomic categories. Within his extraordinary corpus lies a trail of elements that can be used to reconstruct his evolving vision of the concepts that underlie the species and the genus, rarely set in a theoretical framework. While occasionally tipping his hat to the biological and phylogenetic species, Kurtzman espoused a concept founded primarily on genetic distance, even when claiming otherwise. In contrast, his notion of genus incorporated components of both genetic distance and phylogenetic structure, and possibly a size consideration. A phylogenetic approach predominated with higher taxa.

Keywords: Cletus P. Kurtzman; yeast species; yeast genus; genetic distance; phylogenetics; Ascomycetes

INTRODUCTION Kurtzman’s laboratory lived up to the highest standards and implemented forefront methodologies for imaging, ascospore ‘I have never done anything “useful”. No discovery of mine has dissection or nucleic acid analyses. He inherited from Wicker- made, or is likely to make, directly or indirectly, for good or ill, the ham a sense of minutious diligence that continued throughout least difference in the amenity of the world.’ his career (Wickerham, Kurtzman and Herman 1969). His later writings show an immense knowledge of the properties of vari- While this modest confession may be appropriate for some, ous genes in terms of evolutionary rates, concordance with other it holds true neither for its author, mathematician G.H. Hardy genes, or the best primers or cycling conditions to obtain im- (1940), nor for yeast systematist Cletus P. Kurtzman. So much peccable amplifications. The designation NRRL on a sequence could be said about Kurtzman’s monumental opus. In my task of deposit continues to be a certificate of quality. sieving through his writings to unravel his thought on the con- Ihavealluded(Lachance2018) to Clete’s generosity as cepts of species, genus and higher taxa, I was reminded of many a colleague, manifested, inter alia, in naming many taxa in other highlights of his career. Of the greatest importance was his honour of other yeast systematists. These recognitions were implementation and promotion of DNA sequencing, which gave sometimes granted in the face of criticisms, nay disparage- us a barcode identification system for all ascomycetous yeasts ments. Characterisation of nomenclatural choices as ‘point- (Kurtzman and Robnett 1998a). Although the term ‘barcode’ only less’, ‘taxonomically inept’ or ‘bordering on lunacy’ (Barnett appears late in his writings (Kurtzman 2010), Kurtzman (1994a)is 2004) still earned the critic an eponymous genus (Kurtzman, rightly credited as a pioneer of the approach (Hebert et al. 2003), Robnett and Basehoar-Powers 2008) consisting, perhaps ironi- which entails the creation of a comprehensive database of easily cally, of an eclectic brew of species formerly assigned to Pichia, accessible homologous sequences (Kurtzman and Robnett 1991).

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Zygohansenula, Hansenula, Williopsis and Endomycopsis. Of course, entiated the two (Kurtzman, Smiley and Baker 1975), as did dif- challenge and debate, not sycophantry, make for good science. ferences in DNA base composition. Part of Clete’s success stemmed from remaining undeterred by Following an eclipse of nearly three decades, morphology sceptics, although he was terribly displeased when I chose for resurfaced with the remarkable discovery (Kurtzman 2004)of a genus the name Diutina, meaningpermanent, to lament fre- mating and ascus formation in heterothallic strains what were quent name changes (Khunnamwong et al. 2015). Clete may reassigned to the genus Trichomonascus because the asci resem- have savoured the occasional taunt, portending the eventual bled those of Trichomonascus mycophagus. Living material for the split of my favourite genera, Metschnikowia, Starmerella and Wick- latter species was not available, but the ‘nearly identical unique erhamiella (e.g. Mendonc¸a-Hagler, Hagler and Kurtzman 1993; morphology of the sexual states of the new species and T. my- Kurtzman and Robnett 2007; Kurtzman et al. 2010). He settled cophagus leaves little doubt that they are closely related’. More the score posthumously (Kurtzman et al. 2018) with a broad phy- importantly, in the present context, species delineation was logeny of Metschnikowia in which all 30 species of large-spored entirely morphological: ‘Ascospore morphology clearly distin-

Metschnikowia,mymagnum opus, were omitted. ‘Metschnikowia, guishes T. petasosoporus and T. mycophagus, despite lack of ver- Downloaded from https://academic.oup.com/femsyr/article/18/8/foy103/5104380 by guest on 29 September 2021 as presently recognized, may actually represent several gen- ification from gene sequences.’ era. The large-spored species [...] form a separate clade [...].’ Coincidentally, a well-supported phylogeny placing the large- Biological species spored group comfortably inside the genus Metschnikowia nearly In a fleeting foray into the world of Basidiomycetes, Kurtz- appeared in the same journal issue (Lee, Hsiang and Lachance man (1973) discovered mating reactions among strains identi- 2018). fied from growth responses as Cryptococcus laurentii. Although a complete sexual cycle was not documented, the mating pat- Species concepts terns suggested that the various strains represented more than one taxon. The mating strains were later resolved as Papiliotrema Mayden (1997) argued that most species concepts are an attempt flavescens, and the incompatible strains are now named Papil- to approximate the evolutionary concept, which views species iotrema laurentii, Vishniacozyma carnescens, Kwoniella heveanensis as communities that share a common fate over evolutionary or Filobasidium magnum—but not through Kurtzman’s doing (Liu time. Because of the temporal factor, detection of evolutionarily et al. 2016). The same strains were revisited (Baptist and Kurtz- cohesive units is not directly possible and must resort to other, man 1976) using isoenzyme electrophoresis and DNA base com- operational concepts. Darwin (1859) predicted that we would position, with the conclusion that they represented two taxa. one day ‘be freed from the vain search for the undiscovered and Morphology still prevailed: ‘Because the sexually reactive strains undiscoverable essence of the term species’. At a conference in cannot be separated by assimilation tests, we chose not to de- the early 2000s, Clete was apparently confident that the goal scribe them as a new species’. Discontinuities in the isoenzyme had been achieved. He opened the conversation with: ‘What’s and GC data were also used to assign the remaining strains a genus?’, implying, I believe, that the matter of the species was to species bearing three of the specific epithets listed above, a case closed. presage of the genetic species concept that will dominate Kurtz- man’s later work. Morphological species A morphological concept was implicit also in two taxonomic Theorists use the term morphology in the sense of Hennig’s studies of Saccharomycopsis species (Kurtzman and Wickerham (1966) holomorphology, which for yeasts includes both visual 1973; Kurtzman, Vesonder and Smiley 1974), even though het- appearance, growth responses or chemotaxonomic traits, what erothallic mating among strains of Saccharomycopsis crataegen- Kurtzman termed phenotype. Wickerham and Kurtzman (1971) sis and absence of mating with other species were said to be delineated two Saturn-spored Pichia species on scanning elec- a pivotal criterion in setting boundaries. Similarly, Kurtzman tron microscopy of the ascospores, although they did not explic- and Ahearn (1976) examined a group of heterothallic strains itly specify a species criterion. Spore ornamentation was also of Pichia and Candida species that physiologically resembled P. examined in Schwanniomyces (Kurtzman, Smiley and Baker 1972) spartinae, and kept them separate based on mating patterns. in the hope that it would reveal information on relationships The criterion was applied to P. kudriavzevii and P. terricola,which among species. However, two strains assigned to Schwanniomyces were mixed with mating types of several other Pichia or Candida persoonii differed in surface ornamentation, introducing an el- species (Kurtzman and Smiley 1976). Mating between P. kudri - ement of doubt. Growth test responses were added to the cir- avzevii and strains of Candida krusei or C. sorbosa led to the con- cumscription of the species. A similar morphological concept clusion that they are conspecific, and they have been treated as was employed in the delineation of Pichia besseyi (Kurtzman and such in the last two editions of The Yeasts (Kurtzman and Fell Wickerham 1972). 1998; Kurtzman, Fell and Boekhout 2011). As criteria such as mating response, sexual offspring viability As the genospecies concept, discussed next, gained accep- and DNA-based approaches gained momentum, growth charac- tance, doubt began to be cast on the supremacy of reproduc- teristics grew to be less and less important, eventually leading to tive isolation as the golden standard. Kurtzman et al. (1980) their outright rejection. This began with a study of species that noted that spores isolated from a same diploid parent are in form asci by mother-bud conjugation, a so-called Torulaspora- some cases unable to mate, in which case the decision to con- type life cycle (Kurtzman, Smiley and Baker 1975). In that re- sider strains as separate species should be based primarily on spect, Debaryomyces marama differed from other species of De- DNA relatedness: ‘use of fertility as the sole criterion for specia- baryomyces to an extent that it might be better assigned to the tion must be carefully considered’. The statement was qualified, genus Pichia, although it was later shown to be a close relative however: ‘Conversely, defining species strictly on the basis ofa of D. hansenii (Kurtzman and Robnett 1991). Surface ornamen- certain percentage of DNA relatedness may conflict with the bi- tation failed to separate species assigned to Debaryomyces from ological reality of genetic exchange. Almost certainly, consider- those of the Torulaspora group, then assigned to Saccharomyces, able latitude will be found in the definition of species even at the although inner ascospore wall ultrastructure adequately differ- genetic and molecular levels.’ Lachance 3

