Mycologia, 102(1), 2010, pp. 93–107. DOI: 10.3852/07-190 # 2010 by The Mycological Society of America, Lawrence, KS 66044-8897 Aquatic gilled mushrooms: Psathyrella fruiting in the Rogue River in southern Oregon Jonathan L. Frank INTRODUCTION Department of Biology, Southern Oregon University, Ashland, Oregon 97520 Mushrooms with true gills have been observed fruiting underwater in the clear, cold, flowing waters of the upper Rogue River on the western flanks of Crater Lake in Oregon. Aquatic mushrooms first were observed (by R.A. Coffan) in the North Fork of the Rogue River in Jul 2005. Specimens were collected Jul–Sep 2005, 2007 and 2008. These are truly underwater mushrooms and not mushrooms fruiting on wood recently washed into the river. They grow in the main channel, where they are constantly submerged at depths up to 0.5 m. Stipes are erect and attached to substrates including alluvial gravel, silt and woody debris. The Rogue River does not run dry; the habitat is continuously inundated. At the time of year when these aquatic fungi fruit, the land nearby is dry and no similar fruiting bodies occur. Aquatic fungi in freshwater commonly include members of the Oomycota and Chytridiomycota, as well as aquatic hyphomycetes that are anamorphic stages of Ascomycota and Basidiomycota (Shearer et al 2004, Shearer et al 2007). The ascomycete Vibrissea truncorum (Alb. & Schwein.) Fr. fruits submerged on wood in cold running water; its spores are thread-like and dispersed underwater (Mains 1956, Tylutki 1979). Other Ascomycota fruit on submerged wood in lakes in Japan, Thailand and Costa Rica (Minoura and Muroi 1978, Pinruan et al 2004, Ferrer et al 2008). A basidiomycete with a smooth hymenium, Gloiocephala aquatica Desjardin, Martinez-Peck & Rajchenberg, that forms submerged basidiocarps has been reported from lakes and ponds in southern Argentina (Desjardin et al 1995). Basidiocarps of 11 species of homobasidiomycetes occur in marine ecosystems (Hibbett and Binder 2001). These basidiocarps are cyphelloid, minute enclosed cups or spheroids. None are gilled mushrooms. The aquatic gilled mushrooms from southern Oregon appear to represent a novel taxon within the Psathyrellaceae in the large polyphyletic genus Psathyrella (Smith 1972, Padamsee 2008). Based on morphological characters and DNA sequences we propose it as a new species in Psathyrella. Robert A. Coffan Department of Environmental Studies, Southern Oregon University, Ashland, Oregon 97520 Darlene Southworth1 Department of Biology, Southern Oregon University, Ashland, Oregon 97520 Abstract: A species of Psathyrella (Basidiomycota) with true gills has been observed fruiting underwater in the clear, cold, flowing waters of the upper Rogue River in Oregon. Fruiting bodies develop and mature in the main channel, where they are constantly submerged, and were observed fruiting over 11 wk. These mushrooms develop underwater, not on wood recently washed into the river. Substrates include water-logged wood, gravel and the silty riverbed. DNA sequences of the ITS region and a portion of the ribosomal large subunit gene place this fungus in Psathyrella sensu stricto near P. atomata, P. fontinalis and P. superiorensis. Morphological characters distinguish the underwater mushroom from previously described species. Fruiting bodies have long fibrillose stipes with small diameter caps. Immature stages have a thin veil that is soon lost. Gills lack reddish edges. Cystidia are ventricose with subacute apices. Spores