Rhizopogon kretzerae Grubisha, Dowie, sp. nov.
Creators: name: Grubisha, Lisa C.
name: Dowie, Nicholas J.
name: Miller, Steven L.
name: Hazard, Christina
name: Trowbridge, Steven M.
name: Horton, Thomas R.
name: Klooster, Matthew R.
Publisher: name: Zenodo
Year: 2014
Subjects: Biodiversity
Taxonomy
Fungi
Basidiomycota
Agaricomycetes
Boletales
Rhizopogonaceae
Rhizopogon
Rhizopogon kretzerae

description: <i>Rhizopogon kretzerae</i> Grubisha, Dowie, &amp; Mill. sp. nov. and. <b>INDEX FUNGORUM REGISTRATION NUMBER</b> <b>:</b> IF 550254 <b>DIAGNOSIS</b> <b>:</b> Known only from ectomycorrhizal symbioses with <i>Pinus strobus</i> and monotropoid mycorrhizal symbioses with the mycoheterotrophic plant <i>Pterospora andromedea</i> in eastern North America. <i>Rhizopogon kretzerae</i> can be distinguished from <i>R. salebrosus</i>, 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. <b>HOLOTYPE</b> <b>:</b> DAOM 242738 <b>LOCATION</b> <b>:</b> 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 <i>Pterospora andromedea</i> roots, stored with silica. <b>ETYMOLOGY</b> <b>:</b> 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 <i>Rhizopogon vinicolor</i> A.H. Sm. and <i>Rhizopogon vesiculosus</i> A.H. Sm. advanced our understanding of <i>Rhizopogon</i> evolutionary biology and provided inspiration to many mycologists and students. <b>DESCRIPTION</b> <b>:</b> 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. <b>MACROSCOPIC CHARACTERISTICS</b> <b>:</b> 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. <b>MICROSCOPIC CHARACTERISTICS</b> <b>:</b> 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. <b>ECOLOGY AND DISTRIBUTION</b> <b>: <i>Rhizopogon kretzerae</i></b> appears to be re- stricted to areas with <i>Pinus strobus</i> and <i>Pterospora andromedea</i> or <i>P. strobus</i> alone in Northern Michigan, New York, New Hampshire, and southern Quebec. identified <i>R. kretzerae</i> from <i>P. strobus</i> roots within a <i>P. andromedea</i> rootball from Keweenaw County, Michigan. A soil bioassay study has shown that the frequency of <i>R. kretzerae</i> propagules in soil increases in <i>P. strobus</i> areas where <i>P. andromedea</i> is reproductively active with <i>R. kretzerae</i> occurring at a frequency from 6% of soil samples from sites with <i>P. strobus</i> to 14% to 25% (site vs. plot) in soils with both <i>P. strobus</i> and <i>P. andromedea</i> (). <b>COMMENTS</b> <b>: <i>Rhizopogon kretzerae</i></b> is the first record of a <i>Rhizopogon</i> subgenus <i>Amylopogon</i> 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 <i>P. andromedea</i> and <i>R. kretzerae</i> have diameters reaching a maximum of approximately 1.5 inches and are substantially smaller than rootballs of <i>P. andromedea</i> and <i>R. salebrosus</i> in western North America (). Ultrastructure of <i>P. andromedea</i> monotropoid roots has been described in detail by . <b>OTHER SPECIMENS EXAMINED</b> <b>: <i>Pterospora andromedea</i></b> 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, <i>Rhizopogon kretzerae</i> Grubisha, Dowie, &amp; 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 <i>P. andromedea</i> roots. The results from phylogenetic analyses presented here and in previous studies (;) are strong evidence that <i>R. kretzerae</i> 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&gt;99% identity and both came to be known by some variation of the cumbersome phrase “the unnamed species of <i>Rhizopogon</i> subgenus <i>Amylopogon</i> that associates with <i>P. andromedea</i> in the east” (;), which can now be referred to as <i>R. kretzerae</i>. Furthermore, now it is possible to communicate ecological information about <i>R. kretzerae</i> if this species is found in environmental samples not associated with <i>Pterospora</i> in future studies of pines in eastern North America. Likewise, identification of <i>R. kretzerae</i> from soil near <i>P. strobus</i> may indicate the presence of potential candidate sites for <i>Pterospora</i> 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 <i>Amanita muscaria</i> complex (), <i>Tricholoma scalpturatum</i> complex (), and in identifying two lineages of cryptic species in <i>Tricholoma populinum</i> ( ). In this study, <i>R. kretzerae</i> and <i>R. salebrosus</i> 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 <i>Rhizopogon</i> subgenus <i>Amylopogon</i> 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 <i>Pterospora</i> or <i>Rhizopogon</i> 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 <i>Rhizopogon</i> basidiocarps; or (3) at a very low frequency and less likely to be collected. Hypothesis 3 is supported by the low frequency in which <i>R. kretzerae</i> has been identified in soil bioassays () when compared with similar studies on western North America <i>Rhizopogon</i> species (;). In this study, two strategies were implemented to avoid naming an already described species. First, 22 ITS holotype and paratype sequences of <i>Rhizopogon</i> subgenus <i>Amylopogon</i> were retrieved from GenBank for phylogenetic comparison to <i>R. kretzerae</i> (). Second, partial to entire ITS sequences were determined from four <i>Rhizopogon</i> holotypes collected in Michigan that were associated with <i>Pinus</i> spp., but subsequently shown to belong to <i>Rhizopogon</i> subgenera other than subgenus <i>Amylopogon</i>. These results support who had synonymized <i>R. superiorensis</i> with <i>R. succossus</i>. In the case of <i>R. baxteri</i>, Zeller had originally identified the collection Baxter2578 as <i>R. rubescens</i> (see original herbarium card; ). Owing to the high similarity of Baxter2578 with <i>R. rubescens</i> and <i>R. roseolus</i> sequences, it is unclear whether <i>R. baxteri</i> is a unique species; however, this, along with determining the taxonomic status of <i>R. gelatinosus</i> MICH 3268 and holotypes <i>R. gelatinosus</i> MICH 12392 and <i>R. pinicola</i> 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 <i>Rhizopogon</i> collections. <i>Pterospora andromedea</i> 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 <i>Pterospora</i> 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>i</i>) the severe drought, (<i>ii</i>) a simple reflection of the sporadic nature of the flowering pattern in <i>Pterospora</i>, in which the plant is abundant in some years and completely absent in others, similar to the fruiting pattern of some fungi, or (<i>iii</i>) an indication that the Quebec populations are in serious decline, as has been shown in other eastern populations. Continued monitoring of <i>Pterospora</i> 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 <i>Pterospora andromedea</i> and <i>Rhizopogon</i> symbionts. Conservation management plans and scientific studies may now refer to this potentially rare fungal species as <i>Rhizopogon kretzerae</i>, instead of the formerly used name “the undescribed <i>Rhizopogon</i> subgenus <i>Amylopogon</i> species that associates with <i>Pterospora andromedea</i> in the east”.
description: Published as part of <i>Lisa C. Grubisha, Nicholas J. Dowie, Steven L. Miller, Christina Hazard, Steven M. Trowbridge, Thomas R. Horton &amp; Matthew R. Klooster, 2014, Rhizopogon kretzerae sp. nov.: the rare fungal symbiont in the tripartite system with Pterospora andromedea and Pinus strobus, pp. 526-534 in Botany 92</i> on pages 531-533, DOI: 10.1139/cjb-2013-0309, http://zenodo.org/record/246791