As borderline cases continued to be encountered, even inter- ences [exist] between these two species, and on the more prac- fertility began to join the ranks of subordinate properties: ‘As- tical grounds that it [is] important that species used for food fer- sessment of relatedness among yeasts through comparison of mentations possess different species names from those which DNA base sequence complementarity seems to offer a quantita- are known to be mycotoxigenic.’ Three decades later, in an ex- tive means for determining kinship that is superior to present tensive study of relevant Aspergillus species, Frisvad et al. (2019) taxonomic schemes, which can be influenced markedly by confirmed that aflatoxin production does not separate strains changes in a few genes, such as those controlling mating com- along phylogenetic lines, but nonetheless continued to delin- petence or carbon assimilation’ (Kurtzman, Smiley and Johnson eate species on morphological features thought to correlate with 1980). Kurtzman (1984a) nonetheless continued to recognise that toxicity. ‘divergence as shown by lack of fertility between populations Implementation of DNA reassociation also marks the be- should [...] be regarded as the primary criterion for defining ginning of a long series of rejections of growth characteris- species [...]as the ability to hybridize and produce fertile suc- tics as taxonomically useful because they could be polymorphic

cessive generations would seem to be of cardinal importance.’ within a genospecies. The following list also includes characters Downloaded from https://academic.oup.com/femsyr/article/18/8/foy103/5104380 by guest on 29 September 2021 Paraphrasing the Linnaean aphorism that species are the ‘prod- later found dubious in defining genera: melibiose and raffinose uct of nature rather than the creation of the taxonomist’, Kurtz- assimilation, ascus dehiscence (Kurtzman and Smiley 1979), man (1985b) cautioned that yeasts suffered in the application of interfertility (Kurtzman Smiley and Johnson 1980), disaccha- the Dobzhanskian concept because of their propensity to feature ride, oligosaccharide and nitrate utilisation, formation of pseu- ‘unknown sexual stages, lack of complementary mating types dohyphae or true hyphae, ascospore morphology (Kurtzman, and loss of fertility’, making alternative determinations neces- Phaff and Meyer 1983), physiology and morphology (Kurtzman sary. This point was stressed again a decade later when applying 1984b, c), ribitol utilisation (Kurtzman 1987c), coenzyme Q, as- sequence-based species delineation to species assigned to the cospore ornamentation, cellular linoleic acid (Kurtzman and Liu genus Candida (Kurtzman and Robnett 1997). Allusions to the bi- 1990, Kurtzman and Robnett 1991), bipolar budding (Boekhout ological species concept persisted, but with an increased sense et al. 1994; Kurtzman and Robnett 1998a), septal pore and pres- that it is better appraised by molecular approaches than by mat- ence or absence of budding (Kurtzman and Robnett 1995), cold ing experiments. Two strains shared ‘only 30% DNA relatedness’, adaptation (Kurtzman and Robnett 1997) and lactose utilisation indicating that they are ‘biologically separate species’ (Kurtzman (Kurtzman 1998). In contrast, the following characters were 1992a). recognised, based on genetic distance, as potentially useful in the differentiation of species or genera: starch assimilation Genetic species (Kurtzman et al. 1980), septal pores (Kurtzman, Phaff and Meyer Following Mayden (1997), the genetic species concept is meant 1983;Gueho,´ Kurtzman and Peterson 1989), ribitol utilisation to surrogate the biological species based on a measure of ge- and growth at 37◦C (Kurtzman 1987c), xylose in cell wall and netic divergence. In the words of Kurtzman, Johnson and Smiley coenzyme Q (Gueho,´ Kurtzman and Peterson 1990), ogival cells (1979): ‘The unlikelihood of resolving speciation through mat- and acetic acid production (Kurtzman (2005c), methanol util- ing tests prompted us to make comparisons through studies isation (Kurtzman and Robnett 2010), adenine utilisation and of deoxyribonucleic-acid (DNA) base-sequence homology’. In- xylose fermentation (Kurtzman and Robnett 2013b). Note that deed, the online Oxford English Dictionary describes ‘genospecies’ some traits were regarded as useful in some contexts and dubi- as ‘a species defined by the genotypic similarity of its mem- ous in others. bers [...] defined by DNA relatedness’. Although the concept Rejection of a trait was often accompanied by a statement itself is rarely made explicit in Kurtzman’s writings, I recall a that DNA reassociation samples the whole genome, in contrast conversation where a colleague suggested that his sequence to growth characteristics, which are controlled by only a few divergence-based approach to species delineation was phenetic, genes (e.g. Kurtzman 1984b). Kurtzman and Robnett (2003)gen- and not phylogenetic, to which he forcefully countered that eralised: ‘there is now a widespread pattern of disparity between his was a genetic concept, which he soon restated in writing phenotype and genotype as means for classifying yeasts [...] (Kurtzman 2014): ‘A better understanding of the genetic defini- leaving little doubt that phenotype is a poor predictor of genetic tion of a species is needed’. relationships among species’. As it happens, the fate of a derived DNA reassociation became the tool of the trade in the Peoria character state starts with its acquisition in an ancestral species laboratory in the late 1970s, leading to the demonstration that followed by its occasional loss and even reversal in descendent P. carsonii,itsvarietiesandP. vini were indistinguishable by this lineages. A typological outlook that insists on perfectly consis- approach (Kurtzman and Smiley 1979). A first of several papers tent characters is destined to meet with disappointment. dealing with the classification and delineation of species at one time assigned to the genus Saccharomyces (Vaughan Martini and Calibrating DNA reassociation Kurtzman 1985) added credence to the suspicion that S. kluyveri The DNA reassociation scale was an important theme. Kurtz- was distinct from core Saccharomyces species. The study also re- man and Smiley (1979) started with the view expressed by tained S. bayanus as separate from baker’s yeast, and corrobo- other authors that reassociation values of 80% or more indi- rated other reports that several named species were in fact syn- cated conspecificity, while Kurtzman, Johnson and Smiley1979 ( ) onyms of S. cerevisiae. added an exclusion value of 25–30%. Kurtzman thought it im- The classical mycological community experienced conster- portant to correlate DNA reassociation values with mating com- nation when the genetic species concept was extended to fil- patibility as in the case for P. amylophila and P. mississippiensis amentous fungi. Isolates responsible for aflatoxin production (Kurtzman et al. 1980): ‘the isolates proved to be of particu- gave high DNA reassociation values with strains used in food lar interest because they allowed an examination of species fermentations (Kurtzman 1985a; Kurtzman et al. 1986). As a com- parameters through both genetic crosses and DNA reassocia- promise the authors proposed a variety structure for what oth- tion studies’. Mating strains incapable of generating viable as- erwise should be treated as single species without varieties. Pitt cospores gave reassociation values of 20–27%. A meaningful in- (1989) rejected the changes, arguing that ‘morphological differ- terpretation of reassociation values also required reproducibility 4 FEMS Yeast Research, 2018, Vol. 18, No. 8