were observed as wedge-shape rafts released into gas pockets below the caps. Underwater gills and ballistospores indicate a recent adaptation to the stream environment. This particular river habitat combines the characteristics of spring-fed flows and cold, aerated water with woody debris in shallow depths on a fine volcanic substrate. Based on molecular and morphological evidence we conclude that the underwater mushrooms are a new species, Psathyrella aquatica. This report adds to the biodiversity of stream fungi that degrade woody substrates. The underwater environment is a new habitat for gilled mushrooms. Key words: Agaricales, aquatic fungi, ballistospores, Psathyrellaceae, psychrophilic fungi, stream fungi MATERIALS AND METHODS Site.—Submerged basidiocarps were collected underwater in the Rogue River at 42u519420N, 122u309280W, 900 m elevatioin, approximately 45 km downstream from Bound- Submitted 8 Nov 2007; accepted for publication 11 Jun 2009. 1 Corresponding author. E-mail: southworth@sou.edu 93 94 MYCOLOGIA ary Springs, the predominant source of water for the upper reaches of the Rogue River in the Rogue River-Siskiyou National Forest (CES 2006). At this site base flow is relatively high and constant during the summer. Streamflow data (1930–1952) from the nearest USGS gauging station (14327500) 6 km downstream from the site indicate a mean monthly flow in September of 8.4 (SD, 1.6) cubic meters per second (cms) (OWRD 2007). The lowest flow rate recorded 1930–1952 was 5.1 cms, an order of magnitude greater than in nearby streams not fed by springs. Mean monthly high flow for this gauging station was 25.3 cms. Real-time streamflow data from gauging station USGS 14330000, 20 km downstream on the main stem of the Rogue River, showed annual water at 2–14 C with diurnal fluctuations of 1.5 C in winter and 3 C in summer (OWRD 2007). Stream water samples, collected 21 Aug 2007, were analyzed for nitrate, total phosphorus and total organic carbon at Neilson Research Corp., Medford, Oregon (www.nrclabs.com). Collection.—Basidiocarps were photographed in the river, collected and measured in 2005, 2007 and 2008. Some were photographed in situ; others were collected and transported to the lab without exposure to air. Observations and measurements were made on fresh specimens. Pilei were placed over paper to capture spore prints. Specimens were observed with a Leica MZ75 dissecting microscope and Leica DMLB compound microscope. Images were captured with SPOT-RT digital cameras and software. Gill tissue was stained with Melzer’s reagent and treated with 5% KOH and H2SO4. Specimens were compared to descriptions in Smith (1972), Kits van Waveren (1985), Hansen and Knudsen (1992), Breitenbach and Kränzlin (1995), Gibson (2007) and Larsson and Örstadius (2008). Terminology of fruitbody characters follows Largent et al (1977). Nonstandardized color names in lowercase are followed by parenthesized Munsell (1976) alphanumeric color references. Herbaria abbreviations follow Holmgren and Holmgren (1998). We used the following general procedure to classify the underwater mushrooms within genus Psathyrella, which currently includes 414 species known from North America (Smith 1972). We described our collections on the basis of macromorphology, micromorphology, habit, habitat and DNA sequences. We used the keys of Smith (1972) and Breitenbach and Kränzlin (1995) and molecular phylogeny of Padamsee et al (2008) to identify a group of species most similar to our specimens