The phylogenetic relationships of the anaerobic chytridiomycetous gut fungi (Neocallimasticaceae) and the Chytridiornycota. 11. Cladistic analysis of structural data and description of Neocallimasticales ord.nov.

JINLIANCLI, I. BRENTHEATH,] AND LAURENCEPACKER Dep(~rittiet~tof Biology, York Utliversity, North York, Otzt., Cotzodc~M3J IP3 Receivcd May 15, 1992

LI, J., HEATH,I. B., and PACKER,L. 1993. The phylogenetic relationships of the anaerobic chytridiomycetous gut fungi (Neocallimasticaceae) and the Chytridiornycota. 11. Cladistic analysis of structural data and description of Neocalli- masticales ord.nov. Can. J. Bot. 71: 393-407. We investigated the phylogenetic relationships of thc and the chytridiomycetous gut fungi with a cladistic analysis of42 morphological, ultrastructural, and mitotic characters for 38 taxa using both maximum parsimony and distance algorithms. Our analyses show that there are three major clades within the Chytridiomycota: the gut fungi, thc Blastocladiales, and the -Chytridialcs- Monoblepharidales. Conscqucntly. we elevated the gut fungi to the order Neocallimasticales ord.nov. Our results suggest that a modified , including the Monoblepharidales. is a monophyletic group. In contrast the Spizellomycetales are paraphyletic because the Chytridiales arose within them. The separation of the traditional Chytridiales into two orders is thus doubtful. Although the Blastocladiales are closer to members of the Spizellomycetales than the Chytridiales, the cladistic analyses of both structural and rRNA sequence data do not support the idea that the Blastocladiales were derived from the Spizellomycetales. We suggest emendations to the classification of the Chytridiomycota and note which groupings require further analysis. Our phylogeny for the currently recognized species of gut fungi is inconsis- tent with the existing classification. Noncthcless, pending furthcr investigations, we prefer to retain the existing, casily defined genera for which a key is provided. Key word.^: Chytridiomycota, rumen fungi, phylogeny, morphology, ultrastructure, mitosis.

Li, J., HEATH,I. B., et PACKER,L. 1993. The phylogenetic relationships of the anaerobic chytridiomycetous gut fungi (Neocallimasticaceae) and the Chytridiomycota. 11. Cladistic analysis of structural data and description of Neocalli- masticales ord.nov. Can. J. Bot. 71 : 393-407. Les auteurs ont ttudie les relations phylogtnCtiques des Chytridiomycota et des champignons chytridiornycktes intestinaux ii l'aide de l'analyse cladistique de 42 caracttristiques morphologiques, ultrastructurales et rnitotiques chez 38 taxons, en utilisant h la fois la parcimonie maximum et les algorithmes de distance. Les rCsultats montrent qu'il y a trois clades majeurs chez les Chytridiomycota : les champignons intestinaux, les Blastocladiales et les SpizcllornycCtales -Chytridiales-MonoblCpharidales. ConsCquemment les auteurs Clkvent les champignons intestinaux au niveau de I'ordre des N6ocallimasticales ord.nov. Les rksultats suggkrent qu'un groupc Chytridiales modifit. incluant les Monobltpharidales. constitue un groupe monophylttique. Au contraire, les Spizellomycttales sont paraphylttiques parce que les Chytridiales y ont pris naissance. La stparation tradi- tionelle des Chytridiales cn deux ordres apparait donc douteuse. Rien que les Blastocladiales soient plus prks dc certains For personal use only. niernbres des SpizcllomycCtales que des Chytridiales. les analyses cladistiques des donnCes structuralcs aussi bien que des skquences de rRNA ne supportent pas I'idCc que les Blastocladiales soient issues des SpizellomycCtales. Les auteurs suggkrent des arnendements h la classification des Chytridiomycota et notent quels regroupernents nkcessitent une analyse plus poussCe. La phylogCnie proposke par les auteurs pour les cspkces dc champignons intestinaux reconnus diverge des classifications existentes. Cependant, en attendant des Ctudcs plus pousstes, Ics auteurs prCtkrent retenir les genres actuels, faciles h dCfinir, ct en proposent une clC. Mots cl6s : Chytridiomycota, champignons du rumen, phylogCnie, morphologic, ultrastructure, mitose. [Traduit par la rkdaction]

Introduction zoospore ultrastructure, and families were defined on thallus A good taxonomic system should reflect the phylogenetic development. The traditional Chytridiales was split into two relationships among taxa, not just a series of artificial ranks orders, i.e., the Chytridiales sensu Barr and Spizellomycetales Can. J. Bot. Downloaded from www.nrcresearchpress.com by YORK UNIV on 02/13/16 based on character similarity. of the Chytridiornycota Barr, based on ribosome configurations, microbody -lipid com- was originally, of necessity, based on morphological characters. plexes (MLC), and other criteria (Barr 1980). However, this However, these criteria were shown to be quite variable owing system is not perfect. First, the differences between the Spizello- to environmental change (Miller 1976). As a result, the tax- mycetales and Chytridiales were not distinct. There are some onomy was quite artificial. With the advent of routine electron genera that can be assigned to either of the orders since they microscopy, it became possible to utilize zoospore ultrastruc- have characters of both orders, such as the Karlingia complex, ture in taxonomy. These features, as well as thallus develop- , and Zygorhizidium (Barr and Dtsaulniers 1986; ment, were emphasized in a new taxonomic system proposed Montecillo et al. 1980; Beakes et al. 1988). Second, the families by Barr (1980, 1990) and other workers (Lange and Olson were defined by developmental characters largely for con- 1979) because they were thought to be more conserved and are venience, and these characters are not reliable. For example, less influenced by the environment (Barr 1980). Together with sporangial development may be both endogenous and exogenous mitotic characters, they may more accurately reflect the phylo- in a single species, e.g., Entophylctis variabilis, Triparticalcar genetic relationships among species (Barr 1978; Heath 1980, arcticum, Spizellomyces spp., Piromyces communis, and Caeco- 1986; Powell 1978). myces communis (Powell and Koch 1977; Barr 1984a; Barr In this new system, orders and genera were defined on et al. 1989; Wubah et al. 1991b). In the present taxonomic system such variation would place each of these single species 'Author to whom all correspondence should be addressed. into two different families. Furthermore, as the number of Printed In Canada 1 Imprim; au Canada 394 CAN. J. BOT. VOL. 71, 1993 TABLE1. Taxa used in the cladistic analysis

