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Genetic Relationships among Typhula ishikariensis Varieties from Wisconsin
Genetic Relationships among Typhula ishikariensis Varieties from Wisconsin
Weed & Turfgrass Science. 2015. Jun, 4(2): 135-143
JDHHCQ_2015_v4n2_© 2015 The Korean Society of Weed Science and The Turfgrass Society of Korea
This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
  • Received : September 05, 2014
  • Accepted : September 16, 2014
  • Published : June 30, 2015
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Seog-Won Chang
changsw802@hanmail.net

Abstract
Typhula ishikariensis Imai is a causal agent of Typhula snow mold, one of the most important turfgrass diseases in northern regions of the United States. Within Wisconsin isolates, there are three district groups clustered with known isolates of T. ishikariensis var. ishikariensis , var. canadensis and var. idahoensis as identified by RAPD markers. To further investigate the genetic relationship among these groups (varieties), monokaryon-monokaryon and dikaryon-monokaryon mating experiments were conducted. Mating types from var. ishikariensis , var. canadensis and var. idahoensis isolates were paired in all possible combinations. Pairings between var. canadensis and var. idahoensis were highly compatible, while no compatibility was detected between var. ishikariensis and either var. canadensis or var. idahoensis . These results indicate that var. ishikariensis is genetically separated from var. canadensis and var. idahoensis , whereas var. canadensis and var. idahoensis appeared to be genetically related to each other as a taxonomic unit. In the genetic relationship with the known biological species, var. ishikariensis and var. canadensis were genetically related to biological species I and II, respectively. However, var. idahoensis was not compatible with any of the biological species, suggesting that the pathogen may be in the process of biological speciation from var. canadensis .
Keywords
Introduction
Typhula ishikariensis Imai is a causal agent of Typhula snow mold, one of the most important winter diseases on turfgrass and winter cereals (Arsvoll and Smith, 1978 ; Chang et al., 2006a ; Smiley et al., 1994) , and is common areas with much longer snow cover periods than T. incarnata Lasch and T. phacorrhiza Reichard (Hsiang et al., 1999 ; Chang et al., 2006b) . After snowmelt, sclerotia are usually visible in infected plants or on soil where they oversummer. In late autumn, sclerotia produce mycelia or basidiospores serve as role as a primary inoculum (Cunfer and Bruehl, 1973 ; Bruehl and Cunfer, 1975) .
Since T. ishikariensis was first described by Imai (1930) , identification of the fungus mainly relied on morphological descriptions of the sclerotia and fruiting body. However, variation in morphology caused by environmental conditions resulted in taxonomic confusion among researchers, who assigned different names to Typhula species and T. ishikariensis subspecies level, showing the nomenclature of different species, varieties, biotypes or groups among countries and researchers (Bruehl and Cunfer, 1975 ; Arsvoll and Smith, 1978 ; Matsumoto et al., 1982 ; Matsumoto, 1997) . The confusion was solved when morphological characteristics were combined with genetic studies at the species level as a conclusion of Røed (1969) that T. itoana , T. graminum and T. incarnata all were the same species.
Although interfertility experiments have become a valuable tool in the delineation of Typhula species, different forms of T. ishikariensis have been recognized by researchers. Bruehl et al. (1975) used mating experiments to conclude that T. ishikariensis and T. idahoensis were different species. However, Arsvoll and Smith (1978) studied T. ishikariensis isolates from Canada, USA, Norway, Japan and Switzerland on the basis of morphology, cultural characteristics and mating ability. Based on these studies, they rejected T. idahoensis as a separate species and divided T. ishikariensis into three varieties: var. ishikariensis , var. canadensis and var. idahoensis .
In Japan, Matsumoto et al. (1982) identified three forms among Japanese isolates of T. ishikariensis named biotypes A, B, and C. From the results of mating experiments as well as culture morphology, biotype A was considered identical to var. ishikariensis . Biotype C was similar to var. canadensis , while biotype B was considered different from var. idahoensis because of the poor mating between them. However, Matsumoto and Tajimi (1991) later concluded that biotypes B and C were a single biological species adapted to different localities based on more extensive studies.