from one laboratory to the next and in this instance, results ob- ‘understanding of broader relationships must await the appli- tained by the spectrophotometric method used in Peoria were cation of molecular techniques that measure more conserved favourably juxtaposed with those obtained by the chromato- regions of the genome’. In a review published the same year, graphic method of the Davis laboratory. Kurtzman (1987a) pointed out that the correlation between DNA In examining strains assigned to the two varieties of the relatedness and fertility had its limits, and that DNA comple- newly renamed Issatchenkia scutulata, Kurtzman, Smiley and mentarity should not be regarded as an Johnson (1980) found that the varieties formed 21–26% heteroduplex DNA while being able to mate and give rise to 3 to 6% viable infallible indicator of biological relatedness. [...] Wewouldexpect ascospores, which when backcrossed, acquired increased viabil- high relatedness between: (i) Subcultures such as: (a) Single-spore ity. This could be taken to be a textbook example of hybridisa- isolates, (b) mycelial transfers, (c) morphologically distinct sectors tion with the potential for introgressive gene transfer. Instead of a colony, (d) mutants of a parent colony and (e) mating types from heterothallic species; (ii) toxigenic and non-toxigenic strains the authors regarded such cases as of the same species; (iii) domesticated strains and their wild coun-

a nuisance to taxonomists [...]. Although we have retained the terparts from nature; (iv) anamorphs with their teleomorphs; (v) Downloaded from https://academic.oup.com/femsyr/article/18/8/foy103/5104380 by guest on 29 September 2021 original varietal designations because of reduced fertility and low synonymous species, [but not] to resolve: (i) whether species be- sequence complementarity between the varieties, these findings long to a particular genus; (ii) the hierarchy of species within a are of considerable importance to yeast taxonomy, for they show genus. that strains capable of genetic exchange may exhibit DNA relatedness no greater than about 15% above the relatedness observed In a discussion of species of Williopsis and Pichia, between unrelated species. Kurtzman (1991a) stated that comparisons ‘of DNA complementarity and fertility among heterothallic yeasts in several Mating strains in the genus Hansenula formed sterile asci and genera have shown a reasonably linear correlation’, and in had a reassociation value of 19%, which would on its own war- describing Saturnispora ahearnii, Kurtzman (1991b) took a reas- rant separation of varieties into full-fledged species (Kurtzman sociation value of 30% with a close relative to be the decisive 1984b). In contrast, a pair of homothallic varieties was retained criterion for species separation. There were also uncertainties based on 41% reassociation. Pichia bovis and P. o nychi s ,with (Kurtzman 1991a), such that a ‘possible solution to allow impo- 40% DNA reassociation, produced different electrokaryotypes sition of a taxonomic hierarchy on a genetically variable group and formed diploid ascospores, a sign of postzygotic isolation would be to consider all of the strains showing intermediate (Kurtzman 1987b). DNA relatedness as varieties of a single species’. Nearly a The difficulty of identifying a set point that defines species decade later, Kurtzman (1998) tabulated a set of cardinal DNA boundaries may suggest that no such value exists. The prob- reassociation values, where a ‘% nDNA relatedness of 70 or lem may in part hinge on treating % heteroduplex formation as more indicated conspecificity and high expected fertility, values a linear correlate of overall genome sequence identity, which of 40 to 70 were evidence for varieties or sister species with low it is not. This misinterpretation persisted in review papers or no fertility, and values below 40 a sign of separate species (Kurtzman, Phaff and Meyer 1983; Kurtzman 1987a) where re- with no fertility.’ associations of 25% were interpreted as 75% genome sequence divergence (the theorical maximum mismatch between two un- Ignoring the phylogenetic species related sequences). The authors were aware that ‘detectable By 1987 (Kurtzman 1987a, 1989b), it had become amply clear DNA homology presupposes a relatively high degree of nu- that DNA reassociation data were unable to provide useful phy- cleotide complementarity’ (Kurtzman 1984a), but the signifi- logenetic information, but a promising new approach, rRNA cance of this correspondence was not always evident in dis- gene sequencing, would be the foundation for a phylogeneti- cussions of reassociation results. The linearity assumption was cally based species concept. Victory was declared a decade later common (Kurtzman 1987a): ‘some workers have proposed that (Kurtzman 1998): ‘The goal of systematics is to define taxa on the microbial strains must show at least 30% DNA divergence before basis of their natural or evolutionary relationships, and phylo- they are considered separate species’. The problem was later genetic analysis of gene sequences provides the opportunity to acknowledged (Kurtzman 1992b): ‘as evolutionary distances in- achieve this objective’, and again (Kurtzman 2006): a ‘phyloge- crease, a point is reached at which there is insufficient base se- netic species concept based on D1/D2 rDNA analysis generally quence similarity to allow duplexing of paired molecules. It has parallels the biological species concept and serves as a baseline been suggested that sequences must exhibit 75 to 80% or greater for understanding resolution from other gene sequence analy- similarity before reassociation can occur’. Kurtzman (2006)reit- ses’. These articles of faith notwithstanding, truly phylogenetic erated: ‘Measurements of DNA complementarity are commonly considerations remained mostly absent from Kurtzman’s per- expressed as % relatedness. This usage can be misleading be- ception of species. Comments relevant to tree topology only because DNA strands must show at least 75%–80% base sequence gan to appear late as Kurtzman’s attention shifted to the genus similarity before duplexing can occur and a reading is registered (Kurtzman and Robnett 2007). on the scale of % relatedness’. The realisation that an objective Application of DNA sequencing to species delineation (Kurtz- calibration of the DNA reassociation scale might be utopic led to man 1989a; Peterson and Kurtzman 1990) began with the a more relaxed generalisation (Kurtzman 1987b): examination of six short regions of the two larger rRNA genes The picture is less clear when DNA relatedness is between 65–70% to determine their ability to resolve closely related species. The and background. In this range, sufficient genetic divergence has regions were selected following conversations with Carl Woese, generally occurred to have a major effect on fertility. This suggests who provided the required primers (Gueho,´ Kurtzman and that the speciation process is underway and that we must then Peterson 1990). The limited read length afforded by the reverse consider strains showing this diminished relatedness as either va- transcriptase sequencing method made it advisable to search rieties or separate species, depending on what we perceive of their for sequences that had an optimal blend of conservation and chromosomal make-up and other genetic factors. divergence. A preference for the large subunit rRNA gene D2 Kurtzman (1987c) stated that DNA reassociation was unlikely variable domain soon emerged (Gueho,´ Kurtzman and Peterson to be useful beyond closely related species, prophesising that an 1989). The ground-breaking announcement (Kurtzman 1989a) Lachance 5

Figure 2. The delineation of C. infanticola as distinct from C. sorbophila was based on genetic distance and not reciprocal monophyly. Excerpted from Fig. 1, Kurtzman (2007c), with permission.