that we then used for more detailed morphological comparisons. We also selected additional species for DNA sequence comparisons on the basis of morphological characteristics. This iterative approach was employed due to the publication of two large datasets of Psathyrella DNA during the preparation of this manuscript (Padamsee et al 2008, Vašutová et al 2008). Here we use the provisional name, Psathyrella aquatica, to refer to our collections of underwater mushrooms. Herbarium specimens were obtained from the University of Michigan Fungus Collection (MICH) http://www.herb. lsa.umich.edu/Bioinformatics.htm, from the Oregon State University Mycological Collection (OSC) http://ocid.nacse. org/research/herbarium/myco/databases.html, and from M. Padamsee, University of Minnesota (MIN) (TABLE I). In addition to P. aquatica a total of 89 collections in 33 species were examined. Molecular methods.—DNA was extracted from 12 fresh pileus or stipe tissues of Psathyrella aquatica and from herbarium specimens of 28 other Psathyrella species that were related morphologically (subgenus Psathyrella section Psathyrella) or by DNA sequences, as compared to the phylogenetic tree in Padamsee et al (2008) or that were distributed in Oregon and Washington (TABLE I). Tissue samples were stored in buffer (0.1 M Tris, 0.3 M NaCl, 0.04 M EDTA) at 4 C, extracted in 2% cetyltrimethyl ammonium bromide (CTAB) with chloroform. In addition lyophilized CTAB phenol-chloroform extracts of three species, P. aff. brooksii (initially identified as P. brooksii), P. atomata and P. ramicola, were provided by M. Padamsee. Because the ITS region of the specimen originally identified as P. brooksii by Padamsee et al (2008) was found to differ by more than 5% from the P. brooksii holotype we use the nomenclature P. aff. brooksii to refer to specimen Padamsee 098 (MIN) at the recommendation of M. Padamsee (pers comm). DNA was amplified in polymerase chain reactions (PCR) with fungal primer ITS1F (59-ggtcatttagaggaagtaa-39) and universal eukaryote primer TW14 (59-gctatcctgagggaaacttc39) (White et al 1990; Gardes and Bruns 1993, 1996). PCR reactions (20 mL) were performed with 0.6 units GoTaq and 4 mL 53 colorless buffer (Promega), 200 mM each dNTP, 0.3 mM each primer, 2.5 mM MgCl2, and 2 mL undiluted DNA template. An initial 3 min at 93 C was followed by 30 cycles of 30 s at 95 C, 2 min at 54 C, and 3.5 min at 72 C, with a final cycle 10 min at 72 C. When necessary shorter fragments from older herbarium specimens were amplified with fungal primer pairs ITS1F and ITS4 (59-tcctccgcttattga tatgc-39) for the ITS and ITS4r (59-gcaatatcaataagcggagga39) and TW14 for the 28S region; 20 mL PCR reactions were amplified as above with the annealing temperature reduced to 51 C and the extension time reduced to 2 min. The primer ITS4r was designed as the reverse complement of ITS4. PCR products were electrophoresced on 1.5% agarose gels, stained with ethidium bromide (1 mg/mL) and viewed under a Kodak EDAS 290 UV transilluminator. PCR products were purified with QIAquick PCR Purification kits (QIAGEN, Valencia, California), prepared with BigDye Terminator Ready Reaction Mix 3.1 and sequenced in an ABI 310 Genetic Analyzer (Applied Biosystems, Foster City, California) in the Biotechnology Center at Southern Oregon University. Molecular data were obtained by sequencing the internal transcribed spacer (ITS) region, including ITS1, the 5.8S ribosomal DNA gene and ITS2, and part of the 28S ribosomal gene, with