Matsumoto (1997) divided T. ishikariensis isolates from around the world into two biological species as defined by Mayr (1963) who described a biological species as “group of interbreeding natural populations that is reproductively isolated from other groups”. Matsumoto concluded that biological species I (BSI) included T. ishikariensis and T. ishikariensis var. ishikariensis from North America, biotype A from Japan, and groups I and III from Norway, while Biological species II (BSII) included T. idahoensis , T. ishikariensis varieties idahoensis and canadensis , T. ishikariensis group II and biotypes B and C.
The difficulty regarding the taxonomy of T. ishikariensis has also been evident in Wisconsin, where this fungus is one of the most important pathogens on turfgrass (Chang et al., 2006b) . Millet et al. (2001) grouped T. ishikariensis samples according to their morphological characteristics into WIG1 and WIG2. From mating experiments with both BS I and II, the authors concluded that WIG1 and WIG2 isolates were genetically related to BSI and BSII, respectively. However, one of the problems observed from Wisconsin samples was the fact that several isolates could not mate or mated spuriously with any of the BS, thus they could not be classified into either known BS.
Using Random Amplified Polymorphic DNA (RAPD) markers, Jung et al. (2000) investigated the genetic relationship among Wisconsin isolates, known isolates classified as T. ishikariensis var. ishikariensis , var. idahoensis and var. canadensis as well as some of the tester monokaryons from BSI and BSII. The authors found that the T. ishikariensis population in Wisconsin was composed of three genetically distinct groups, which were named groups A, B and C. Interestingly, group A and BSI were clustered with T. ishikariensis var. ishikariensis . Group B and some isolates of the BSII were clustered with T. ishikariensis var. canadensis , while group C and some BSII isolates were located within var. idahoensis . The objective of this study was to examine the genetic relationship among the three T. ishikariensis varieties from Wisconsin isolates as well as the taxonomic relationships with known BS using interfertility experiments.
Materials and Methods
- Fungal isolates
A total of 105 isolates of T. ishikariensis varieties from Wisconsin were randomly selected from previously identified collections ( Fig. 1 A and B) (Chang et al., 2006b) and 39 isolates are used for mating are listed in Table 1 . Based on morphological and DNA fingerprinting data (Chang et al., 2006b) , all isolates selected out of 39 isolates were pathogenic on three bentgrass species ( Agrostis sp.) (Chang et al., 2006a) . In order to test mating compatibility with Wisconsin isolates, three tester monokaryons representing T. ishikariensis Biological Species (BS) I and BS II were obtained from Dr. Naoyuki Matsumoto, National Institute of Agro-Environmental Sciences, Tsukuba, Japan ( Table 1 ).
PPT Slide
Lager Image
Snow mold damage by Typhula ishikariensis var. ishikariensis on creeping bentgrass putting green (A) and the fungus’ mycological characteristics (B~F). Old sclerotia of var. ishikariensis in field are black color (B), wheras fresh sclerotia produced on PDA medium after 4 weeks at 10℃ are usually brown color (C). The sporocarps were produced after 4 weeks on non-sterilized field soil in a growth chamber at 9/4℃ (day/night) with 8 photoperiods. Arrow heads indicate basidiocarp. (D and E). Basiospores and their germination (F). Stained monokaryotic and dikaryotic state of T. ishikariensis mycelia are described in Chang and Jung (2008). Scale bar = 2 μm.