Order and " References Spizellomycetales Neocallimasticaceae Neocallimastix" Barr et al. 1989; Heath et al. 1983; Heath and Bauchop 1985; Munn et al. 1987, 1988; Orpin and Munn 1986; Webb and Theodorou 1991; Wubah et al. 1991a Piromyces" Barr et al. 1989; Breton et al. 1991; Li et al. 1990; Munn et al. 1988 Caecomycesh Gold et al. 1988; Munn et al. 1988; Wubah et al. 1991b Orpinomyces Barr et al. 1989; Breton et al. 1989; Li et al. 1991 Spizellomycetaceae Spizellomyces" Barr and Hadland-Hartmann 1979; Barr 1984a, 19846 Triparticalcar Barr 1984a; Barr and Allan 1981; Chong and Barr 1973 Gaertneriomycesb Barr 1981b; Barr 1984a Kochiomyces Barr 1984a; Barr and Allan 1981 Karlingia A Barr and Hartmann 1977; Barr and DCsaulniers 1986 Karlingia B Barr and Hartmann 1977; Barr and DCsaulniers 1986 Karlingia C Barr and DCsaulniers 1986 Karlingia D Barr and DCsaulniers 1986 brassicae Lange and Olson, 1976a, 19766 Olpidium radicale Barr and Hadland-Hartmann 19786; Lange and Olson 19786 Held 1975 Chong and Barr 1974; Heath 1986'; Powell and Koch 1977 Caulochytriaceae Caulochytrium Olive 1980; Powell 1981b Chytridiales Chytridiaceae " Barr and Hartmann 1976 Rhizoclosmatiutn Barr and Hartmann 1976 Rhizophydiutnb Barr and Hadland-Hartmann 1978a; Chong and Barr 1974; Heath 1986' Allochytrium Barr 1986; Barr and DCsaulniers 1987 Polyphagus Powell 1981a Cladochytriaceae Nowakowskiella Heath 1986c; Lucarotti 1981 Cladochytrium Lucarotti 198 1 Harpochytriaceae For personal use only. Harpochytriutn Gauriloff et al. 1980a; Heath 1986'; Travland and Whisler 1971 Synchytrium endobioticutn Lange and Olson 1978a Synchytrium macrosporurn Montecillo et al. 1980 Uncertain Zygorhizidium affluens Beakes et al. 1988 Zygorhizidium planktonicum Beakes et al. 1988 Monoblepharidales Monoblepharidaceae Monoblepharella Dolan and Fuller 1985; Fuller and Reichle 1968; Gauriloff et al. 1980b; Heath 1986c; Reichle 1972 Oedogoniomycetaceae Oedogoniotny ces Gauriloff et al. 1980b; Reichle 1972 Blastocladiales

Can. J. Bot. Downloaded from www.nrcresearchpress.com by YORK UNIV on 02/13/16 Blastocladiaceae Blastocladiella Heath 1986'; Reichle and Fuller 1967 Blastocladia Lingle and Barstow 1983 Allomycesb Fuller and Olson 1971; Olson 1973 Catenariaceae Catenaria Chong and Barr 1974; Heath 1986'; Olson et al. 1978 Phy sodermataceae Physodermab Lange and Olson 1980; Lowry and Sparrow 1978; Olson and Lange 1978 Coelomomycetaceae Coelomomycesb Lucarotti and Federici 1984; Travland 1979 Uncertain Sorochytrium Dewell and Dewel 1990 Saprolegniales Saprolegniaceae Saprolengia Barr and Allan 1985; Holloway and Heath 1977; Heath 1978, 1986c

"Orders and families based on Barr (1980, 1990). h~asedon a number of species. 'This reference was used as a secondary source for the mitotic data. Original references are contained in it and in Heath (1980). LI ET AL. 395

TABLE2. Structural characters of the Chytridiomycota used in the cladistic analysis

1. Carpicity 6. Simple spur 0. Holocarpic 7. Complicated system 1. Eucarpic 8. Electron dense arc with fibrous materials 2. Centricity 9. Striated, fibrous rhizoplast 0. Monocentric 17. Organization of mitochondria 1. Polycentric 0. Several, disperse 2. Colonial I. Several, at the periphery of nuclear cap area 3. Sporangial development 2. Several, posterior 0. Endogenous 3. Several petal-like arrangement I. Exogenous 4. Single central 5. Single posterior, surrounding the kinetosome . . 4. Thallus morphology 0. Without rhizoids or hyphae 6. Single, within nuclear cap I. Sori 7. A single, large, posterior with several small 2. Rhizoids 8. Absent 3. Hyphae 18. Type of MLC (cf. Powell 1978) 5. Rhizoid morphology 0. Absent 0. Without rhizoids 1. Type 3 1. Branched 2. Type 4 2. Bulbous 3. Type 1B 3. Uniaxial with a disklike holdfast 4. Type 1A 6. Hypha morphology 5. Type 2 0. Without hyphae 19. Subtype IB of MLC . . 1. Extensively branched 0. Absent 2. Unbranched filament I. Subtype LB1 3. Very delicate, interconnecting with sporangia 2. Subtype lB2 4. Dichotomously branched 20. Subtype 1A of MLC 7. Crystal body in thallus 0. Absent 0. Absent I. Subtype 1Al 1. Present 2. Subtype 1A2 8. Oxygen requirement 3. Subtype IA3 0. Primarily aerobic 21. Subtype 4 of MLC (cf. Beakes et al. 1988) 1. Primarily anaerobic 0. Absent 9. Type of flagellum 1. Subtype 4A 0. Heterokont 2. Subtype 4B 1. Whiplash 3. Subtype 4C 10. Number of flagella 22. Type of kinetosomes For personal use only. 0. Biflagellate 0. Two kinetosomes, both functional I. Mainly uniflagellate 1. Two kinetosomes, one functional, one nonfunctional 2. Polyflagellate 2. One kinetosome, functional 11. Transition zone helix 23. Angle between the functional and vestigal kinetosomes 0. Absent 0. Nonfunctional kinetosome absent 1. Present 1. Right angle 12. Prop 2. Parallel 0. Absent 3. Variable 1. Present 4. Anterior 13. Ribosome configuration 24. Relationship between the functional kinetosome and nonfunctional 0. Aggregates and (or) helices kinetosome 1. Disperse 0. Nonfunctional kinetosome absent 2. Loosely packed by membrane 1. No connection between the kinetosomes Can. J. Bot. Downloaded from www.nrcresearchpress.com by YORK UNIV on 02/13/16 3. Nuclear cap 2. Kinetosomes connected by electron opaque or fibrous bridge 14. Nucleus position 25. The veil-like sheath on the side of nonfunctional centriole 0. Variable 0. Nonfunctional kinetosome absent 1. Anterior 1. Absent 2. Center 2. Present 3. Posterior 26. Configuration of endoplasmic reticulum 15. Nucleus shape 0. Dispersed in cytoplasm and not specific patterns 0. Spherical or irregular 1. Endoplasmic reticulum stacks and extensive sheets in 1. Cone-shaped cytoplasm 2. With an elongated beak 2. Partially delimiting ribosomes and not traversing 16. Type of MTOC or rhizoplast ribosomal area 0. No specific structure 3. Partially delimiting ribosomes and traversing ribosomal 1. Compound spur with three arms area 2. Striated disc 4. Fully delimiting a nuclear cap 3. Electron opaque structure 27. One or more obvious Golgi apparatus 4. Electron dense plate 0. Absent 5. Trisegmented spur 1. Present 396 CAN. J. BOT. VOL. 71, 1993