Candida pseudorhagii (Kurtzman 2005b) was justified purely by Figure 1. In terms of genetic distance, C. germanica is closest (12 pairwise substi- ‘five noncontiguous nucleotide positions located in both theD1 tutions) to C. dendronema. Phylogenetically, the closest relatives of C. germanica and D2 domains’. Species in the Blastobotrys (Kurtzman 2007a), Downloaded from https://academic.oup.com/femsyr/article/18/8/foy103/5104380 by guest on 29 September 2021 are P. nakazawae and P. philogaea. Excerpted from Fig. 1, Kurtzman, Robnett and Sugiyamaella (Kurtzman 2007b), Wickerhamiella (Kurtzman 2007c) Yarrow (2001b), with permission. and Ogataea (Kurtzman 2011b) clades were described based on pairwise distances, independently of phylogenetic support, even wasaptlytitled‘Estimationofphylogeneticdistances...’,a first though the authors stipulated that the species were circum- indication that Kurtzman’s interpretation of phylogenies devi- scribed by maximum parsimony analysis. An example is given ated from the Hennigian (cladistic) mindset adopted by most in Fig. 2. other systematists. Exceptionally, P. lachancei was described entirely on phyloge- Genetic distance and phylogenetic relationship are often netic structure (Phaff, Starmer and Kurtzman 1999), being ‘lo- confused in the yeast taxonomic literature and it is appropriate cated basal to P. rhodanensis and P. jadinii’, with no allusion what- to rectify some common misinterpretations. (1) Phylogenetic re- soever to nucleotide differences. latedness is determined hierarchically from the number of an- Disregard for the phylogenetic species concept should not be cestral nodes separating taxa, unlike genetic distances, which viewed as a failure. A species concept based solely on mono- are calculated from pairwise substitutions (p-distances) or in- phyly is often impractical, as it leaves open the question of ferred from summing branch length in a scaled tree (patristic inclusivity (Taylor et al. 2000). One solution is the apomorphic distances). In the description of Candida germanica (Kurtzman, variant of the phylogenetic concept (Wheeler and Meier 2000), Robnett and Yarrow 2001b), both approaches were used. Candida where a defining property is specified. This is rarely possible dendronema and P. philogaea were both cited as the closest rela- with the yeasts. Another solution is the determination of ge- tives, the first based on 12 pairwise substitutions and the second nealogical concordance, where an evolutionary coherent entity with no specified justification, but evidently based on tree topol- (a real species) is recognised by its superimposable monophyly ogy. Figure 1 shows P. philogaea and P. nakazawae to be equally for two or more independent genes, which demonstrates ge- related phylogenetically to C. germanica, whereas the shortest netic independence among species. Kurtzman’s later multigene patristic distance is to C. diddensiae. (2) Phylogenetic species are approach approximates this by way of a purely genetic species delineated based on reciprocal monophyly as inferred from tree concept that usually coincides with that based on genealogical topology. This normally requires that the taxa being compared concordance. However, when faced with discordant gene phylo- be represented each by at least two individuals. (3) With the ex- genies between C. infanticola and C. sorbophila, Kurtzman (2007c) ception of linkage methods such as UPGMA, phylogenetic tree concluded that one of the two might be a hybrid species. Simi- reconstruction optimises criteria other than pairwise distance. larly, Kurtzman et al. (2005) interpreted the near identity of RNA Maximum parsimony, minimum evolution and its algorithmic polymerase II sequences in Kazachstania pintolopesii and K. het- counterpart, neighbour-joining, search for the shortest tree, and erogenica as a case of non-concordance attributable to hybridis- maximum likelihood fits the data a probabilistic model of sub- ation. An analogous conclusion was drawn for Komagataella ulmi stitutions. These methods seldom generate trees that perfectly and Ko. phaffii, whose mitochondrial SSU rRNA sequences were reflect pairwise distances for non-sister taxa. (4) Given data that nearly identical, unlike other genes examined (Kurtzman 2012b). fulfil certain ideals such as a steady molecular clock, absence of sampling error and (most improbably) the infinite allele model Calibrating sequence divergence (Kimura and Crow 1964), all methods should generate essentially Determination of a cardinal genetic distance that could serve the same tree. as a universal criterion for the delineation of species from se- While showing a clear preference for maximum parsi- quence divergence required examination of pairs of closely re- mony analysis, Kurtzman nearly always gave precedence to lated species, as detected by DNA reassociation values below pairwise divergence data, presented either in the form of 70%. Would sequences of a genetic species be polymorphic? aligned sequences (e.g. Kurtzman 1990) or distance matrices (e.g. Would the sequences of closely related species be sufficiently di- Kurtzman and Liu 1990). Species circumscription rarely men- vergent? Would rates of divergence be constant across species? tioned tree topology. To be sure, delimiting species from ge- Gueho,´ Kurtzman and Peterson (1990), Kurtzman and Liu (1990) netic distance does not preclude reciprocal monophyly. For ex- and Kurtzman and Robnett (1991) found polymorphisms of no ample, C. ontarioensis and C. tammaniensis (Kurtzman and Rob- more than two substitutions in well-defined species. Liu and nett 1998b) belonged to two separate subclades within a larger Kurtzman (1991) noted a single nucleotide polymorphism in a clade, demonstrating their distinct phylogenetic status. The cir- species of Willopsis and a 4–6 nucleotide indel in another. cumscription of Ogataea saltuana (Peter´ et al. 2011a) also met Peterson and Kurtzman (1991) focused on species of Pichia both criteria. Conversely, many delineations were based purely and Issatchenkia known to be close relatives. Starting with the on sequence divergence, as for example, that of Tetrapisispora assumption that the biological species concept is ‘the only ob- fleetii (Kurtzman, Statzell-Tallman and Fell 2004). Phylogeneti- jective definition of a species’ (especially when augmented by cally the strain could have been added to Tetrapisispora phaffii. DNA reassociation data), the authors examined putative sister 6 FEMS Yeast Research, 2018, Vol. 18, No. 8

pairs. All species examined were monomorphic in their D2 se- a sound induction is a statistical evaluation of the variation in quences and the sequences diverged between species, but the polymorphism. Other than the brief range of p-values given by authors showed concern that species pairs with similar fertili- Peterson and Kurtzman (1991), a rigorous evaluation has yet to ties or DNA reassociation values ranged in D2 divergence from be made. Kurtzman and Robnett (1997) listed 17 predictions of 1.0 to 5.1%. A large study of Candida species (Kurtzman and conspecificity for pairs or triplets of Candida or other species. Robnett 1997) demonstrated that most polymorphic species Of these, only four were eventually rejected in the light of addi- rarely varied by more than two nucleotides, but that ‘strains with tional evidence; four were accepted on additional data such as as little as 40% nDNA complementarity may be indistinguishable DNA reassociation or ITS sequences, and most remaining cases, from one another by sequence differences in region D1/D2’. prediction became fact with no new information (Lachance et al. In their seminal article on the ascomycetous yeasts, 2011). To give one example, in the description of Kuraishia molis- Kurtzman and Robnett (1998) compared some 500 species and chiana,Peter´ et al. (2005) initially considered a strain to be con- varieties of which over 100 were represented by at least two specific in spite of three substitutions and one insertion com-