forward primers ITS1F, ITS1 (59-tccgtaggtgaacctgcgg-39), ITS3 (59-gcatcgat gaagaacgcagc-39) and ITS4r, and reverse primers ITS4, TW13 (59-ggtccgtgtttcaagacg-39) and TW14. Sequences were edited with Chromas 1.45 (McCarthy 1998); contigs were assembled in Sequencher 4.7 (Gene Codes Corp. Ann Arbor, Michigan) and compared to other fungal ITS and 28S sequences in GenBank with BLAST (Altschul et al 1990). Clustal X was used to generate FRANK ET AL: AQUATIC PSATHYRELLA 95 TABLE I. Collections of Psathyrella species examined, with collector, number, herbarium, collection date, state in which collected (USA), habitat and GenBank numbers for ITS and 28S regions Psathyrella species Collector No./ herbarium P. aquatica 4 Jul 2005 OR 4 Jul 2005 OR 4 Jul 2005 OR 4 Jul 2005 OR 4 Jul 2005 OR 4 Jul 2005 OR 4 Jul 2005 OR 4 Jul 2005 OR P. aquatica R.A. Coffan (D. Southworth 1086)/SOC R.A. Coffan (D. Southworth 1087/SFSU R.A. Coffan (D. Southworth 1088)/OSC R.A. Coffan (D. Southworth 1089)/OSC R.A. Coffan (D. Southworth 1090)/MICH R.A. Coffan (D. Southworth 1091)/MICH R.A. Coffan (D. Southworth 1092)/MICH R.A. Coffan (D. Southworth 1093)/MICH J.L. Frank 1334/MICH 14 Aug 2007 OR P. aquatica J.L. Frank 1335/MICH 14 Aug 2007 OR P. aquatica 14 Aug 2007 OR 14 Aug 2007 OR P. aquatica R.A. Coffan (D. Southworth 1096)/MICH R.A. Coffan (D. Southworth 1097)/MICHa J.L. Frank 1336/MICH 21 Aug 2007 OR P. aquatica J.L. Frank 1337/SFSU 21 Aug 2007 OR P. aquatica 21 Aug 2007 OR P. aquatica R.A. Coffan (D. Southworth 1100)/MICH D. Southworth 1101, /MICH 21 Aug 2007 OR P. aquatica J.L. Frank 1347/OSC 21 Sep 2007 OR P. aquatica J.L. Frank 1348/OSC 21 Sep 2007 OR P. aquatica D. Southworth 1261/MICH 4 Sep 2008 OR P. alluviana A.H. Sm. P. alluviana A.H. Sm. P. alluviana A.H. Sm. P. alluviana A.H. Sm. P. alnicola A.H. Sm. P. alnicola A.H. Sm. P. alnicola A.H. Sm. P. alnicola A.H. Sm. A.H. Smith 19272/MICHa 30 Sep 1944 OR A.H. Smith 28232/MICH 25 Oct 1947 OR Meadow A.H. Smith 23782/MICH 27 Sep 1946 OR A.H. Smith 30217/MICH 17 Aug 1948 WA A.H. Smith 70222/MICHa 6 Sep 1964 ID On debris, vine maple forest On debris of Betula papyrifera Under Alnus A.H. Smith 70223/MICH 6 Sep 1964 ID Under Alnus E. Trueblood 162/MICH 7 May 1957 ID E. Trueblood 2280/MICH 19 Sep 1963 ID P. aquatica P. aquatica P. aquatica P. aquatica P. aquatica P. aquatica P. aquatica P. aquatica Date State GenBank ITS Habitat Underwater in Rogue River Underwater in Rogue River Underwater in Rogue River Underwater in Rogue River Underwater in Rogue River Underwater in Rogue River Underwater in Rogue River Underwater in Rogue River Underwater in Rogue River Underwater in Rogue River Underwater in Rogue River Underwater in Rogue River Underwater in Rogue River Underwater in Rogue River Underwater in Rogue River Underwater in Rogue River Underwater in Rogue River Underwater in Rogue River Underwater in Rogue River GenBank 28S the the EU664989 the the EU664990 the EU664991 EU664994 the EU259194 EU259195 the EU259196 the the the the the EU259192 EU259193 FJ899609 FJ899627 the the the the the the the Coniferous forest near rotting Populus 96 TABLE I. MYCOLOGIA Continued Psathyrella species Collector No./ herbarium Date P. alnicola A.H. Sm. P. alnicola A.H. Sm. P. alnicola A.H. Sm. E. Trueblood 2680/MICH 16 Jun 1967 ID Juniperus E. Trueblood 1439/MICH 5 Jun 1961 ID Coniferous forest E. Trueblood 1458/MICH 5 Jun 1961 ID Debris along creek, Salix and Alnus leaves P. atomata (Fr.) Quel. P. atomata (Fr.) Quel. P. atomata (Fr.) Quel. J.S. Hopple 