28. Organization of microtubules 35. Configuration of NAOs 0. Not detected 0. Centrioles oriented at 180" 1. Radiating around nucleus and forming a posterior fan I. Centrioles oriented at approximately 90" 2. In 9 groups of 3, radiating around nucleus 2. Variable, either centrioles oriented at 90' or discs 3. Bundle to rumposomes 36. Spindle development 4. Along spur to nucleus 0. Poles concomitant with spindle during spindle formation 5. Radiating into cytoplasm and around nucleus I. Poles migrate prior to spindle formation 6. Dual and complex system 37. Nuclear envclope behaviour 7. Cone-shaped basket around nucleus 0. Remains intact througho~~tmitosis 29. Electron dense area at the base of flagella 1. Develops polar fenestrae 0. Absent 38. Perinuclear endoplasmic reticulum 1. Present 0. Absent 30. Circumflagellar ring I. Present 0. Absent 39. Telophasc behaviour of nuclcar cnvclope 1. Present 0. Nuclear envelope niedianly constrictcd 31. Number of struts I. A new cnvclope formed both insidc and independently from 0. None the old envelope I. One strut 2. Nuclear envelope constrict in two places near thc NAOS so 2. Two struts that the interzone is cut off fronl thc daughter nucleus 3. Three struts 40. Metaphase plate 32. Connective 0. Absent 0. Absent 1. Present I. Present 4 1. Arrangement of nlctaphase chromoson~es 33. Skirt 0. Uncondensed 0. Absent I. Distinguished separate chromosome I. Present 2. Fused mass of chromatin 34. Posterior dome 42. Nucleolus behaviour 0. Absent 0. Persistent I. Present I. Dispcrsivc 2. Discardivc

species studied has increased, the data base has expanded sig- In some cases several species in a genus were identical for the selected nificantly, which makes subjective analysis of the data more characters, in which case the genus was the unit used. In other cases, isolates or species within a genus differed. so that these were the units difficult.-.. . - .. .. . For personal use only. There are now numerous cladistic methods of data analysis ""byzed(Table 3). designed to aid in phylogenetic reconstructions, but they have In a more detailed analysis of the gut fungi alone. we used an expanded sct of 2 1 more specific characters (Table 4) from 10 species yet be the Chytridiomycota. the advent of and four genera (Table 5). Ultrastructural data for Anaerorrzyces (Breton DNA sequence data has provided criteria that are et al. 1989) and Rur~zinornyces(Ho and Bauchop 1990) were unavail- available for some Chytridiomycota (Bowman et al. 1992; Li able, hence these taxa were not included, and Heath 1992; DorC and Stah1 1991) but that have yet to be Morphological critcria such as opcrculation and sporangium and correlated with structural data. In addition to these opportunities zoospore size and shape were not analyzed since they are very variable, for clarifying chytrid phylogeny, controversy has developed con- eve; in an isolate (Miller 1976). In contrast, developmental charac- cerning the chytridiomycetous gut fungi (Heath 1988; Bauchop ters, such as sporangial development, are more stable (Whiffen 1944; 1989). Although they were formally assigned to the Neocalli- Barr 1980) and were included. The ultrastructural characters were masticaceae in the Spizellomycetales, they differ from both the predominantly from zoospores since they are conserved both within Spizellomycetales and the Chytridiales in a number of ways and between isolates (Powell 1978; Barr 1978, 19810). The kineto- Can. J. Bot. Downloaded from www.nrcresearchpress.com by YORK UNIV on 02/13/16 such that their assignment to the Spizellomycetales is question- some and its root system and the MLC, which are considered to be good phylogenetic indicators because they are stable, easily identi- able (Billon-Grand et al. 1991; Heath and Bauchop 1985; Li fied, and refractory to fixation-induced changes (Powell 1978; Barr et al. 1990, 1991; Munn et al. 1987; Yarlett et al. 1986). 1978, 198 la), were emphasized. Vaguely defined organelles such as We have discussed a limited rRNA sequence analysis of the y bodies, various vesicles, and inclusions were excluded, since their Chytridiomycota in a previous paper (Li and Heath 1992). homology among the chytrids could not be established. A cladistic analysis of the available structural data in conjunc- It is very difficult to justify ordering character states for many tion with this work may further clarify the phylogeny and tax- characters in the chytridiomycetous fungi. Without a fossil record, a onomy of the Chytridiomycota. This paper focuses on such an poor knowledge of character function, and little analysis of onto- analysis of the structural data and integrates the results from genetic variation, any proposed ordering of states is pure speculation. both approaches in producing a revised phylogeny and tax- Consequently, ail characters were treated as unordered. Some charac- ters, such as thallus morphology (characters 4-6), type of MLC onomy of the Chytridiomycota. (characters 18 -2 l), and type of kinetosomes (characters 22 - 25), form character state trees that were coded by nonredundant linear Methods coding (O'Grady and Deets 1987; Wiley et al. 199 1). Question marks Taxa, character- seiectior~s,arzd coding indicate that the data were unavailable. We selected 38 taxonomic units representing genera, species, and isolates from the major groups of the Chytridiomycota for our analy- Outgroclp nrznlysis sis of the division (Table 1). The units were selected on the basis of Although the origin of the Chytridiomycota is uncertain, they are the 42 characters used (Table 2) and the availability of adequate data. probably monophyletic (Barr 1983; Li and Heath 1992). The Oomycota TABLE3. Coded structural data of the Chytridiomycota for 42 characters (see Table 2) Taxon Snproleg11 ia 1113010000 OOIOO7OOOO 0000011600 0000000000 00 Neocallirnasrix 1002101112 I103268000 0200000101 2111210000 20 Pirornyces 1002101111 I?O3068000 0200000101 3111?????? ?? Orpinornyces 1113011112 lIO2068000 0200000101 loll?????? ?? Cnecornyces 1002201111 1?03068000 0200000101 3111?????? ?? Syr~chyrriurner~dobioricurlr 0211000011 1112000502 0121101500 OOOO?????? ?? Syr~chyiriurnrnncrosporum 0211000011 1111000310 0121101300 OOOO?????? ?? Spizellornyces 1002100011 1111263401 0132210500 OOOO?????? ?? Tripnrricnlcar 1002100011 1111053401 0112110400 OOOO?????? ?? Gaerineriornyces 1002100011 1113083401 0132111500 OOOO?????? ?? Kochiornyces I002100011 1113013401 0132111500 OOOO?????? ?? Er~rophlycris I012100011 1112083402 0111100500 0000111111 2? Olpicliurr~brassicae 00I0000011 1111090401 0132101000 OOOO?????? ?? Olpidiurn radicale 00I0000011 1111000401 0132101510 OOOO?????? ?? Rozella 0000000011 1111094403 0131101500 OOOO?????? ?? Cnulochyrriurn 1000000011 111109040? 01?1101000 OOOO?????? ?? Karlingia A 1002100011 11110904OL 0132101000 OOOO?????? ?? Karlingia B IOO~~OOO~I1123090401 013213?000 OOOO?????? ?? r Karlingia C 1002100011 11220014OI 0122131000 OOOO?????? ?? 3 Karlingin D 1002100011 1122201402 012213?000 OOOO?????? ?? > r Zygorhidiurn aflnens 1002100011 1122006402 0122130010 OOOO?????? ?? Zygorhidiurn plar~kronicurn 1002100011 111IOO4320 0132100000 OOOO?????? ?? For personal use only. Chyrridiurn 1002100011 1122041320 0122221310 OOOO?????? ?? Rhizoclosrnariurn 1002100011 I121041320 0122121310 OOOO?????? ?? Rhizophydiurn 1002100011 I122204320 0122120300 0000111121 12 No~~akowskiella 111301OOII I122001320 0122121310 0000111121 ?? Clndochyrriurn 111301OOII I122001320 0122121310 OOOO?????? ?? Allochyrriurn 1012100011 1122001320 0122131300 OOOO?????? ?? Polyphagus 10121000I1 I122021310 0122130500 0000110121 12 Hnrpoch yrriurn 101230001I I123020500 0122130510 0000111001 11 1013020011 I122022100 0122121510 OOOO?????? ?? 1113010011 1122022100 0122121510 0000111121 12 1012100011 1133135200 1141140200 00001100?? ?O 1113040111 1133135000 0141140200 OOOO?????? ??