strains. In most cases, no more than two nucleotide posi- pared to the type, but ultimately excluded it from the species on Downloaded from https://academic.oup.com/femsyr/article/18/8/foy103/5104380 by guest on 29 September 2021 tions were polymorphic. Pairs with less than 30% DNA re- the basis of a reported 4% difference in GC content. The strain is association generally had ‘greater than 3 nucleotide differ- nowlabelled‘Kuraishia molischiana’ in the CBS catalogue, but as ences and would be recognized as separate species’. Cali- Kuraishia cf. molischiana intheNCBIdatabase. bration on the biological species was often realised vicari- Systematists of the moulds have embraced a different ap- ously through DNA reassociation. The authors drew the mo- proach, subordinating sequence data to a pragmatic vision of mentous conclusion that ‘it is predicted that strains show- the species (e.g. Samson and Varga 2009; Frisvad et al. 2019). All ing greater than 1% substitutions in the ca. 600-nucleotide information is combined to yield a cohesive taxon. Barcode se- D1/D2 domain are likely to be different species and that strains quences such as the ITS region are often invariant across species with 0–3 nucleotide differences are either conspecific or sis- thus defined, and practitioners resort to more rapidly evolving ter species’. Other papers published at around the same time genes (e.g. calmodulin), until the desired degree of subdivision often contained a more dogmatic variant: ‘Strains differing is obtained. Although not explicitly stated, this would seem to by no more than three substitutions (0.5%) are generally con- be the approach used by Kurtzman (2005c) in the case of some specific whereas strains with six (1%) or greater substitutions Kregervanrija and Saturnispora species. Two species of the first are separate species’, sometimes with the qualification that a genus differed by four D1/D2 substitutions, and thus failed to ‘few exceptions have been found’ (Kurtzman and Dien 1998). meet the 1% cut-off. Addition of ITS sequences elicited 41 dif- With time, qualifications became vanishingly faint and the ferences (substitutions and indels), causing the two taxa to be prediction commensurately bold: ‘strains with greater than ‘regarded as genetically separate’. Saturnispora besseyi and Sa. 1% sequence divergence are separate species’ (Kurtzman 2000; ahearnii differed by three indels in the D1/D2 domains, but the Kurtzman, Robnett and Yarrow 2001b); ‘Conspecificity [...] can ITS region had 24 differences, causing the taxa to be treated as be ruled out since we found differences in the respective se- ‘closely related species rather than as divergent members of the quences at 24 nucleotide positions’ (Fonseca et al. 2000); ‘As- same species’. As multigene analyses became the norm, so did comycetous yeasts that differ from one another by greater than a tendency to give more weight to loci that supported splitting 1% substitutions in the variable 600-nucleotide D1/D2 domain (Kurtzman 2007b). Saitoella coloradoensis was described based on of large subunit rDNA have been shown to represent different four D1/D2 differences, two ITS substitutions, but substantially species’ (Kurtzman, Robnett and Yarrow 2001a); ‘all ascomyce- greater divergence for two other genes (Kurtzman and Robnett tous yeasts differing from one another by greater than 1% sub- 2012). Kuraishia piskuri was considered distinct from C. ogatae stitutions in this domain have been shown, so far without ex- based on four substitutions and one indel in the D1/D2. In the ception, to be different species’ (Kurtzman 2001a); ‘strains dif- same study, two other species pairs were kept separate in spite fering by more than 1% substitutions in this rDNA domain of only two differences in the D1/D2 sequences because of ‘no- represent separate species’ (Kurtzman and Droby 2001; Kurtz- table divergence for EF-1a, RPB1,andRPB2’ genes. This broader man 2001b); ‘strains differing from one another by more than view led Kurtzman (2014) to redefine the species criterion ‘based 1% substitutions in domain D1/D2 represent separate species’ on the prediction that strains of a species diverge in D1/D2 and (Janisiewicz, Tworkoski and Kurtzman 2001; Kurtzman, Robnett ITS sequences by no more than 1%’, concluding that a ‘better and Basehoar-Powers 2001; Kurtzman 2004); ‘strains that differ understanding of the genetic definition of a species is needed from one another by 1% or greater nucleotide substitutions in and this can only come when additional strains of a species and theD1andD2domainsofthe26SrRNAgenehaveproventobe thoseofrelatedspeciesareanalysedusingmultiplegenes[...] separate species on the basis of the results from genetic crosses and whole genomes’. and from the extent of nuclear DNA complementarity’ (Kurtz- How is one to deal with exceptions? In reassigning P. man et al. 2005). Kurtzman and Robnett’s (1998) guidelines have monospora to Saccharomycopsis, Kurtzman (1999) noted many served as the decisive argument in the delineation of the major- similarities with Saccharomycopsis synnaedendra, but the two dif- ity of yeast species described in the 21st century. fered‘by8 nucleotides[...].Becausestrainsshowingthismuch divergencehaveneverbeenfoundtobeconspecific[...],thetwo No exceptions, some exceptions, many exceptions: polyphasic species isolates are regarded as sister species that would be expected to delineation? be genetically isolated from one another’. Uncharacteristically, The danger existed, when searching for a critical divergence the possibility was raised that some conspecific strains yet to be value used to delineate species, of engaging in some logical cir- discovered might differ by more than 1% substitutions. Would cularity. Syllogism: (1) several yeasts known to be conspecific such an exception be fatal? based on the biological concept have no more than x substitu- Kurtzman (2005a) justified the creation of Komagataella phaf- tions in the D1/D2 domains; (2) yeasts a and b have fewer than x fii on its divergence of six substitutions and some indels from substitutions; therefore, (3) yeasts a and b are conspecific. What existing species. ‘Each of these species shows greater than 1% is needed to transform this perhaps hasty generalisation into substitutions with its nearest neighbour, providing the genetic Lachance 7

basis for predicting that each taxon is a separate species, which caused us to overstate our respective positions. Indeed, he never is based on earlier findings that strains showing 1% or greater claimed that a sample size of one was better or desirable. Kurtz- non-contiguous substitutions represent separate species’. The man, Smiley and Baker (1972) warned that ‘differences found mention of a prediction signalled a return to the original phrase- between[...]strains[...]emphasizetheneedtoexaminemore ology of Kurtzman and Robnett (1998), whereas the stipulation than one strain of a species.’ Likewise, I have on occasion con- of non-contiguity was a novel element of stringency. The re- ceded that single-strain descriptions may be justified (Lachance newed caution may have been triggered by the report of a sub- and Kurtzman 2013). Kurtzman (2001a, 2010) noted that in the stantial polymorphism in the D1/D2 domains of Clavispora lusi- fourth edition of The Yeasts (Kurtzman and Fell 1998), 205 out of taniae, ‘the only well-documented exception to this prediction’ 725 species described were supported each by a single strain, (Kurtzman 2005a). The new qualification became a regular ad- hence that ‘nearly one-third of yeast biodiversity would be un- dendum (Kurtzman 2006, 2007b, c, 2010, 2011c, 2012a, b, 2014; recognized if species represented by a single strain were not de- Kurtzman et al. 2015). Thirty-seven mating strains of Cl. lusita- scribed’. This incorrectly assumes that the only alternative to

