Rhizopogon kretzerae Grubisha, Dowie, & Mill

Lisa C. Grubisha, Nicholas J. Dowie, Steven L. Miller, Christina Hazard, Steven M. Trowbridge, Thomas R. Horton & Matthew R. Klooster, 2014, Rhizopogon kretzerae sp. nov.: the rare fungal symbiont in the tripartite system with Pterospora andromedea and Pinus strobus, Botany 92, pp. 526-534 : 531-533

Rhizopogon kretzerae Grubisha, Dowie, & Mill . sp. nov.

and.

INDEX FUNGORUM REGISTRATION NUMBER: IF 550254

DIAGNOSIS: Known only from ectomycorrhizal symbioses with Pinus strobus and monotropoid mycorrhizal symbioses with the mycoheterotrophic plant Pterospora andromedea in eastern North America. Rhizopogon kretzerae can be distinguished from R. salebrosus , presently the closest identified species, by two indels found in the ITS1 and 12 fixed nucleotide differences, three in the ITS1 and nine in the ITS2.

HOLOTYPE: DAOM 242738

LOCATION: United States of America, Michigan, Ottawa National Forest, 46.64271°N, 89.17873°W. Collection number: Miller 207, Collected: 4 August 2011 by N.J. Dowie and S.L. Miller. DAOM 242738 is a specimen from silica-dried, dead culture isolated from Pterospora andromedea roots, stored with silica.

ETYMOLOGY: Named in honor of Dr. Annette Kretzer (State Univer- sity of New York, Syracuse, New York, USA), whose pioneering work on the population genetics of Rhizopogon vinicolor A.H. Sm. and Rhizopogon vesiculosus A.H. Sm. advanced our understanding of Rhizopogon evolutionary biology and provided inspiration to many mycologists and students.

DESCRIPTION: Macroscopic and microscopic observations were made from a live culture of isolate Miller 207 but not the dried specimen that was deposited as the holotype because these notes were made after the dried holotype specimen was deposited at DAOM.

MACROSCOPIC CHARACTERISTICS: Mycelial mat whitish at first (circa one week), aerial hyphae developing shades of pale brownish grey, older cultures (circa three weeks) pale reddish grey to gray- ish red, brownish orange, light brown or brown, often with patches of lighter color scattered across the colony (). Under U.V. light, light purple with dark purple patches; reverse brown, uniform, with radiating fans at the margin; mycelial mat floccose when first isolated, becoming low, dense, felty with age and with repeated transfers; margin irregular, whispy, aerial mycelium sparser toward edge, hyphae submerged at edge of colony when younger, submerged area overgrown on surface with fine hyphae in age, darker brown-like reverse appearing as a dark halo sur- rounding mycelial mat, minute knots of hyphae scattered to abundant at margin and throughout colony, concolorous with mycelium or lighter in color; maximum growth rate 3.0–3.4 cm in one month at 25 °C; odor strong, sweet, and pleasant.

MICROSCOPIC CHARACTERISTICS: Hyphae of interior of mycelial mat mostly 3–7 um, thick-walled, smooth, frequently swollen at sep- tations or forming vesciculose, catenulate cells, 6–33 µm wide, thin-walled, easily collapsing; branching mostly simple, dendritic hyphae common; septae simple; clamps absent. Hyphae of ex- terior of mycelial mat mostly 3–10 µm, cylindrical, branching simple, dendritic hyphae common, few swollen catenulate cells present, moderately thick-walled, heavily encrusted overall or in discreet patches with granular, pale brown pigment in KOH, in Melzer's Reagent pigment darker brown (dextriniod), soluble and floating free from the hyphal wall and appearing large globular or amorphous; septae simple; clamps absent.

ECOLOGY AND DISTRIBUTION: Rhizopogon kretzerae appears to be re- stricted to areas with Pinus strobus and Pterospora andromedea or P. strobus alone in Northern Michigan, New York, New Hampshire, and southern Quebec. identified R. kretzerae from P. strobus roots within a P. andromedea rootball from Keweenaw County, Michigan. A soil bioassay study has shown that the frequency of R. kretzerae propagules in soil increases in P. strobus areas where P. andromedea is reproductively active with R. kretzerae occurring at a frequency from 6% of soil samples from sites with P. strobus to 14% to 25% (site vs. plot) in soils with both P. strobus and P. andromedea () .

COMMENTS: Rhizopogon kretzerae is the first record of a Rhizopogon subgenus Amylopogon species that occurs in eastern North Amer- ica (i.e., east of the Mississippi River in US;;). Presently, there are no known annotated herbarium collections of the basidiocarp. Monotropoid mycorrhizal rootballs of eastern P. andromedea and R. kretzerae have diameters reaching a maximum of approximately 1.5 inches and are substantially smaller than rootballs of P. andromedea and R. salebrosus in western North America (). Ultrastructure of P. andromedea monotropoid roots has been described in detail by

.

OTHER SPECIMENS EXAMINED: Pterospora andromedea monotropoid mycorrhizal roots DAOM 242739, Location: Canada, province of Quebec, 45.4947°N, 76.3786°W; Collection number: Quebec A21, Collected: 26 July 2012 by M.R. Klooster and L.C. Grubisha; DAOM 242740, Location: Canada, province of Quebec, 45.4947°N, 76.3786°W; Collection number: Quebec A22, Collected: 26 July 2012 by M.R. Klooster and L.C. Grubisha; DAOM 242741, Location: Canada, province of Quebec, 47.2900°N, 79.4566°W, Collection number: Quebec D16, Collected 27 July 2012 by M.R. Klooster and L.C. Grubisha; DAOM 242742, Location: Canada, province of Quebec, 47.3166°N, 79.4533°W, Collection number: Quebec E22, Collected: 27 July 2012 by M.R. Klooster and L.C. Grubisha; DAOM 242743, Location: Canada, province of Quebec, 45.4672°N, 75.8069°W, Collection number: Quebec H11, Collected: 27 July 2012 by M.R. Klooster and L.C. Grubisha. All root samples were dried in silica gel.