Can. J. Bot. Downloaded from www.nrcresearchpress.com by YORK UNIV on 02/13/16 I113010011 1133135200 1141140200 0000110021 22 1113040011 1133137200 2141140200 OOOO?????? ?? 1113030011 1133135200 1141140200 OOOO?????? ?? 0211000011 1133136200 3131140700 OOOO?????? ?? 1110000011 1133135200 1200040700 OOOO?????? ??

NOTE:? indicates missing data. 398 CAN. J. BOT. VOL. 71, 1993 TABLE4. Morphological and ultrastructural characters of the gut fungi used in the cladistic analysis

1. Thallus morphology 13. Shape of free hydro,oenosome 0. Monocentric 0. Without hydrogenosomes 1. Polycentric 1. Tubular 2. Sporangial development 2. Morc or less spherical 0. Endogenous 14. Megahydrogenosome 1. Exogenous 0. Absent 3. Subsporangial swelling I. Present 0. Absent 15. Nuclear shape I. Present 0. Pear shape with a concave end towards the kinetosome 4. Sporangiophore I. More or less spherical 0. Absent 2. With a beak towards the kinetosome I. Present 3. Cone-shaped 5. Rhizoid morphology 16. Circumflagellar ring 0. Branched 0. Without circumflagellar ring I. Coenocytic hyphae I. Uniform 2. Single bulbous 2. With a C-shaped vertical elongation 3. Several bulbous 3. inconspicuous or absent in one side 6. Rhizoid with restriction 17. Number of struts 0. Absent 0. Without struts I. Present I. One strut 7. Crystal bodies 2. Two struts 0. Absent 3. Three struts I. Present 18. Connective 8. The mechanism of zoospore release 0. Absent 0. Through multiple differentiated pores I. Present I. Through an undifferentiated area 19. Shape of skirt 2. Through one differentiated pore 0. Without skirt 9. Number of flagella 1. Smooth 0. Mainly uniflagellate 2. With a saclike structure 1. Polyflagellate 20. The relationship of microtubules and megatubules in the 2. Biflagellate posterior dome 10. Nonfunctional kinetosome 0. Without posterior dome 0. Present I. Microtubules parallel the dome I. Absent 2. Microtubules intersect the dome 1 1. Ribosomal configuration 21. Sporangia reaction to Sophorn jnpor~ica lectin 0. Free posterior aggregates 0. Positive For personal use only. I. Ribosomes dispersed 1. Negative 2. Free anterior aggregates 3. Anterior aggregates embedded in uniform substratum 4. Nuclear cap 12. Position of nucleus in the zoospore 0. Anterior 1. Posterior 2. Central 3. Variable

are related to the Chrysophyta and Xanthophyta (Beakes 1989) and set, similar options were used except that the BRANCH AND BOUND