T. ishikariensisvarieties isolates used in this study.
Isolates Classification (stage) Year and location collected

93-32-MI var. ishikariensis (BS Ia) (monokaryon ) Unknown, Russia (Matsumoto and Tajimi, 1990)
8-2 Biotype B (BS II) (monokaryon) Unknown, Japan (Matsumoto et al., 1982, 1997)
4-3, S-5 Group II (BS II) (monokaryon) 1995, Norway (Matsumoto et al., 1996)
NE 9. 4. 5 var. ishikariensis (dikaryon) 2001, Big Sand GC, Vilas, WI, U.S.A
NE 10. 5. 1 var. ishikariensis (dikaryon) 2001, Big Stone Golf & SB, Oneida, WI, U.S.A
NE 23. 6. 1 var. ishikariensis (dikaryon) 2001, DeSmidt’s GC & CC, Marinette, WI, U.S.A
NE 63. 11. 1 var. ishikariensis (dikaryon) 2001, Merrill GC, Lincoln, WI, U.S.A
NE 73. 17. 2 var. ishikariensis (dikaryon) 2001, Peck’s Wildwood GC, Vilas, WI, U.S.A
NE 73. 17. 3 var. ishikariensis (dikaryon) 2001, Peck’s Wildwood GC, Vilas, WI, U.S.A
NE 85. 6. 2 var. ishikariensis (dikaryon) 2001, Riveredge CC, Marathon, WI, U. S. A
NW 10. 8. 2 var. ishikariensis (dikaryon) 2001, Botten’s Green Acres, Douglas, WI, U.S.A
NW 10. 9. 2 var. ishikariensis (dikaryon) 2001, Botten’s Green Acres, Douglas, WI, U.S.A
NW 26. 9. 3 var. ishikariensis (dikaryon) 2001, Fox Run GC, Burnett, WI, U.S.A
NW 33. 2. 3 var. ishikariensis (dikaryon) 2001, Hayward G&TC, Sawyer, WI, U.S.A
NW 69. 8. 5 var. ishikariensis (dikaryon) 2001, Spider Lake Golf Resort, Sawyer, WI, U.S.A
NE 27. 4. 1 var. canadensis (dikaryon) 2001, Edgewater GC, Lincoln, WI, U. S. A
NE 31. 5. 5 var. canadensis (dikaryon) 2001, Four Season GC, Marinette, WI, U.S.A
NE 42. 13. 1 var. canadensis (dikaryon) 2001, High Cliff GC, Calumet, WI, U.S.A
NE 57. 2. 4 var. canadensis (dikaryon) 2001, Maple Hills GC, Shawano, WI, U.S.A
NE 68. 17. 2 var. canadensis (dikaryon) 2001, Nicolet GC, Forest, WI, U.S.A
NE 90. 11. 3 var. canadensis (dikaryon) 2001, Sandalwood GC, Oconto, WI, U.S.A
NE 95. 6. 5 var. canadensis (dikaryon) 2001, Spread Eagle GC, Oconto, WI, U.S.A
NE 104. 14. 4 var. canadensis (dikaryon) 2001, Trout Lake G & CC, Vilas, WI, U.S.A
NE 110. 1. 3 var. canadensis (dikaryon) 2001, Wander Springs, Outagamie, WI, U.S.A
NW 7. 6. 1 var. canadensis (dikaryon) 2001, Barronett Hills GC, Barron, WI, U.S.A
NW 10. 6. 5 var. canadensis (dikaryon) 2001, Botten’s Green Acres, Douglas, WI, U.S.A
NW 14. 13. 5 var. canadensis (dikaryon) 2001, Chippewa Valley GC, Dunn, WI, U.S.A
NW 48. 1. 1 var. canadensis (dikaryon) 2001, Neillsville GC, Clark, WI, U.S.A
NW 57. 4. 1 var. canadensis (dikaryon) 2001, Pine Meadow GC, Douglas, WI, U.S.A
NW 73. 18. 1 var. canadensis (dikaryon) 2001, Tagalong, Washburn, WI, U.S.A
SE 79. 12. 4 var. canadensis (dikaryon) 2001, Quit Qui Oc GC, Sheboygan, WI, U.S.A
SE 86. 27. 3 var. canadensis (dikaryon) 2001, Rolling Meadows GC, Fond Du Lac, WI, U.S.A
SW 8. 10. 4 var. canadensis (dikaryon) 2001, Castle Rock GC, Juneau, WI, U.S.A
NE 4. 10. 5 var. idahoensis (dikaryon) 2001, Antigo Bass Lake, Langlade, WI, U.S.A
NE 31. 2. 3 var. idahoensis (dikaryon) 2001, Four Season GC, Marinette, WI, U.S.A
NE 34. 4. 10 var. idahoensis (dikaryon) 2001, Gateway, Vilas, WI, U.S.A
NE 45. 6. 5 var. idahoensis (dikaryon) 2001, Homestead GC, Wood, WI, U.S.A
NE 95. 3. 5 var. idahoensis (dikaryon) 2001, Spread Eagle GC, Oconto, WI, U.S.A
NW 7. 2. 1 var. idahoensis (dikaryon) 2001, Barronett Hills GC, Barron, WI, U.S.A
NW 9. 7. 2 var. idahoensis (dikaryon) 2001, Bloomer Memorial GC, Chippewa, WI, U.S.A
NW 33. 18. 5 var. idahoensis (dikaryon) 2001, Hayward G&TC, Sawyer, WI, U.S.A
NW 45. 16. 4 var. idahoensis (dikaryon) 2001, Mellen CC, Ashland, WI, U.S.A
aClassification given by the author who described the isolate.