Discussion

In this study, Rhizopogon kretzerae Grubisha, Dowie , & Mill., sp. nov. was described. This is also the first report of the distribution of this rare fungus in Quebec from five locations covering approximately 430 km. Since there are no known basidiocarp collections, the species description was based on ITS sequence data, ecological information, and a morphological description of the culture isolated from P. andromedea roots. The results from phylogenetic analyses presented here and in previous studies (;) are strong evidence that R. kretzerae is a species according to the phylogenetic species concept. If a basidiocarp is collected at some future time, the present description should be amended. Prior to this study, two GenBank environmental sequences ( AF442136 , DQ426677 ) had a>99% identity and both came to be known by some variation of the cumbersome phrase “the unnamed species of Rhizopogon subgenus Amylopogon that associates with P. andromedea in the east” (;), which can now be referred to as R. kretzerae . Furthermore, now it is possible to communicate ecological information about R. kretzerae if this species is found in environmental samples not associated with Pterospora in future studies of pines in eastern North America. Likewise, identification of R. kretzerae from soil near P. strobus may indicate the presence of potential candidate sites for Pterospora restoration efforts.

Nucleotide divergence estimates provided a quantitative measure for comparing how sequences between species differ at the molecular level. In previous fungal studies, these statistics provided additional molecular evidence in support of speciation events in the Amanita muscaria complex (), Tricholoma scalpturatum complex (), and in identifying two lineages of cryptic species in Tricholoma populinum (

). In this study, R. kretzerae and R. salebrosus are separated by the fewest number of fixed nucleotide differences (12) that support an apparent sister-group relationship in the unrooted phylogeny (). A phylogeny with an outgroup was not shown due to the large number of gaps and ambiguously aligned positions that needed to be removed for analyses, which would have removed too many informative positions within the ingroup. Phylogenetic assessments of species in Rhizopogon subgenus Amylopogon based on multiple nuclear loci are currently underway (N.J. Dowie, unpublished data).

The lack of historic collections of basidiocarps in herbaria or collection by contemporary researchers of Pterospora or Rhizopogon may stem from a variety of reasons. This species may no longer reproduce sexually in nature due to severely altered environmental conditions (e.g., fire suppression), or alternatively, basidiocarps are produced but (1) at a time when no one has searched for them (e.g., early or late in the fruiting season); (2) are deeper in the soil profile than is typical for finding Rhizopogon basidiocarps; or (3) at a very low frequency and less likely to be collected. Hypothesis 3 is supported by the low frequency in which R. kretzerae has been identified in soil bioassays () when compared with similar studies on western North America Rhizopogon species (;).

In this study, two strategies were implemented to avoid naming an already described species. First, 22 ITS holotype and paratype sequences of Rhizopogon subgenus Amylopogon were retrieved from GenBank for phylogenetic comparison to R. kretzerae () . Second, partial to entire ITS sequences were determined from four Rhizopogon holotypes collected in Michigan that were associated with Pinus spp., but subsequently shown to belong to Rhizopogon subgenera other than subgenus Amylopogon. These results support

who had synonymized R. superiorensis with R. succossus . In the case of R. baxteri, Zeller had originally identified the collection Baxter2578 as R. rubescens (see original herbarium card;

). Owing to the high similarity of Baxter2578 with R. rubescens and R. roseolus sequences, it is unclear whether R. baxteri is a unique species; however, this, along with determining the taxonomic status of R. gelatinosus MICH 3268 and holotypes R. gelatinosus MICH 12392 and R. pinicola MICH 12404 will be left for investigation in future studies as it is beyond the scope of the current study. The ITS2 primers introduced here (R370F and R536R) may be useful in future studies of historic Rhizopogon collections.

Pterospora andromedea populations in Quebec were reproductively active in July 2012 despite a widespread drought in southeastern Canada. We found plants at five out of seven sites visited based on 10 historic Pterospora locations listed on the collecting permit provided by Quebec's provincial government. Four of five sites had more than 10 plants, but only one site had more than 20 plants. Stems from 2011 were present at all sites that had flowering plants in 2012, but were equal or fewer in number to those counted in 2012. However, the total number of stems present in 2012 at these five sites was lower than in previous records (24–270) provided on the collecting permit. The low number to complete absence of plants observed in 2012 could have been the result of: (i) the severe drought, (ii) a simple reflection of the sporadic nature of the flowering pattern in Pterospora , in which the plant is abundant in some years and completely absent in others, similar to the fruiting pattern of some fungi, or (iii) an indication that the Quebec populations are in serious decline, as has been shown in other eastern populations. Continued monitoring of Pterospora sites, historic and contemporary, is necessary to gain a better understanding of the ecology and reproductive biology of this species.

This work is the first installment of a series of conservation genetics and phylogeographic studies of eastern and western populations of Pterospora andromedea and Rhizopogon symbionts. Conservation management plans and scientific studies may now refer to this potentially rare fungal species as Rhizopogon kretzerae , instead of the formerly used name “the undescribed Rhizopogon subgenus Amylopogon species that associates with Pterospora andromedea in the east”.