niae had been found to vary by up to 32 substitutions in the D2 naming those 205 species from single isolates was not to de- Downloaded from https://academic.oup.com/femsyr/article/18/8/foy103/5104380 by guest on 29 September 2021 domain, a two-base substitution in the D1 domain and two small scribe them at all. The matter is relevant to the species concept. deletions (Lachance et al. 2003). The polymorphisms were inde- A species is an evolving community. Whereas a single strain may pendent of each other and of mating type. Both versions of the serve as representative, it cannot document reciprocal mono- large polymorphism were found in each of five fertile strains, phyly, precluding application of a phylogenetic species concept. but this was apparently overlooked by Kurtzman (2011c), who As stated by Kurtzman, Robnett and Yarrow (2001b), single iso- worried that ‘viability of the ascospores was not tested’. late descriptions weaken our understanding of variability and The recognition that exceptions exist led to a renewed pru- ecology. The main argument in their favour is that ‘detection and dence and eventually to outright questioning of the D1/D2 dis- description of these new species adds to the overall knowledge tance formula (Kurtzman, Albertyn and Basehoar-Powers 2007): of yeast biodiversity [by providing] additional genetic markers in ‘Kurtzman & Robnett (1998) suggested that strains differing by their respective phylogenetic clades’. I counter that strains, not no more than three nucleotides in this c. 600-nucleotide se- species, are the unit of information in databases, such that bio- quence are likely to be conspecific. However, they made the diversity information is not contingent on the creation of Lin- point that this was a prediction’. Kurtzman (2011c, see also naean binomials. Kurtzman (2010) noted, however, that ‘many Kurtzman and Sugiyama 2015) even stated that the guidelines culture collections are reluctant to accession material that may developed by Kurtzman and Robnett (1998) were treated as a never be described’, which is a valid concern. prediction because exceptions had been found earlier. Kurtzman (2011c) added that ‘single gene analyses can lead to incorrect in- What’s a genus? terpretations’ and that other genes should be used as well, concluding that the Classis et Ordo est sapientiae, Genus et Species naturae opus. Linnaeus (1766) tells us that the class and the order are the work practice of simply counting nucleotide differences between strain of science, the genus and species that of nature. Although the pairs to determine if the strains are the same or different species claim has been made that a genus, like a species, has a finite is somewhat subjective. A better approach is to examine multiple lifespan in evolutionary time, delimited by two events analogous gene trees to estimate genetic separation. It is clear that multi- to speciation and extinction (Reed and Hughes 2002), it is doubt- ple strains of a large number of species need to be compared to ful that many contemporary systematists regard the genus as a provide a stronger basis for data interpretation. An additional approach would be to identify species from whole genome sequence natural kind (Dupre´ 2002). Besides, the life of the typical yeast comparisons. Whether this proves practical for rapid diagnostics genus is much too short to be expressed in units of geologi- is uncertain, but it is a concept worth testing because of the ease cal time! Jocularity aside, most would agree that an ideal genus of determining whole genome sequences for microorganisms. should be a homogeneous collection of related species, monophyletic and characterised by a defining feature (apomorphy). Cryptic sibling and sister species Kurtzman’s early views aligned with this definition, but over Some species concepts are purely technical and have little to do time, patterns of genetic relatedness took centre stage, eventu- with species delineation. That is the case for sibling or cryp- ally giving way to a more phylogenetic concept. tic species, those which are virtually impossible to distinguish The question of what is a genus must be nearly as old as the ques- (holo)morphologically. Peterson and Kurtzman (1991), however, tion of what is a species. The definition offered in the Dictionary of used the expression ‘sibling species’ simply to designate close Fungi [...]statesthatthereare‘nouniversallyapplicable criteria relatives. Conversely, P. toletana and P. xylosa were said to be by which genera are distinguished, but in general the emphasis is indistinguishable except by molecular approaches (Kurtzman now on there being several discontinuities in fundamental charac- 1992a), but not labelled sibling species. This unorthodox usage ters’. The discontinuities that systematists now look for are those was eventually abandoned (Kurtzman 1998) but the more correct in phylogenetic trees. term ‘sister species’ was somewhat redefined to mean ‘closely (Kurtzman 2011a; see also Kurtzman and Robnett 2013b) related species that have only recently become genetically isolated from one another’ (Kurtzman and Robnett 1998). Sister- Early efforts were aimed at refining the quality of morpho- hood for taxa at higher ranks was applied in the conventional logical data used in generic definitions, with the belief that manner to refer to adjacent taxa in a tree (Kurtzman and Rob- high-quality data can be phylogenetically meaningful. An early nett 1995, 1997; Kurtzman 1998). example is the use of inner spore wall structure to reassign P. melissophila to Debaryomyces (Kurtzman and Kreger-van Rij Single-strain species descriptions 1976). Similarly, Kurtzman, Smiley and Johnson (1980)re- Clete and I exchanged opposing views on species descriptions assigned certain Pichia species to the resurrected genus Is- based on a single isolate (Fell, Kurtzman and Lachance 2000; satchenkia. ‘On the basis of ascospore surface ultrastructure, type Kurtzman 2010; Lachance 2011), a debate that may in itself have of ascus, and chemical structure of the ubiquinone present, it 8 FEMS Yeast Research, 2018, Vol. 18, No. 8

appears that the species assigned to Issatchenkia represent a phylogenetically distinct group.’ But a definition based on genetic distance soon became increasingly appealing. Kurtzman, Phaff and Meyer (1983) noted that rRNA-DNA reassociation had the potential to provide genus level relatedness values, but the approach was not adopted by yeast systematists due to its technical onus. In the absence of an early genetic distance metric applica- ble to a whole genus, but on the finding of 68% DNA reassociation between Hansenula mrakii and P. sargentensis and 75% between H. minuta and P. lindneri, Kurtzman (1984c) transferred all Hansenula species to the genus Pichia.Itfollowedthattheas-

similation of nitrate is ‘unsuitable for separating either species Downloaded from https://academic.oup.com/femsyr/article/18/8/foy103/5104380 by guest on 29 September 2021 or genera. [...] The definition of yeast species and genera has been principally on the basis of morphological and physiologi- Figure 3. Phylogram of Lipomyces and Myxozyma species where L. anomalus was cal characters, and many of these appear to be unreliable indi- regarded as sufficiently divergent to consider reassignment to a separate genus, cators of kinship.’ Kurtzman warned that this was only the be- demonstrating the emphasis on genetic distance and not phylogenetic hierarchy. Excerpted from Fig. 4, Kurtzman and Liu (1990), with permission. ginning, as many yeast genera, like Pichia, ‘should be regarded as groupings of taxa with similar phenotypic characteristics rather than as clusters of species having a common phylogenetic ori- species previously assigned to Issatchenkia. All the while, they gin’. Although the demise of Hansenula met with some uneasi- lamented the absence of a phenotypic characteristic, other than ness (e.g. Barnett 2004), multigene analyses (Kurtzman, Robnett ascospore ultrastructure, to support the exclusion of Issatchenkia and Basehoar-Powers 2008; Kurtzman and Robnett 2010)eventu- terricola, which was indicated by ‘the considerable divergence’ ally demonstrated that species formerly assigned to Hansenula of the species. A similar concern was expressed (Kurtzman and did not constitute a natural assemblage. Instead, they were dis- Robnett 1991) regarding inclusion of S. kluyveri among core Sac- tributed across two non-sister clades each consisting of many charomyces species. Although the species were known to ex- species formerly assigned mostly to Pichia as understood before hibit some mating cross-reactivity and the phylogeny provided 1984. In terms of nomenclature, members of the two clades were no evidence of polyphyly, a larger genetic distance favoured its assigned to seven genera in total. The retention of the name exclusion. Likewise, Liu and Kurtzman (1991)sawlargepair- Hansenula for some of these species might have appealed to wise distances among certain species of Williopsis as a sign that some, but the cogent arguments made by Kurtzman, Robnett each may represent a separate genus, despite their monophyly. and Basehoar-Powers (2008) for abandoning the name are com- Conversely, the large distances separating Schizosaccharomyces pelling. species were not thought to support a previous proposal for a three-way split (Kurtzman and Robnett 1991). Focusing on Signatures branch length, Kurtzman and Liu (1990) stated that ‘L. anoma- An early pursuit was that of signature sequences, namely con- lus is the most divergent member of Lipomyces’, when in fact the served motifs that could eventually be used to assign species tree showed it to occupy a core position in the genus, albeit on to a genus. Gueho,´ Kurtzman and Peterson (1989) searched for alongbranch(Fig.3). ‘Once there is a clearer understanding of such ‘nucleotide signatures for genera as well as higher or- rates of divergence among yeasts, L. anomalus may prove to be ders of classification’. Synapomorphic positions were identi- better placed in its own genus.’ It is not clear whether the au- fied for the genera Sterigmatomyces and Fellomyces together and thors worried that this would render the remaining genus pa- for Malassezia, but not for Trichosporon’, confirming the ‘hetero- raphyletic, something that was disallowed outright for Debary- geneous nature of the genus’. Kurtzman and Robnett (1991) omyces by Kurtzman and Robnett (1991), resulting in the (tempo- tried to identify, among species of Saccharomyces and Debary- rary) annexation of Schwanniomyces. omyces, ‘genus-specific signature nucleotides’, with mixed re- With an emphasis on distance at the exclusion of topology, sults. Mendonca-Hagler, Hagler and Kurtzman (1993) looked for the next step was to discover ‘definitive guidelines [...] upon signatures in Metschnikowia species and noted that they ‘are ev- which to base generic separations’ (Kurtzman and Liu 1990). But ident for the genus Metschnikowia if M. hawaiiensis is removed, the problem of rate heterogeneity seemed insurmountable. To and the genus is even more homogeneous if M. lunata is also remedy this, Kurtzman and Robnett (1991) estimated ‘phyloge- removed’, leading them to conclude that ‘inclusion of M. hawai- netic distances’ by a maximum likelihood procedure. But varia- iensis and perhaps M. lunata in the genus Metschnikowia is ques- tions in evolutionary rates led Kurtzman (1993) to state that the tionable’. The reasoning is not entirely free of circularity if one ‘definition of yeast taxa solely on number of nucleotide differ- is to remedy the absence of a signature by removing offending ences is unreliable because branch lengths and possibly branch- species till the desired state of uniformity is attained. ing order can be affected’.