Can. J. Bot. Downloaded from www.nrcresearchpress.com by YORK UNIV on 02/13/16 molecular sequence data showed that the Oomycota separated from algorithm replaced the HEuRlsTrc search algorithm. For both dam Sets, the Chytridiomycota in early evolutionary history (Gunderson et al. the 50% majority-rule consensus tree option was used to represent the 1987). Both the Chytridiomycota and Oomycota have vegetative and phylogeny. The mean pairwise distances between taxa calculated by asexual stages that can be compared. Therefore we selected one of PAUP (Tables 6 and 7) were used as input data for FITCH. In the FITCH the best-studied genera of the Oomycota, Saprolegnia, as an outgroup algorithm, the global search option was used. The PAUP program was for the Chytridiomycota. For similar reasons, Spizellomyces punctatiis run either on a Macintosh Plus with 4 megabytes of RAM or a Macintosh was selected as an outgroup in the gut fungi analysis. LC computer with 10 megabytes of RAM and a math coprocessor. The latter was approximately 10 times faster than the former. The FITCH Tree corzstruction program was run on a VAX 4000-300 mainframe computer. The PAUP (phylogeny analysis using parsimony) version 3.0 (Swofford 1989) and the FrTcH (Fitch-Margoliash algorithm) program from the PHYLIP package version 3.4 (Felsenstein 1991) were used to construct Results phylogenetic trees. In PAUP,both HEURISTIC and BRANCH AND BOUND Taxonomy and phylogeny of the Chytridiornycota algorithms were used in finding the most parsimonious trees. For the A total of 19 055 equally parsimonious trees was generated chytridiomycetous data set, the following options were used: MAXTREE, by PAUP with the NNI option. The tree length was 181, with a 20000; optimization, ACCTRAN;typeset, ALL UNORDERED;wtset, consistency index and a retention index of EQUAL WEIGHTS; exset, ALL INCLUDED; ancStateS, STANDARD; rooting, (CI) of 0.56 (RI) OUTGROUP;HEURISTIC search algorithm (stepwise addition sequence, 0.77. A 50% majority-rule consensus tree was generated from RANDOM;branch-swapping, NNI, MULPARS,COLLAPSE, zero-length the above equally parsimonious trees and had a tree length of branches; STEEPEST DESCENT was not in effect). For the gut fungi data 182, CI of 0.555, and RI of 0.771 (Fig. 1). Two kinds of pair- C polypha& C IlIanJochvtrimn -100 s -* 100 c ZY~Y - S Karl~nmaC 6 0 s IGXtine - 6 o S Gaertnenomyces 100 Csgachiamm -100 S mparticalcar S GntoDhlvctis -6 7 S Spizellonwxs -5 0 S Karlineia A 67 S Caulochytrium s Qlpidimk SRazella -100 -100 B Soroch-yhmm B 100- 100 B 100 BCatenarla 100°C B -m. - B- B Coelomomvces s chPimK0- SCaecomvces S Pirom~. I S Neocall~rnast~x

For personal use only. S Saprolegnia

FIG. 1. A majority-rule consensus tree from 19055 parsimony trees generated by PAUP using NNI option, showing the phylogenetic relation- ships of the genera in the Chytridiomycota. The letters indicate orders from Table 1. The numbers above each node represent percent occurrence of that particular node among the total number of trees. The branch lengths do not represent the evolutionary distance.

TABLE5. Coded structural data of the chytridiomycetous gut fungi for 21 characters (see Table 4)

Neocallirnastix patriciarurn Neocallirrlastix frontalis Neocallirnastix hurleyensis Pirornyces mae Can. J. Bot. Downloaded from www.nrcresearchpress.com by YORK UNIV on 02/13/16 Pirornyces durnbonica Pirornyces rhizinj7ata Pirornyces cominunis Caecoinyces equi Caecornyces communis Orpinomyces joyonii Spizelloinyces plrnctatlrs Blastocladiella einersonii Catenaria anglrilllrlae Saprolegnia ferax

NOTE:'? indicates missing data.

wise evolutionary distances between taxa were generated by the tree constructed with the mean distance is shown (Fig. 2). PAUP, absolute distance and mean distance (Table 6). The phylo- The phylogenetic trees generated by both methods had simi- genetic trees constructed by FITCH with the two sets of dis- lar major groupings. Basically, there were three major distinct tances had the same topology and similar branch lengths. Only groups: the gut fungi, the Blastocladiales, and the Spizello- 400 CAN. J. BOT. VOL. 71. 1993

TABLE6. Pairwise distances generated by PAUP

12 3 4 5 6 7 8 9 10 11 12 1. Saprolegrzia 0 0.667 0.548 0.429 0.548 2. Neocnllirnasti,r 28 0 0.071 0.214 0.095 3. Pirorrtyces 23 3 0 0.214 0.024 4. Orpirzornyces 18 9 9 0 0.214 5. Caecornyces 23 4 1 9 0 6. Spizellornyces 21 19 19 24 20 7. Triparticnlcnr- 20 21 19 24 20 8. 19 21 19 25 20 9. Kochionzyces 19 21 19 25 20 10. Knrlirlgin A 19 21 19 24 20 1 1. Karlirzgia B 20 20 18 24 19 12. Knrlirzgin C 21 22 20 24 2 1 13. Karlirzgia D 22 20 20 24 2 1 14. Olpidium brnssicr~e 18 25 23 23 23 15. Olpirliu~nrndicnle 19 26 24 24 24 16. Rozella 20 24 22 24 22 17. Cauloclzytri~rrrz 16 21 19 2 1 19 18. Erzrophlyctis 27 26 19 2 1 20 19. Ctzyfridiur~z 22 23 2 1 25 22 20. Rtzi~oclosnza~ium 22 23 2 1 26 22 2 1. Rltizophydiurn 31 27 20 24 2 1 22. Nowakowskiella 23 33 26 20 26 23. C1ndocll)lrriurn 17 28 26 20 26 24. Allochytrium 20 23 2 1 23 22 25. Polyphagus 28 28 20 22 2 1 26. Sy~zchytriumendobiotic~trn 18 26 24 22 24 27. Synchytriu~n~~zacrosporurn 18 26 24 23 24 28. Zygorlzizidiu~nnfluerzs 23 22 20 24 2 1 29. Zygorllizidiu~nplarzk~o~zicu~n 2 1 20 18 23 19 30. Hnrpochyrriurn 26 27 20 23 20 3 1. Oedogo~ziorr~yces 18 26 24 2 1 24 32. Mo~zoblephnrellr~ 24 34 25 19 25 33. Blnstocladielln 24 22 20 24 2 1 34. Blnstocladia 18 22 2 1 18 2 1 For personal use only. 35. Caterzaria 24 29 24 20 24 36. Allomyces 19 25 24 2 1 24 37. Physoderrna 19 25 24 2 1 24 38. Sorochytriunl 22 25 24 24 24 39. Coelo~nomyces 17 20 19 18 19

NOTE: Thc lower part shows absolute distances. and thc uppcr part shows rncan distances.