- Determination of mating types
Fifteen dikaryotic isolates from T. ishikariensis var. ishikariensis , 15 from var. canadensis and 45 from var. idahoensis were used to obtain monokaryons from basidiospores. A larger number of isolates from T. ishikariensis var. idahoensis were chosen due to the failure of this variety to produce sporocarps in preliminary experiments. To obtain monokaryotic isolates the procedures used by Bruehl and Cunfer (1975) were followed. At least 20 sclerotia per isolate were placed on non-sterilized soil in pots ( Fig. 1 C, D, and E). The pots were placed into plastic trays containing water to assure constant moisture and maintained in a growth chamber at 5 C with 8 h photoperiods at 3.45 mEm -2 s -1 (ca. 300 lux).
Once the most sporocarps arose and reached their mature stage (about one week after emerging), they were collected. Each was individually placed on the lid of a petri dish containing 1.5% water agar (WA) medium, and incubated at 10 C. After 4-5 days, the basidiospores showered onto the WA were suspended in distilled-sterilized water and spread out onto the media surface. The plates were incubated at 10 C for 4-7 days or until germinating spores were observed. Following that, a single germinated basidiospore was transferred from WA to PDA (Potato Dextrose Agar) medium and incubated at 10 C until colony formation (10-15 days) ( Fig. 1 F). The colonies were then examined for the presence or absence of clamp connections, the absence of which is an indication of the monokaryotic state.
At least 12 monokaryons per dikaryotic isolate were selected. These monokaryons were paired in all combinations in order to identify the four mating types. Pairings were made following the methodology used by Bruehl et al. (1975) with slight modifications. Plugs of mycelia from two monokaryons were placed 1 cm apart in a petri dish containing PDA. After 15 days at 10 C, hyphae from the junction zone of both colonies were examined under the microscope. Presence with numerous and normal clamps connections indicated a compatible pairing as a positive reaction ‘+’, meaning two isolates were considered to have a closer relationship. When the pairing was incompatible no clamp connections were observed and the reaction was noted as negative ‘-’. When no compatible reaction was noticed between two isolates, the isolates were considered to be not genetically related. An uncertain relationship with very few clamp connections or abnormal growth of mycelium was noted as ‘±’.
The assignment of the mating type within each isolate was arbitrary, based on the tetrapolar incompatibility system present in Typhula spp. (Bruehl et al., 1975 ; Chang and Jung, 2008) . Positive or compatible reactions can only be possible between two monokaryons carrying different alleles in both A and B factors (i.e. A 1 B 1 + A 2 B 2 ). On the contrary, mating cannot occur when there is at least one allele in common (i.e. A 1 B 1 + A 1 B 2 ).
- Monokaryon-monokaryon pairing
Four monokaryons representing the four mating types from each dikaryon were selected. Mating experiments were made by taking the four mating types from each isolate and pairing them in all combinations. Pairings and identification of mating reactions were done per the methods of Bruehl et al. (1975) explained in the previous section.
To investigate the compatibility between Wisconsin monokaryotic isolates and known tester monokaryons, four mating types of each variety were paired with the monokaryons representing T. ishikariensis BS I and BS II. Compatible reactions meant the isolates were genetically compatible and considered as the same BS.
- Dikaryon-monokaryon pairing
Thirty dikaryons from three varieties were selected and mated with four mating types of each variety. Pairings and identification of mating reactions were done per the methods of Bruehl et al. (1975) with slight modifications. Five mm diameter agar discs with mycelia were cut from the margin of actively growing PDA cultures of both monokaryotic testers and dikaryotic isolates and placed approximately 1 cm apart on PDA plates. After 3 to 4 days of colony contact (ca. 15 days incubation at 10 C), a 5 mm agar disc was cut from the monokaryon colony 1 cm behind the colony junction and transferred to a new PDA plate. The mycelia growing from the junction piece was microscopically examined for the presence of clamp connections one week later. Identification of mating reactions was done as the methods of Bruehl et al. (1975) explained in the previous section.
Results
- Determination of mating types
Seventy-five isolates from three T. ishikariensis varieties were examined to obtain monokaryons. From T. ishikariensis var. ishikariensis , five out of fifteen isolates (33%) produced sporocarps, but only two of them (13.3%) produced monokaryons. Seven isolates out of fifteen (46.6%) from T. ishikariensis var. canadensis produced sporocarps. From them, five isolates (33%) produced basidiospores that developed into monokaryotic colonies. Five isolates out of forty-five (11%) from T. ishikariensis var. idahoensis produced sporocarps, but only two (4.4%) produced basidiospores that developed into monokaryons ( Table 2 ).