A genetic genus concept The rise (and periodic fall) of a phylogenetic genus concept In the application of sequence analysis to species delineation, The importance of monophyly was made explicit by Kurtz- the frequent representation of a species by a single strain pre- man and Robnett (1998a): ‘we make no proposals for redefin- cluded the use of reciprocal monophyly as a fundamental cri- ing genera, but we suggest that many present genera are not terion. This might in part explain Kurtzman’s focus on genetic monophyletic and that additional genes must be compared distance, and not tree topology, in the pursuit of an operational before yeast classification can have a solid phylogenetic ba- species concept. As most yeast genera are polytypic, a distance- sis’. Kurtzman and Dien (1998) made similar remarks. As was based approach to generic circumscription was not necessary. the case with the strictly phylogenetic delineation of P. lachan- Indeed, Peterson and Kurtzman (1990) noted the monophyly of cei (Phaff, Starmer and Kurtzman 1999), Boekhout et al. (1994) Lachance 9

He noted that some clades were well defined both phylogenetically and phenotypically, such that they could be used as models to calibrate less cohesive genera. The model genera included Saccharomyces (sensu stricto), Torulaspora and Zygosaccha- romyces,aswellasEremothecium from the sister family Eremoth- eciaceae. Transmutation to a purely phylogenetic genus concept was not complete however. A table specifying genetic distances within and between genera was presented. Within-genus heterogeneity was determined from pairwise distances between the most distant species pair of each genus. These were compared with the inner patristic distances separating the roots of each genus, which are bound to be short, such that the table did

not strengthen the case for the proposed genera as much as it Downloaded from https://academic.oup.com/femsyr/article/18/8/foy103/5104380 by guest on 29 September 2021 could have. Kurtzman (2005c) gave explicit precedence to phylogenetic relationships in his discussion of the genera Kregervanrija and Saturnispora, which he nonetheless characterised as ‘separated by just 105 substitutions’. Strength of topology grew in importance, becoming a cardinal criterion in classification. How- ever, application of the criterion was not always uniform. Kurtzman, Robnett and Basehoar-Powers (2008) recognised that many species formerly assigned to Hansenula fit clearly in a large clade that received 100% bootstrap support. However, the clade was further subdivided into subclades that were interpreted as individual genera (Wickerhamomyces and Lindnera) despite less than robust support. The low signal-to-noise ratio incited the authors to perform multiple analyses on alternative subsets of Figure 4. Phylogram of Debaryomyces species presented in support of the poten- data where highly variable positions are removed. In an at- tial creation of a new genus for D. melissophus, D. carsonii and an undescribed species. Both the three-species assemblage and the entire genus are mono- tempt to sort out species that share co-enzyme Q-9, Kurtzman phyletic. Excerpted from Fig. 2A, Kurtzman (1998), with permission. and Suzuki (2010) combined large and small subunit rRNA gene data. Various clades were identified, some on better evidence (Yamadazyma, Millerozyma, Meyerozyma, Priceozyma, monotypic Babjeviella), others on less solid ground (Debaryomyces and explored species currently assigned to Brettanomyces and Hanse- Schwanniomyces, Scheffersomyces with P. spartinae included). Addi- niaspora from a fully phylogenetic perspective. Explicit reference tion of one or more protein-coding genes might have been well was made to tree topology, monophyly, homoplasy and boot- advised, for some generic assignments may have been hasty. strap support as significant factors in generic delineation. The Curiously, the analysis did not include the Metschnikowiaceae, authors concluded that the two clades containing bipolarly bud- which also share Q-9 (and Q-8) ubiquinones and are now known ding species (Eeniella nana and all Hanseniaspora species) should to be embedded in the Debaryomycetaceae (Kurtzman 2011a). not be regarded as closely related. This paradigm shift heralded As more difficult genera began to be tackled, a strict phylo- an increased use of phylogenetic language in discussions of gen- genetic approach was not always conclusive, and a genetic con- era, although with occasional semantic liberties. For example, cept stealthily resurfaced. An attempt to position the new mono- Kurtzman (1998) remarked that if ‘the circumscription of genera typic genus Spencermartinsella amidst other ascosporic genera as is to be based on monophyly of their assigned species, the [...] well as many neighbouring asexual species did not result in a se- three species and other members of their clade represent a new ries of discontinuous clades that were readily assignable to dis- genus’. However, as the three species in question, now assigned crete genera (Peter´ et al. 2011b). Instead, the authors constructed to Priceozyma (Kurtzman and Suzuki 2010), were a sister clade to a phylogram from a single representative of each neighbour- the others, their separation would have no effect on monophyly ing genus or equivalent clade. The result was a simplified tree (Fig. 4). devoid of inner branching structure, where genera ‘clustered A multigene analysis (Kurtzman and Robnett 2003) led to ma- about equidistantly from each other, supporting the proposal jor nomenclatural changes in generic assignments in the Sac- [of] a new genus’. The use of a phylogenetic inference method charomycetaceae. Fourteen clades were resolved that became to generate a distance-based tree is nothing less than a tour de the basis for restructuring the classification of relatives of Sac- force. In contrast, the new genus Diddensiella, also in the Tri- charomyces ‘based on natural relationships, thus providing ge- chomonascaceae, formed a clearly resolved clade, well isolated netic homogeneity and predictiveness to taxon names’. In so from other members of the family (Peter´ et al. 2012). doing, Kurtzman (2003) cautioned that Abolition of the dual nomenclature system for fungi (Mc- Neill 2012) created new challenges, as the task of renaming anamorphs needed undertaking. The rigour with which this was somewhat divergent phylogenetically defined genera will be fur- done varied. In some cases, monophyly may have been the main ther divided as additional species are discovered, and that mono- criterion, and inclusivity was established on an obvious phyloge- typic genera established for isolated species will expand in size as more species are found. Consequently, genera defined phylo- netic discontinuity, as was the case for the genera Martiniozyma genetically from presently known species will be subject to future (Kurtzman 2015), Groenewaldozyma (Kurtzman 2016), Teunomyces modification, but establishing a phylogenetic framework now will or Suhomyces (Kurtzman, Robnett and Blackwell 2016). In others, provide direction to future work. clear gaps were lacking, and some subjectivity was apparent, as 10 FEMS Yeast Research, 2018, Vol. 18, No. 8

of the four clades appears to represent a separate genus, despite differing in number of included species’. The reason for this specification is not clear. The separation of Wickerhamomyces and Lindnera into two sister genera may have been prompted by the wish to restrict genus size (Kurtzman, Robnett and Basehoar- Powers 2008). An urge to cut taxa across phylogenetic gaps, more than Figure 5. Phylogram presented in support of the transfer of a Candida species to size considerations, might also explain some choices. Although the new genus Deakozyma. Candida incommunis was left out despite strong boot- Boekhout et al. (1994) rejected a proposal to separate some strap support, demonstrating an emphasis on genetic distance and not phylo- species of Hanseniaspora into a second genus (Kloeckeraspora) genetic topology. Excerpted from Fig. 1, Kurtzman and Robnett (2014b), with permission. on the basis of an apparent dichotomy, Kurtzman and Robnett (2003) and Kurtzman (2003) regarded Saccharomycodes as a sep-