139/DUKE P. atomata (Fr.) Quel. P. atomata (Fr.) Quel. P. atomata (Fr.) Quel. P. atomata (Fr.) Quel. P. atomata (Fr.) Quel. P. atomata (Fr.) Quel. P. atomata (Fr.) Quel. P. atomata (Fr.) Quel. P. brachycystis A.H.Sm. P. aff. brooksii State Habitat NC GenBank ITS GenBank 28S FJ899610 N.S. Weber 2949/MICH 12 Jun 1972 ID N.S. Weber 2948/MICH 12 Jun 1972 ID A.H. Smith 8565/MICH 22 Jun 1938 MI On damp soil under Salix along creek On dirt under Salix and low herbs along small stream On wet soil A.H. Smith 78106/MICH 15 Sep 1969 MI On soil under weeds A.H. Smith 43070/MICH 23 Sep 1953 MI Grass A.H. Smith 74433/MICH 4 Jul 1967 MI On wet soil A.H. Smith 74436/MICH 4 Jul 1967 MI On wet soil C.H. Kauffman/MICH 47963 22 Jul 1912 MI Lawn K. McKnight F1009/MICH 30 Jul 1955 UT Soil in Populus-Abies forest K. McKnight F1565/MICH 21 Aug 1956 UT T. E. Brooks 1605/MICHa 1 Sep 1946 KS Terricolous M. Padamsee 098/MIN 3 Jan 2003 WA EU664992 P. brooksii A.H. Sm. P. calvinii A.H. Sm. P. calvinii A.H. Sm. P. calvinii A.H. Sm. T. E. Brooks 1594/MICHa 2 Sep 1946 KS On wood chips under Cornus Terricolous next to pile of corn cobs C.H. Kaufman/MICH 11891a 9 Sep 1923 WY A.H. Smith 34788/MICH 13 Jul 1950 WY On soil by road FJ899611 R. Leach 6/MICH Jan 1944 CA On sandy soil covered by grass along creek P. candolleana (Fr.) Maire. P. carbonicola A.H.Sm. P. caudata (Fr.) Quel. P. caudata (Fr.) Quel. P. caudata (Fr.) Quel. J.M. Trappe 19657/OSC 17 Apr 1997 OR S. Carpenter CH-186/OSC 22 Sep 1980 WA Smith A.H. 35985/MICH 19 Sep 1950 MI C.H. Kauffman/MICH 32909 28 Sep 1922 OR S. Lundell 1770/MICH 29 Sep 1940 Sweden EU664993 FJ899612 Blow-down area near dead forest On dung and debris near barn On grassy slope close to farmyard EU664995 FRANK ET AL: AQUATIC PSATHYRELLA TABLE I. 97 Continued Psathyrella species Collector No./ herbarium Date State Habitat P. coloradensis A.H. Sm. P. conopilea (Fr.) Pearson & Dennis P. filamentosa A.H. Sm. P. fontinalis A.H. Sm. A.H. Smith 51659/MICHa 3 Aug 1956 CO T. O’Dell 174/OSC 1 May 1990 OR A.H. Smith 78074/MICHa 13 Sep 1969 MI On mud flats A.H. Smith 25644/MICHa 12 Jul 1947 MI P. fontinalis A.H. Sm. P. fontinalis A.H. Sm. P. fontinalis A.H. Sm. P. gracilis (Fr.) Quel. P. cf. gracilis A.H. Smith 25652/MICH 12 Jul 1947 MI On black muck in low area among elm and ash Muck A.H. Smith 28751/MICH 15 Jun 1948 MI Muck A.H. Smith 28753/MICH 15 Jun 1948 MI Muck S. Pittam 170/OSC 5 Jun 2000 OR J.L. Frank 1307/SOC 27 Apr 2007 OR S.M. Zeller/OSC 5951 16 Nov 1921 OR J.M. Trappe 22479/OSC 3 Dec 1997 OR A.H. Smith 3563/MICH 12 Aug 1950 WY W. Gruber 5/MICH Jan 1944 CA J.M. Trappe 19674/OSC 4 Dec 1997 OR A.H. Smith 30388/MICHa 21 Aug 1948 WA A.H Smith. 29526/MICH 28 Jul 1948 WA A.H. Smith 29602/MICH 29 Jul 1948 WA A.H. Smith 29642/MICH 30 Jul 1948 WA A.H. Smith 30175/MICH 16 Aug 1948 WA On humus A.H. Smith 30239/MICH 18 Aug 1948 WA On debris A.H. Smith 30241/MICH 18 Aug 1948 WA On Alnus debris A.H. Smith 4990/MICHa 2 Jul 1936 MI A.H. Smith 25280/MICH 23 Jul 1947 MI A.H. Smith 28182/MICHa 24 Oct 1947 OR A.H. Smith 18305/MICHa 02 Jun 1942 MI P. hydrophila (Fr.) Maire P. hydrophila (Fr.) Maire P. intermedia (Pk.) A.H. Sm. P. intermedia (Pk.) A.H. Sm. P. marcescibilis (Britz.) Singer P. nitens A.H. Sm. P. nitens A.H. Sm. P. nitens A.H. Sm. P. nitens A.H. Sm. P. nitens A.H. Sm. P. nitens A.H. Sm. P. nitens A.H. Sm. P. opacipes A.H. Sm. P. opacipes A.H. Sm. P. oregonensis A.H. Sm. P. parvicystis A.H. Sm. GenBank ITS On debris GenBank 28S FJ899628 FJ899613 FJ899614 Terrestrial