Production of sporocarps and monokaryons fromT. ishikariensisvarieties isolated in Wisconsin.
Variety Number of dikaryons Number of dikaryons producing sporocarps Number of dikaryons that produced monokaryons

Var. ishikariensis 15 5 2
Var. canadensis 15 7 5
Var. idahoensis 45 5 2
In all cases, more than 30 monokaryons were obtained from each isolate that produced the monokaryotic colony. The sets of four mating types with the exception of two isolates were obtained from one dikaryon of T. ishikariensis var. ishikariensis , from four dikaryons of var. canadensis , and from two dikaryons of var. idahoensis . The result demonstrates that the most isolates represented the tetrapolar incompatibility mating system ( Table 3 ). However, T. ishikariensis var. ishikariensis NE 63.11.1 yielded three mating types and monokaryons from var. canadensis NW48.1.1 could not be assigned to any of the mating type because several monokaryons failed to mate with any other. In each case, four monokaryons representing the four mating types were selected. For those isolates whose compatibility groups could not be assigned, four monokaryons were chosen based on their ability to mate (i.e. two monokaryons unable to mate with any other monokaryon and two monokaryons able to mate with the rest). The reason monokaryons were selected in this way is that it permitted us to know whether those isolates incapable to mate with monokaryons coming from the same dikaryon, were also unable to mate with monokaryons from other dikaryons. The inability of mating could suggest the presence of infertility factors.
Mating types of each T. ishikariensis variety identified from dikaryon isolates.
Isolate Variety Number of monokaryons mated Number of mating types identified

NE63.11.1 var. ishikariensis 12 3
NE85.6.2 var. ishikariensis 12 4
NE27.4.1 var. canadensis 12 4
NW14.13.5 var. canadensis 12 4
NW48.1.1 var. canadensis 14 0
NW57.4.1 var. canadensis 12 4
NW73.18.1 var. canadensis 12 4
NE34.4.10 var. idahoensis 13 4
NW33.18.5 var. idahoensis 13 4
- Genetic relationships among T. ishikariensis varieties by monokaryon-monokaryon pairing
Mating types from five isolates were selected to mate among varieties ( Table 4 ). All four mating types with the exception of NE 63.11.1 were compatible with mating type carrying different alleles of same isolates. Among T. ishikariensis var. canadensis , 34 positive reactions, 12 negative reactions, and 1 uncertain reaction were found. In pairing among three varieties, no compatible reactions were observed between monokaryons from T. ishikariensis var. ishikariensis and var. canadensis or var. idahoensis , while monokaryons of var. canadensis and var. idahoensis were compatible with one or more monokaryons from the both variety. The pairing of T. ishikariensis var. idahoensis with var. canadensis produced 38 positive reactions, 6 negative reactions and 4 uncertain reactions.
Pairings among four mating types of Wisconsin dikaryons representingT. ishikariensisvarieties.
Isolatea Mating type NE63.11.1 (var. ishikariensis) NW14.13.5 (var. canadensis) NW57.4.1 (var. canadensis ) NW73.18.1 (var. canadensis ) NE34.4.10 (var. idahoensis )

A1 A1 A2 A2 A1 A1 A2 A2 A1 A1 A2 A2 A1 A1 A2 A2 A1 A1 A2 A2

B1 B2 B1 B2 B1 B2 B1 B2 B1 B2 B1 B2 B1 B2 B1 B2 B1 B2 B1 B2

NE63.11.1 (var. ishikariensis) A1B1b - - - +
A1B2 - + + -
A2B1 - + + -
A2B2 + - - -
NW14.13.5 (var. canadensis) A1B1 -c - - - - - - +
A1B2 - - - - - - + -
A2B1 - - - - - + - -
A2B2 - - - - + - - -
NW57.4.1 (var. canadensis ) A1B1 - - - - + + + + - - - +
A1B2 - - - - + + + + - - + -
A2B1 - - - - + + + + - + - -
A2B2 - - - - + + + + + - - -
NW73.18.1 (var. canadensis ) A1B1 - - - - + + + ± - - + - - - - +
A1B2 - - - - + - + + - - + + - - + -
A2B1 - - - - + + + + + - - - - + - -
A2B2 - - - - + - + - + + - - + - - -
NE34.4.10 (var. idahoensis ) A1B1 - - - - + + - - + + + + + + + + - - - +
A1B2 - - - - + + + + + + + + + + ± + - - + -
A2B1 - - - - + + + + + + + ± + + - ± - + - -
A2B2 - - - - + - + - + + + ± + + - + + - - -
aName of dikaryon from which the mating type was selected. bHypothetical assigned alleles. Numbers 1 and 2 are used to refer to as different alleles but are not intended to represent any specific genotype. c+ indicates compatible reaction, -indicates no compatible reaction, ± indicates an uncertain reaction where few clamp connections were observed.