mycetales -Chytridiales -Monoblepharidales. Monoblepharella Phylogeny of chytrirliottzyceto~tsgut fifilrzgi and Oedogoniotnyces, the two Monoblepharidales genera, were A total of 162 equally parsimonious trees with length of 41, clustered with No~vakowskiellaand Cladochytriurn, two poly- C1 of 0.79, and RI of 0.74 were generated by PAUP with the centric genera of the Chytridiales in the trees generated by branch and bound option for the gut fungi data set. The 50% Can. J. Bot. Downloaded from www.nrcresearchpress.com by YORK UNIV on 02/13/16 both algorithms. In the parsimony tree, most Chytridiales, majority-rule consensus tree generated from these equally par- excluding S)azclzytrium spp., formed a distinct group including simonious trees was the same as one of the most parsimonious the Monoblepharidales, whereas the Spizellomycetales did not trees (Fig. 3). Similarly, the phylogenetic trees constructed by form a monophyletic group. In contrast, two clusters corre- FITCH with the two sets of distances (Table 7) had the same sponding roughly to the Chytridiales and Spizellomycetales topology and were similar in branch length. Only the tree con- were found in the distance tree. The most notable anomalies structed with the mean distance is shown (Fig. 4). in these trees were (i)Soroclzytriurn, which clustered with the Both phylogenetic trees clustered the two polyflagellate genera Blastocladiales in the distance tree but not in the parsimony (Orpinonzyces and Neoca1li1nasti.r) together (Figs. 3 and 4). tree, (ii)Synclzytrium spp., which consistently clustered within, The relationships between the uniflagellate genera were unclear. or closer to, the Spizellomycetales than the Chytridiales, and Pirotnyces dumbonica and Caecomyces equi consistently clus- (iii) Karlirzgia C and D, which consistently clustered with the tered together. The remaining species of Pirotnyces are ply- Chytridiales rather than the Spizellomycetales. The Blasto- tomous. However, C. cotnmunis was consistently isolated and cladiales were a well-defined group. They are closer to members closer to the polyflagellate species than to the other uniflagel- of the Spizellomycetales than the Chytridiales in the parsimony late species (Figs. 3 and 4). tree and show similar affinities to the Spizellomycetales and Because the outgroup chosen for these analysis (Spizello- the Chytridiales in the distance tree. All the gut fungi formed rnyces) is a more derived genus than the gut fungi (Figs. 1 a single clade, which was the first group separated from the and 2), we also analyzed the data set using PAUP with Blasto- other chytrids in both trees. cladiella, Catenaria, and Saprolegnia, either singly or in various LI ET AL. 40 1

based on structural characters of the Chytridiornycota For personal use only.

combinations, as outgroups. These results (not illustrated) were idales is not obvious, especially in the parsimony tree, which variable, some were similar to Fig. 3, others closer to Fig. 4 disagrees with Barr's (1980) separation of the traditional Chy- and others placed Orpinotnyces as the earliest branch in the tridiales. This was not totally unexpected (as previously recog- group, remote from Neocallitnastix. None generated mono- nized; Barr 1980; Barr and DCsaulniers 1986; Beakes et al.

Can. J. Bot. Downloaded from www.nrcresearchpress.com by YORK UNIV on 02/13/16 phyletic clusters for Piromyces and Caecomyces. 1988) since some genera, such as the Karlitzgia complex, Synchytrium, and Zygorlzizidiutn, have characters of both orders. However, in the distance tree, the traditional Chytridiales Discussion are divided into two groups, most members of which do cor- Taxonomy and phylogeny of the Chytridiotnycota respond to Barr's (1980) Chytridiales and Spizellomycetales, Barr (1980, 1990) split the traditional Chytridiales into two and in the parsimony tree most of Barr's Chytridiales do occur orders, the Spizellornycetales and the Chytridiales sensu Barr in a single group. However, some revisions are necessary. and proposedthat there were two evolutionary lines among the Karlingia types C and D are close to the Chytridiales, while Chytridiomycota, one from the Chytridiales to the Monoblephar- previously they were assigned to the Spizellomycetales (Barr idales, the other from the Spizellomycetales to the Blastocladi- and DCsaulniers 1986). On the other hand, Synchytrium is close ales. Our cladistic analysis shows that the Spizellomycetales, to the Spizellornycetales and probably deserves ordinal status Chytridiales, and Monoblepharidales form a monophyletic of its own. Previously it was assigned to the Chytridiales (Barr group, separate from the Blastocladiales and Neocallimasti- 1980, 1990), but it was pointed out that its relationship with caceae. This finding is consistent with the limited rRNA based other chytrids was unclear (Barr 1980, 1981~).Overall, our phylogeny that also clustered the Spizellornycetales and the analyses do support a taxonomy that recognizes a modified Chytridiales, separate from the Blastocladiales and gut fungi Chytridiales as a group, but the best treatment of the para- (Bowman et al. 1992; Li and Heath 1992). The distinction phyletic Spizellomycetales requires further investigation, pos- between the Spizellomycetales, Chytridiales, and Monoblephar- sibly by use of rRNA sequence analysis of a greater range of 402 CAN. J. BOT. VOL. 71. 1993 TABLE6 (concluded)

28 29 30 3 1 32 33 34 35 36 37 38 39 1. Snprolegnin 2. Neocallinznstix 3. Pirotnyces 4. Orpinotnyces 5. Caecotnyces 6. Spizellotnyces 7. Triparticalcnr 8. Gaertneriomyces 9. Koclziomyces 10. Karlingia A 11. Karlingia B 12. Karlingia C 13. Karlingia D 14. Olpidiutn brassicae 15. Olpidium rndicale 16. Rozella 17. Caulochytriutn 18. Entophlyctis 19. Chytridiutn 20. 2 1. Rhizophydiutn 22. Nowakowskiella 23. Cladochytrium 24. Allocl~ytrium 25. Polyphagus 26. Synchytriutn endobioticuttl 27. Synchytriutn tnacrosporutn 28. Zygorhizidium afluetzs 29. Zygorhizidium planktonicurn 30. Hnpochytrium 3 1. Oedogoniomyces 32. Monoblepharella 33. Blastocladielln 34. Blastocladia For personal use only. 35. Catenaria 36. Allotnyces 37. 38. Sorochytrium 39. Coelotnomyces