in oak woodland Rotting wood FJ235146 Under Pseudotsuga menziesii On moss in spring FJ899615 Growing from rotting wood FJ899616 FJ899629 FJ899630 FJ899617 On debris FJ968757 FJ899631 On debris FJ968757 FJ899632 On rotten conifer wood On muck under aspen 98 TABLE I. MYCOLOGIA Continued Psathyrella species P. parvicystis A.H. Sm. P. parvicystis A.H. Sm. P. parvicystis A.H. Sm. P. praeatomata A.H. Sm. P. prona (Fr.) Gillet P. prona (Fr.) Gillet P. prona (Fr.) Gillet P. prona (Fr.) Gillet P. prona (Fr.) Gillet P. prona (Fr.) Gillet P. prona (Fr.) Gillet P. prona (Fr.) Gillet P. prona (Fr.) Gillet P. prona (Fr.) Gillet P. prona (Fr.) Gillet P. quercicola A.H. Sm. P. quercicola A.H. Sm. P. quercicola A.H. Sm. P. rainierensis A.H. Sm. P. ramicola A.H. Sm. P. rogueiana A.H. Sm. P. subincarnata A.H. Sm. P. subolivacea A.H. Sm. P. subolivacea A.H. Sm. P. subolivacea A.H. Sm. P. superiorensis A.H. Sm. P. superiorensis A.H. Sm. Collector No./ herbarium Date State Habitat GenBank ITS A.H. Smith 26004/MICH 27 Jul 1947 MI On mud in roadway V. Potter 4834/MICH 16 Jun 1948 MI E.B. Mains 6134/MICH 21 Aug 1941 MT On & along side of hardwood limb On wet soil A.H. Smith 9-28-69/MICHa 28 Sep 1969 MI On mud J.M. Trappe 19678/OSC 17 Apr 1997 OR L.R. Hesler 19032/MICH 9 Jul 1949 NC H.C. Beardslee Jr. 683/MICH 19 Jun 1901 OH A.H. Smith 13859/MICH 29 May 1939 WA A.H. Smith 14080/MICH 5 Jun 1939 MI A.H. Smith 14193/MICH 9 Jun 1939 MI A.H. Smith 14782/MICH 5 Jul 1939 MI J.B. Flett/MICH 33314 8 Apr 1941 MI W.B. Cooke 19881/MICH 9 Jun 1947 MI W.B. Cooke 19882/MICH 9 Jun 1947 MI A.H. Smith 35271/MICH 29 Jul 1950 WY A.H. Smith 55689/MICHa 15 Nov 1956 OR On Quercus stump A.H. Smith 55690/MICH 15 Nov 1956 OR On Quercus log A.H. Smith 55377/MICH 10 Nov 1956 OR On mossy Quercus log A.H. Smith 30929/MICHa 5 Sep 1948 WA FJ899619 P.B. Matheny 871/WTU 1 Oct 1946 WA FJ899620 A.H. Smith 55708/MICHa 16 Nov 1956 OR A.H. Smith 63594/MICHa 21 Jul 1961 MI A.J. Smith 11042/MICHa 23 Sep 1938 MI A.J. Smith 4991/MICH 2 Oct 1936 MI C. Nimke 150/MICH 11 Oct 1971 MI J.F. Ammirati 2251/MICHa 14 Aug 1968 MI A.H. Smith 32107/MICH 24 May 1949 MI GenBank 28S FJ899618 Horse dung and rich soil On manure pile and soil in farm yard On straw and dung pile On dung and soil in farmyard On dung and straw pile On grassy area by road On manure in apple orchard On manure in apple orchard On clay soil along logging road On mud under Pinus FJ899621 FJ899633 FJ899623 FJ899634 Scattered, on Quercus leaves On chip dirt Scattered in drying drainage pond On humus and sawdust FJ899622 FRANK ET AL: AQUATIC PSATHYRELLA TABLE I. Continued Psathyrella species P. tenera Peck P. tenera Peck P. uskensis A.H.Sm. P. velutina (Fr.) Singer a 99 Collector No./ herbarium Date State Habitat GenBank ITS GenBank 28S A.H. Smith 65853/MICH A.H. Smith 29601/MICH A.H. Smith 73377/MICH 19 Aug 1962 29 Jul 1948 14 Sep 1966 ID WA WA On mud On wet earth On mud FJ899624 FJ899635 FJ899625 FJ899636 S. Carpenter CH-186/OSC 22 Sep 1982 WA Mount St Helens FJ899626 FJ899637 Holotype. alignments of the 28S region. Alignments were edited manually with BioEdit (Thompson et al 1997, Hall 1999). Sequences generated in this study have been deposited in GenBank (TABLE I). A total of 27 sequences were aligned, 12 that we generated and 15 from GenBank. All were in Psathyrella sensu stricto Clade A v, except Psathyrella melleipallida and P. tephrophylla from Clade A iv, which were used as outgroup (Padamsee et al 2008). All but one were from North America; the European