Mating types from four isolates were selected to mate with the known BS testers ( Table 5 ). The four mating types from T. ishikariensis var. ishikariensis isolate NE63.11.1 were compatible with the tester monokaryon of BSI, but not compatible with most of the BSII testers. Only one monokaryon produced an uncertain reaction ‘±’ with 4.3,5.2, forming very few clamp connections and rhizomorphous hyphae. Two or more mating types from the two isolates of T. ishikariensis var. canadensis successfully mated with the two BS II testers. T. ishikariensis var. canadensis isolates were not compatible with BS I, with the exception of one monokaryon from isolate NW14.13.5, which paired uncertainly with the BS I tester 92-32-MI. Monokaryons from T. ishikariensis var. idahoensis did not mate with any of the BS testers.
Pairings among mating types of Wisconsin dikaryons representingT. ishikariensisvarieties and tester monokaryons representing the biological species I and II.
Tester monokaryon Biological species, mating type NE63.11.1 (var. ishikariensis) NW14.13.5 (var. canadensis) NW57.4.1 (var. canadensis) NE 34.4.10 (var. idahoensis)

A1B1a A1B2 A2B2 A2B2 A1B1 A1B2 A2B1 A2B2 A1B1 A1B2 A2B1 A2B2 A1B1 A1B2 A2B1 A2B2

92-32-MI I, A1B1 +b + + + ± - - - - - - - - - - -
8-2 II, A2B1 - - - - + - - + + - + - - - - -
4.3,5.2 II, A2B2 - - ± - + - + + + + + + - - - -
aHypothetical assigned alleles. Numbers 1 and 2 are used to refer to as different alleles but are not intended to represent any specific genotype. b+ indicates compatible reaction, - indicates no compatible reaction, ± indicates an uncertain reaction where few clamp connections were observed.
- Genetic relationships among T. ishikariensis varieties by dikaryon-monokaryon pairing
Thirty dikaryotic isolates of T. ishikariensis varieties were paired with four monokaryotic isolates of each variety ( Table 6 ). All T. ishikariensis var. ishikariensis mated positively with at least two mating types of var. ishikariensis testers. No compatible reactions, however, were observed between dikaryons from T. ishikariensis var. ishikariensis and monokaryons of var. canadensis or var. idahoensis . In addition, monokaryons of T. ishikariensis var. ishikariensis were not compatible with dikaryons of var. canadensis or var. idahoensis . All dikaryons of the T. ishikariensis var. canadensis mated positively with at least two mating types of var. canadensis testers. In contrast with T. ishikariensis var. ishikariensis , four dikaryons of var. canadensis showed compatible reactions with two or more tester monokaryons from var. idahoensis . In the case of T. ishikariensis var. canadensis monokaryons, all mating types were mated with four var. idahoensis dikaryons with the exception of NW 45.16.4. Three out of seven dikaryotic isolates from T. ishikariensis var. idahoensis gave compatible reactions with at least two var. idahoensis testers. Six of T. ishikariensis var. idahoensis dikaryotic isolates showed the compatible reactions with two more tester monokaryons from var. canadensis .
Dikaryon-monokaryon mating reactions of dikaryoticT. ishikariensisvarieties isolates with monokaryotic tester isolates of Wisconsin.