the species for which we have ultrastructural data. Based on zoospore ultrastructure, the Harpochytriales were At a lower level, Barr (1980, 1990) used thallus develop- abandoned, Oedogoniomyces was transferred to the Monobleph- ment to define families but pointed out that the families in the aridales, and Harpochytrium was transferred to the Chytridiales Chytridiomycota should also be defined on zoospore ultra- (Barr 1990; Gauriloff et al. 1980a, 1980b). Our analysis sup- structure (Barr 1988). Only two of Barr's families are relatively ports these moves. It shows that Oedogoniomyces and Mono- Can. J. Bot. Downloaded from www.nrcresearchpress.com by YORK UNIV on 02/13/16 well defined in the parsimony tree. One is the Cladochytria- blepharella are closely related to the two polycentric genera of ceae, which consists of Cladochytrium and Nownkowskiella, Chytridiales (i.e., Cladochytrium and Nowakowskielln), which and the other is the Spizellomycetaceae, which needs revisions. supports the Chytridiales-Monoblepharidales line proposed It should include the exogenous Entophlyctis and exclude the by Barr (1981a). The ordinal status of the Monoblepharidales endogenous Karlingia complex. The remaining families do not seems unwarranted, and they should perhaps be assigned as a form monophyletic groups. family of the Chytridiales. However, it may be justifiable to Olpidium, Rozella, Caulochytrium, and Karlingia A form a retain them as an order since the sexual life cycle of at least monophyletic group in the distance tree but not in the parsi- Monoblepharella, which is completed by fertilization of a non- mony tree. These species are related by a striated rhizoplast motile female gamete by a motile male gamete, is very distinct and dispersed ribosomes in the zoospores, although they belong from the Chytridiales (cf. Alexopoulos and Mims 1979). to different families according to Barr (1980, 1990). However, The Blastocladiales is a well-defined group except for Soro- the frequencies of the branch node of these species in the 50% chytrium. Although it was assigned to the Blastocladiales by majority-rule consensus tree are not very high (50 - 67 %), Dewell and Dewell (1990), it does not consistently cluster with which indicates that their relationships are ambiguous. Simi- the rest of the order. Both distance tree analysis and rRNA larly, the Chytridiaceae form a monophyletic group in the sequence analysis (Li and Heath 1992; Bowman et al. 1992) distance tree, while the parsimony tree shows that they are show that the Blastocladiales have similar relationships with paraphyletic. Clearly, more data are needed to clarify these the Spizellomycetales and Chytridiales. In the parsimony tree, problems. the Blastocladiales are closer to members of the Spizellomyce- LI ET AL ) M Monoblepharella 0.1 M Oedogonlomyces I I I C llkukxhy! - I C Nowakowskiella. c Pd3rphgm C Allochytrlum c C Chytxm C Rhizoclos~ - C Harpochytr~um S Karlrn< - S Rarlmm C Zygorhizldlmn af C Zyporhizidium pl S Gaertneriomyces S -vces S Tnwarticalcar S S~uellomyces s I3..&2rrhly! - S RarllnPlaA - SRozella S Caulochytr~mn S Upidmmra -- I s a- C S-wch-&mmm c sb- s -* B Blastocla~ B Physoderma B Catenaria B Allomvces BBlasotocladla B Cwlomomyxxs B Sorochvtnum kI!s s QrPinQmy- S I!lmw!! S Caecomyces SNeocallunastxx S Saprole- For personal use only. FIG 2 An unrooted d~stancetree generated by the FITCH -MARGOLIASH algorithm from the PHYLIP package, showing the phylogenetic rela- I t~onshipsof the genera In the Chytr~d~omycotaThe letters ind~cateorders from Table 1 The branch lengths represent the evolut~onaryd~stance between genera

tales than to those of the Chytridiales. In either case, the cladistic analysis does not support the idea that the Blastocladiales were derived from the Spizellomycetales. In the Blastocladiales, the relationships between the genera are unclear, with little support for existing families. Originally, the gut fungi were assigned to the Spizellomyce- tales as the Neocallimasticaceae (Heath et al. 1983) based on

Can. J. Bot. Downloaded from www.nrcresearchpress.com by YORK UNIV on 02/13/16 zoospore ultrastructure. Later Heath and Bauchop (1985) found that the mitotic characters of Neocallimastix, the type genus, were different from those of other chytrids; consequently they concluded that the gut fungi had a distant relationship with the FIG. 3. A majority rule concensus tree from the 162 most parsi- chytrids and noted that the taxonomic position of the gut fungi monious trees generated by PAUP using the BRANCH AND BOUND was problematic (Heath 1986). Munn et al. (1987) suggested algorithm and showing the phylogenetic relationships of the gut fungi. that a new order with affinities to both the Chytridiales and The numbers above each node represent the percent occurrence of Spizellomycetales might be justified for the gut fungi. The that particular node among the total number of trees. The branch cladistic analysis of structural data, as well as rRNA data (Li lengths do not represent the evolutionary distance between genera. and Heath 1992; Bowman et al. 1992; DorC and Stahl 1991), show that the gut fungi are monophyletic and clearly members helictica; kinetosomata circumvallata ab apparatu perkineto- of the Chytridiomycota (Li and Heath 1992), but they are not somatic0 includenti proprium anulum circumflagellarem, lim- closely related to the existing orders. Consequently, a new bum et calcar; mitochondria, microcorpis et guttulae consociata order, the Neocallimasticales, is established for all the chytridio- et rumposomata absunt; microcorpora (hydrogenosomata) et mycetous gut fungi. cupola posterior praesunt. Neocallimasticales ord.nov. Polycentric or monocentric thallus, uniflagellate or poly- Thallus monocentricus vel polycentricus; zoosporae uniflagel- flagellate zoospores; ribosomes in zoospores occur in aggregates latae vel polyflagellatae; ribosomata zoosporae aggregata et and helices; kinetosomes surrounded by perikinetosomal appara- CAN. J. BOT. VOL. 71. 1993

FIG.4. An unrooted distance tree generated by the FITCH-MARGOLIASH algorithm from the PHYLIP package, showing the phylogenetic rela- tionships of the gut fungi. The branch lengths represent the evolutionary distance between species.

TABLE7. Pairwise distances generated by PAUP based on structural characters of the gut fungi

1 . Neocallirnastix pnrriciarltttz 2. Neocallimasrix frontalis 3. Neocallimasrix hurleyensis 4. Piromyces rnae 5. Pirotnyces d~tmborzica 6. Pironzyces r/~izinflata 7. Pirotnyces conzrnltnis 8. Cuecornyces eqlti 9. Cnecotnyces corrltnltnis 10. Orpirzornyces joyotlii 1 1. Spizellornyces pltrlctatlcs