arate clade from Hanseniaspora in spite of strong bootstrap sup- Downloaded from https://academic.oup.com/femsyr/article/18/8/foy103/5104380 by guest on 29 September 2021 in the transfer of several Candida species to Kuraishia or Naka- port (98%) for a single clade. Although a phylogeny of the genus sawaea (Kurtzman and Robnett 2014a). Reassignment of other Wickerhamiella showed it to be cohesive, with a bootstrap of 100% Candida species to Ambrosiozyma (Kurtzman and Robnett 2013a) for the genus as a whole and lower values for its component was done entirely on the basis of monophyly, although only clades, Kurtzman and Robnett (2007) thought that the discov- one representative of the sister clade (Ogataea) was included in ery ‘of additional species in both Wickerhamiella and Hansenias- the dataset, thereby missing an opportunity to demonstrate re- pora will help determine if each represents a single genus or two ciprocal monophyly. A similar approach was used in the cre- closely related genera’. They emphasised that the ‘type species ation of three genera to accommodate orphan Candida species of Wickerhamiella, W. domercqiae, and the anamorphic species (Kurtzman and Robnett 2014b). Neighbouring genera were rep- Candida versatilis, represent members of a subclade that is basal resented by single species, although the authors indicated that to more recently described species of the genus [. . . ]’. Likewise, phylogenetic separation had been documented in other studies. the genus Starmerella has long been the object of a yearning While this was true for Middelhovenomyces spp., the species as- to split. ‘Even with the addition of many new lineages to the signed to Danielozyma fell close to the Metschnikowiaceae, even Starmerella clade, the two subclades originally recognized are though previous work had placed them near Sporopachydermia still evident’ (Kurtzman et al. 2010). (Kurtzman and Robnett 2007). This does not weaken the case for a new genus, quite to the contrary. The third, monotypic genus, Deakozyma, formed a well-supported outlying clade with its sis- Apomorphies ter C. incommunis (Kurtzman and Robnett 2014b), as shown in Kurtzman (2005) remarked that ‘phylogenetically defined genera Fig. 5. A justification for not including the latter species inthe may not always be recognized from phenotype, and that gene new genus was not given, but it is not wild speculation to infer sequence analysis represents the primary means for separating that it had to do with genetic distance. both species and genera’. Other articles reiterated this observa- In the end, a phylogenetic genus concept was never com- tion (e.g. Kurtzman and Robnett 2007). Was this meant to sug- pletely supplanted by a genetic concept. Kurtzman and Robnett gest that a defining characteristic is not desirable? Although the (2014b) saw them as complementary: search for characteristics that are free of exceptions is destined Genus circumscription from molecular criteria is affected by many to fail, a marked synapomorphy such as the twin needle-shaped factors, which include the number of genes analyzed, possible dif- ascospores of Metschnikowia can hold a good genus together as ferences in substitution rates for the genes under study and per- it sails through squalls of phylogenetic torment. haps the number of species included in the analysis. Consequently, there is no simple distance formula for defining a genus, as was also found for definition of yeast (Kurtzman and Robnett 1998a) Paraphyletic genera and mold species (Taylor et al. 2000). Most phylogenetically circum- Paraphyletic taxa are generally reviled, but a candid examina- scribed yeast genera appear as strongly supported clades in phylogenetic trees (e.g., Kurtzman 2011). tion of how evolutionary novelty arises shows that paraphyly is anything but rare (Lachance 2016). Following a multigene analysis of the Lipomycetaceae, Kurtzman, Albertyn and Basehoar- Genus size Powers (2007) favoured the retention the genus Dipodascopsis,‘at the risk of establishing a paraphyletic genus’. The deviation from In a strictly phylogenetic system, there should be no restriction cladistic orthodoxy, however, was intended to be provisional, on the number of species appropriate to a meaningful genus, awaiting the discovery of new taxa that will restore monophyly. although an abundance of monotypic genera would render the category redundant and therefore useless. Conversely, the study Higher categories of genera containing hundreds of species can be impractical (Frodin 2004). Such considerations probably were of some impor- Like species and genera, families and higher taxa were first tance in justifying certain nomenclatural choices. Kurtzman and organised, in the Linnaean tradition, on the basis of growth Robnett (2007) deemed the division of the large genus Candida traits thought to reflect phylogeny. In discussing the morphology into numerous small genera undesirable, but favoured the trans- of the newly described Cephaloascus albidus, Kurtzman (1977) fer of a few Candida species to the hitherto monotypic genus favoured assignment of the genus to the family Endomycetaceae Trigonopsis, as it allowed the retention of a well-known name for instead of the Ascoideaceae because ‘a more natural classifica- a phylogenetically well-defined taxon. Although size per sewas tion would seem to be served’. He was referring to the forma- not cited as a defining factor, it was sometimes mentioned asa tion of unique, erect ascophores. He also wondered whether the constitutive element of the definition of a genus. In discussing genus is better assigned to the Endomycetaceae or the Saccha- Kregervanrijia and relatives, Kurtzman (2005c) stated that ‘each romycetaceae, noting that ‘C. albidus shares characteristics with Lachance 11

both families and appears to be an intermediary form connect- the utilisation of 1-butanol shared by Tortispora and Trigonop- ing the two families’. The type of septal pore was deemed to be sis, and a general similarity in the growth responses patterns meaningful, and the sharing of a simple central pore supported of all three genera. Here, the family concept sat at the heart an affinity with Saccharomycopsis. As prescient as Kurtzman may of a fragile trinity of morphological, genetic and phylogenetic often have been, multigene phylogenies (e.g. Kurtzman 2014) models. eventually placed Ascoidea as a sister genus to Saccharomycop- sis, while Cephaloascus took on an early-emerging position in the Debaryomycetaceae-Metschnikowiaceae clade. Epilogue Discussions of taxa at the rank of family or above took ‘It would not surprise me at all if my dad was talking to you while on a new life with the advent of sequence-based phylogenies. you’ve been working on your manuscript! And, if you feel like Kurtzman and Liu (1990) noted a sharp discontinuity between you are struggling within your thoughts, my guess is that my species of Lipomyces or Myxozyma and an eclectic mix of Saccha- dad is trying to convince you that HE is right.’ romyces, Debaryomyces and Pichia species, leading them to pro- (Mary Heffernan, nee´ Kurtzman, personal communication). Downloaded from https://academic.oup.com/femsyr/article/18/8/foy103/5104380 by guest on 29 September 2021 pose recognition of the family Lipomycetaceae for the first two genera and Saccharomycetaceae for the others. Kurtzman (1993) identified four major clades among Ascomycetes that showed Acknowledgements morphological affinities to the yeasts. One consisted of typical yeasts, another contained yeast-like fungi such as Eremascus, Preparing this review was made particularly easy thanks to the another joined together Taphrina and Protomyces, and a fourth United States Government policy that publications of their re- was restricted to Schizosaccharomyces. He noted that within typ- searchers be widely accessible. I thank Mary Heffernan for her ical yeasts, Lipomyces and Dipodascopsis appeared distinct from encouragement and permission to quote a private communica- the rest. All this began to answer the long-time question posed tion. I appreciate extensive, insightful, helpful comments from by early yeast systematists whether the yeasts were ‘primitive three anonymous reviewers. fungi or reduced forms of more evolved taxa’. The Endomyc- etaceae were at the time understood to include both budding Funding and mycelial forms. Sequence analyses showed that in fact, ‘with the exception of Schizosaccharomyces, the ascomycetous I am grateful for 36 years of continuous funding by the Natural yeasts and yeastlike species appear to be a monophyletic group Sciences and Engineering Research Council of Canada. that is distinct from the so-called higher ascomycetes’. With Conflict of interest. None declared. the qualification that the phylogeny presented in the paper was provisional because of restricted sampling, Kurtzman asserted that the orders Endomycetales, Protomycetales, and Taphrinales REFERENCES within the subclass Hemiascomycetidae are artificial groupings, that yeasts and mycelial fungi are not subgroups of the En- Baptist JN, Kurtzman CP. Comparative enzyme patterns in domycetales and that the Protomycetales are a synonym of Cryptococcus laurentii and its taxonomic varieties. Mycologia the Taphrinales. He also proposed the emendation of the order 1976;68:1195–203. 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