Varietya Isolate NE 63.11.1 (var. ishikariensis)a NW 14.13.5 (var. canadensis) NE 34.4.10 (var. idahoensis)

A1B1b A1B2 A2B1 A2B2 A1B1 A1B2 A2B1 A2B2 A1B1 A1B2 A2B1 A2B2

Var. ishikariensis NE 9.4.5 +c ± - + - - - - - - - -
NE 10.5.1 + + + + - - - - - - - -
NE 23.6.1 + - - + - - - - - - - -
NE 73.17.2 - + + ± - - - - - - - -
NE 73.17.3 - - + + - - - - - - - -
NW 10.8.2 + + + + - - - - - - - -
NW 10.9.2 + ± - + - - - - - - - -
NW 26.9.3 + + + + - - - - - - - -
NW 33.2.3 + - - + - - - - - - - -
NW 69.8.5 + - ± + - - - - - - - -
Var. canadensis NE 31.5.5 - - - - + - ± + - - - -
NE 42.13.1 - - - - - + + - + - - +
NE 57.2.4 - - - - ± + + - - - - -
NE 68.17.2 - - - - - + + - - - - -
NE 90.11.3 - - - - + + + + - - - -
NE 95.6.5 - - - - - + + ± - - - -
NE 104.14.4 - - - - + + + + - - - -
NE 110.1.3 - - - - + - - + + - - +
NW 7.6.1 - - - - - + + - + ± - +
NW 10.6.5 - - - - - + + ± + - - +
SE 79.12.4 - - - - + - - + - - - -
SE 86.27.3 - - - - + + + + - - - -
SW 8.10.4 - - - - + + + + - - - -
Var. idahoensis NE 4.10.5 - - - - + - - + - - - -
NE 31.2.3 - - - - + - + + + ± - +
NE 45.6.5 - - - - + + + + - - - -
NE 95.3.5 - - - - + + + + - - - -
NW 7.2.1 - - - - - + + - + ± - +
NW 9.7.2 - - - - - + + - + + + +
NW 45.16.4 - - - - - - - - - - - -
aName of dikaryon from which the mating type was selected. bHypothetical assigned alleles. Numbers 1 and 2 are used to refer to as different alleles but are not intended to represent any specific genotype. c+ indicates compatible reaction, -indicates no compatible reaction, ± indicates an uncertain reaction where few clamp connections were observed.
Discussion
Incompatibility between monokaryons derived from basidiospores of Typhuls species is determined by complementation of multiple alleles at two loci (Cunfer, 1974) . The incompatibility alleles have been used as convenient genetic markers for taxonomy of Typhula spp. (Kiyomoto and Bruehl, 1976) . Within T. ishikariensis varieties, the incompatibility system using monokaryons or dikaryons are an important key to solve the taxonomic confusion as well as genetic relationships within and among the fungi (Bruehl et al., 1975 ; Matsumoto, 1997 ; Matsumoto et al., 1982 ; Matsumoto and Tajimi, 1990) .
The production of monokaryons from dikaryotic isolates of T. ishikariensis in this study was low ( Table 2 ). Some isolates never produced sporocarps, some formed sporocarps but not basidiospores and others produced basidiospores that never germinated or germinated but did not develop into a colony. In total, only 33% of the dikaryons from T. ishikariensis var. canadensis produced monokaryons, but this amount was higher than for var. ishikariensis and var. idahoensis , with only 11% and 4.4% monokaryon production from fertile dikaryons, respectively. This phenomenon was similar to a report by Bruehl et al. (1975) that many dikaryons did not produce sporocarps, produced few basidiospores or mostly spores that did not germinate, or produced spores that germinate, but failed to develop further. In addition, these relative proportions were observed by Bruehl et al. (1978) , who explained that sterility was common in T. idahoensis and less common in T. ishikariensis . The authors also found that 90% of a collection of 2000 T. idahoensis isolates from Idaho, Utah, Montana and Wyoming were infertile, concluding that T. idahoensis from those regions were nearly asexual.
Even though in our study T. ishikariensis var. canadensis appears to be more fertile than var. ishikariensis and var. idahoensis ( Table 2 ), this may be due to environmental conditions rather than intrinsic fertility of the fungi. Each of the varieties favors different habitats (Chang et al., 2006b ; Matsumoto et al., 1982) . Although we used a constant condition (5 C with 8 h daylength) for the incubation of all three varieties, a specific environment may be necessary for the production and development of the sexual stage in basidiomycetes (Bruehl and Cunfer, 1975 ; Dahlberg and Van Etten, 1982 ; Kawakami et al., 2004) . Kawakami et al. (2004) also reported that environmental factors including fluctuating temperature (10/5 C; day/night), high humidity, and high intensity are conductive to sporocarp formation in T. ishikariensis biotype A and B.