NOTE: The lower part shows absolute disranccs. and the upper part shows mean distancc For personal use only. tus including characteristic circumflagellar ring, skirt, and such is not the case. For example, on one set of characters spur; mitochondria, microbody-lipid complex, and rumpo- Orpirzornyces consistently is most distant from all other genera somes absent; microbodies (hydrogenosomes) and posterior (Figs. 1 and 2), but with the narrower set of characters its dome present. position varies substantially depending on the outgroup chosen. HABITAT:Anaerobically inhabit the digestive system of herbi- Each of the tested outgroup species is a valid choice, and con- vorous animals. sequently we are unable to determine the most valid tree. The NOTE:The order includes a single family, the Neocallimasti- rRNA sequences are similarly confusing, placing Orpinotnyces caceae and five or six genera. either with Pirornyces, remote from Neocallirnastix (DorC and We note that all of the above characters, excluding thallus Stahl 199 1; Li and Heath 1992) or with Neocallimastix, more type and number of flagella are apomorphic. remote from Pirornyces (Li and Heath 1992). The three spe- The interrelationships between the orders of the Chytridio- cies of Neocallir,zastix frequently form a single cluster (with or mycota requires further clarification because in addition to the more distant from 0rpinohyces) on morphological characters Can. J. Bot. Downloaded from www.nrcresearchpress.com by YORK UNIV on 02/13/16 questions about the Spizellomycetales -Chytridiales - Mono- (Figs. 3 and 4), suggesting that it may be a good genus, but blepharidales group, the present morphological data suggest that Pirornyces and Caecomyces, while consistently mostly separate the Blastocladiales are more closely related to this assemblage from Neocallimastix and Orpitzotnyces, do not form two separate than are the Neocallimasticales, whereas the limited molecular clusters along current generic lines. As noted previously, physio- data suggest the opposite (Li and Heath 1992). logical characters seem unlikely to help resolve these questions (Philips and Gordon 1988), and based on the Orpinomyces- Taxonomy and phylogerzy of gut fingi Neocallimastix- Pirotnyces examples above, it is doubtful that At present, the taxonomy of the chytridiomycetous gut fungi rRNA analyses will be unequivocal. Indeed, we have analyzed is based on simple but undoubtedly artificial characters such data from DorC and Stahl (1991. Table 2) with an artificial that genera are defined on the basis of thallus morphology, outgroup by using FITCH and NEIGHBOR algorithms from the rhizoid type, and number of flagella alone, and species are PHYLIP package. The results show that Caecomyces is the sister mostly based on details of zoospore ultrastructure. In principle group to the remaining three genera, and the phylogenetic rela- the present analysis of all available morphological and ultra- tionships among Neocallir7zastix, Pirotnyces, and Orpinomyces structural characters should provide a more natural and consis- are controversial. tent grouping of the isolates, which should be comparable with Although the current taxonomic scheme may not be ideal, the limited rRNA sequence data previously published; however, for example, rhizoid morphology is variable within a single LI ET AL. 405

isolate (Gold et al. 1988; Wubah et al. 1991b), it is easy to and a maximum of four versus many (> 10) flagella per zoo- use. For example, no isolates producing predominantly myce- spore, and monocentric thalli are clearly distinct from poly- lioid rhizoids have ever been shown to produce bulbous rhizoids, centric ones. Consequently at present, we prefer to retain these there does seem to be a clear break between a minimum of one existing genera and present a key to aid in their identification.

Key to current genera of the chytridiomycetous gut fungi 1. Polycentric thalli ...... 2 1. Monocentricthalli ...... 3 2. Predominantly polyflagellate ...... Orpitiott~yces 2. Predominantly uniflagellate ...... Atzaerotriyces* 3. Predominantly polyflagellate ...... Neocallitt~czsrix 3. Predominantly unitlagellate ...... 4 4. Mostly with bulbous rhizoids, occasionally with myceloid rhizoids ...... Caecott~yces 4. Mostly with extensively branched rhizoids, never with bulbous rhizoids ...... Pironlyces *Both Anaerotnyces and Rutnitlornyces have polycentric thalli and uniflagellate zoospores. They were described independently by Breton et al. (1990) and Ho and Bauchop (1990) and appear to be synonymous.

In conclusion, cladistic analysis of structural data shows that Barr, D. J. S. 1990. Chytridiomycota. 111 Handbook of Protoctista. (i) the Chytridiales, Spizellomycetales, and Monoblepharidales Eclirecl by L. Margulis, J. 0. Corliss, M. Melkonian, and are closely related, and their separation into individual orders D. J. Chapman. Jones and Bartlett Publishers, Boston. pp. 454 -466. is questionable; (ii) the Blastocladiales are a monophyletic group Barr, D. J. S., and Allan, P. M. E. 1981. Ultrastructure of Kochio- separate from both the Spizellomycetales and Chytridiales, rnyces and Triparricalcat- zoospores (Spizellomycetales, Chy tridio- with no special relationship with the Spizellomycetales; and mycetes). Can. J. Bot. 59: 649-661. Barr, D. J. S., and Allan, P. M. E. 1985. A comparison of the (iii) gut fungi are distinct from both the Spizellomycetales and flagellar apparatus in Plzytoph~hora,Saprvlegtzia, Thra~rsrocl~yrrirot~, Chytridiales on morphological, ultrastructural, mitotic, and and Rl~izicliotnyces.Can. J. Bot. 63: 138- 154. molecular characters. Consequently a new order, the Neocalli- Barr, D. J. S., and DCsaulniers, N. L. 1986. Four zoospore subtypes masticales, is established for them. in the Rhizophlyctis-Kmlitzgia complex (). Can. J. Bot. 64: 561 -572. Acknowledgements Barr, D. J. S., and DCsaulniers, N. L. 1987. Allod1y~riciirrtnlrtreutn For personal use only. spa.: morphology, physiology and zoospore ultrastructure. Mycolo- This work was supported by a grant from the Natural Sciences gia, 79: 193- 199. and Engineering Research Council of Canada to I.B.H. We Barr. D. J. S., and Hadland-Hartmann, V. E. 1978a. Zoospore sincerely thank Dr. P. Swarney for the Latin diagnosis and ultrastructure in the genus Rhizophycliutn (Chytridiales). Can. J. E. C. Gardonio for use of the Macintosh LC. Bot. 56: 2380-2404. Barr, D. J. S., and Hadland-Hartmann, V. E. 1978b. Zoospore Alexopoulos, C. J., and Mims, C. W. 1979. Introductory mycology. ultrastructure of Olpicli~ttnc~tcurbitacectr~tt~1 (Chytridiales). Can. J. John Wiley & Sons, New York. Bot. 56: 887-900. Barr, D. J. S. 1978. Taxonomy and phylogeny of chytrids. BioSystems, Barr, D. J. S., and Hadland-Hartrnann, V. E. 1979. Zoospore ultra- 10: 153-165. structure of Pl~lyctocl~y~rirtttzplrtrigibbosutn (Chytridiales). Can. J. Barr, D. J. S. 1980. An outline for the reclassification of the Bot. 57: 48-53. Chytridiales, and for a new order, the Spizellomycetales. Can. J. Barr, D. J. S., and Hartmann, V. E. 1976. Zoospore ultrastructure Bot. 58: 2380-2394. of three Chytric/illtn species and Rl~izoclosmariutnglobos~tm. Can.

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