The mating experiments were successful in characterizing the T. ishikariensis subspecies at the variety level ( Table 4 and 6 ). Most mating types mated monokaryons carrying different alleles obtained from the same isolate, demonstrating that most isolates have the typical tetrapolar incompatibility. In particular, at least 10 alleles of A and 10 of the B locus were identified within T. ishikariensis var. canadensis , suggesting that this population exists as an active interbreeding unit (Kiyomoto and Bruehl, 1976 ; Bruehl and Machtmes, 1979) . Upon examining the genetic relationships among T. ishikariensis varieties, var. canadensis and var. idahoensis were found to be closely related to each other. All T. ishikariensis var. canadensis monokaryons mated with mating types of var. idahoensis and the resulting dikaryons had normal and numerous clamp connections, indicating that the varieties were genetically compatible as a taxonomic unit. These trends were also shown in the data from dikaryon-monokaryon mating. The two varieties in the pairing mated with each other, although the recovery of parental allele was different among isolates. This may be associated with recovery of both alleles of a parental haploid nucleus, recovery of one parental allele, and recovery of one or two different alleles from the other nucleus (Bruehl et al., 1975) .
Monokaryons from T. ishikariensis var. ishikariensis did not mate with those of var. canadensis or var. idahoensis at all, although the sample size was very small. These trends were confirmed with the data from dikaryon-monokaryon mating. Di- or mono-karyons from T. ishikariensis var. ishikariensis did not mate with mono- or di-karyons of var. canadensis or var. idahoensis at all. These results indicate that T. ishikariensis var. ishikariensis is incompatible with both varieties, which is a sign that a reproductive barrier might be present. Similar results have been found in interactions among isolates of T. ishikariensis varieties with different origins (Arsvoll and Smith, 1978 ; Bruehl et al., 1978 ; Bruehl and Machtmes, 1980 ; Matsumoto, 1997) . Interestingly, however, our result was very different with that of Jung et al. (2000) , which found that the genetic relationship between T. ishikariensis var. ishikariensis and var. idahoensis appears to be closer than that of var. canadensis and var. idahoensis . A possible interpretation of this discrepancy is that their genetic map was constructed using RAPD markers, which are random and short primers and therefore may not be linked to mating factors.
Our results support the hypothesis that T. ishikariensis var. ishikariensis is distinctly separated from the other two varieties. The concept of a biological species in general establishes “speciation” as the process by which a genetically cohesive group of interbreeding individuals diverges into two or more genetically distinct groups of individuals (Bock, 1986) , in spite of its other characteristics. If this concept is applied to T. ishikariensis varieties, it is possible to say that T. ishikariensis var. ishikariensis and other varieties are different species, or the two groups might be undergoing speciation.
As stated by Petersen and Hughes (1999) , interbreeding populations may diverge genetically over time until speciation occurs. During that process, strong reproductive barriers often accompany the differentiation into species, with many factors such as ecological and geographical separation contributing to those barriers. In this sense, the habitat of T. ishikariensis varieties is very important. Based on ecological habitats of three varieties, T. ishikariensis var. canadensis and var. idahoensis favored the more unstable environment (Matsumoto et al., 1982 ; Chang et al., 2006b) , have low growth ability (Bruehl and Machtmes, 1980) , and have a limited host range (Matsumoto and Tajimi, 1993) compared with var. ishikariensis . Thus, even though the three varieties are able to grow in the same area, T. ishikariensis var. canadensis and var. idahoensis might not occur at the same time as var. ishikariensis which could be an ecological barrier for interbreeding. If this barrier has been present for a long time, reproductive isolation might have occurred leading to genetic divergence.
Interestingly, pairings with the known BS also provided a possibility that there are three biologically different groups in Wisconsin. T. ishikariensis var. ishikariensis and var. canadensis were genetically related to BS I and BS II, but var. idahoensis was not related to either BS I or BS II isolates. Similarly, Millet et al. (2001) found that several isolates from Wisconsin could not mate or mated spuriously with any of the BS. This phenomenon suggests that T. ishikariensis var. idahoensis may represent a third group with genetically different traits from var. canadensis in only North America as described by Arsvoll and Smith (1978) . Otherwise, the fungi may be in the process of biological speciation from var. canadensis . Further experiments need to be done to understand more about three varieties.
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