taxon (an unidentified species) closest to P. aquatica was included (Vašutová et al 2008). Phylogenetic trees built with parsimony and maximum likelihood with 1000 bootstrap replicates and 1000 jackknife replicates were generated from 28S sequences using PAUP 4.10b10 (Swofford 2002). Consensus trees with 50% majority rule were generated with a tree-bisection-reconnection branch swapping algorithm. All characters were given equal weight; gaps were treated as missing. Concensus trees were examined to confirm branch positions. TAXONOMY Psathyrella aquatica J.L. Frank, Coffan, & Southworth, sp. nov. FIGS. 1–10 Mycobank: MB511824 Basidiomata 4.5–10 cm alta, immersa. Pileus 0.8–1.5 cm latus, brunneolus vel brunneigriseus. Basidioporae ellipsoideae, leves, brunneae, 10–14 3 6–8 mm, poro germinali. Cystidia hymeniales: cheilocystidia pleurocystidiaque similaria, ventricosa, 25–45 3 10–18 mm. Lamellae adnatae. Stipes textura porrecta. Macromorphology. Basidiomata (FIGS. 1–4, 6) immersed, 4.5–10 cm tall; pileus 0.8–1.5 cm diam, broadly parabolic to campanulate, light brown to brownish gray (10YR 7/2–6/1), sometimes with central orangebrown (10YR 5/4) disk, sometimes mottled or striate, smooth, hygrophanous; pileus context thin above gills, light tan to orange-brown; odor not distinctive; lamellae adnate, thin, light tan, densely speckled with dark brown spores, extending to pileus margin, lamellulae in two ranks and extending from one-half to onefourth of the radius; stipe 4.0–9.5 cm long, diameter expanding from 1.0–2.2 mm at apex to 1.8–3.2 mm at base, white to pale yellow, hollow, lacking annulus, fibrous, surface fibrillose covered with wefty white to gray-white mycelium, and with cottony rhizomorphs and mycelial tomentum emanating from base. Micromorphogy. Basidiospores (FIG. 7) 10–14 3 6– 8 mm, ave. 12.3 3 6.9 mm, elliptical with a germ pore, smooth, dark reddish brown in water and in Melzer’s, fading to gray-brown in KOH and to lilac in H2SO4, spore print purple-black; basidia (FIG. 8) 4-spored, clavate, 32–40 3 10–13 mm, hyaline; cheilocystidia (FIG. 10) 25–45 3 10–18 mm ventricose, apex subacute to elongate, thin walled, colorless, hyaline; pleurocystidia (FIG. 8, 9) 25–40 3 10–13 mm, ventricose, apex subacute, scattered, thin-walled, colorless, hyaline; caulocystidia 32–40 3 10–13 mm, cylindrical to ventricose, in fascicles, apex obtuse; pileipellis cellular, suprapellis a single layer of spherical to isodiametric, inflated cells, 25–35 mm diam, on 30–50 3 3–5 mm peduncles that extend into the pileus trama, clamp connections absent; pileus trama thin-walled hyphae 8– 15 mm diam, interwoven; stipe hyphae 35–70 3 8– 14 mm, parallel; clamp connections present in mycelium at stipe base, absent elsewhere. Habit. Basidiomes were observed in below rapids and areas of turbulence. Specimens were anchored at depths up to 0.5 m, most over an area of approximately 200 m2, with two specimens collected 1 km upstream. The pileus of one specimen was above water; all others were submerged. One specimen was growing in an eddy behind woody debris in the main river channel; all others were in moving water. No basidiomata were observed in slack water. The lateral distance from submerged basidiomata to the nearest stream bank or gravel bar was 20–340 cm. In Aug and Sep 2007 epigeous fungi, including Collybia sp., Russula spp., Alnicola sp. and Lycoperdon sp., were observed in adjacent terrestrial areas. Specimens were attached to a substrate of sticks (FIG. 5) or gravel or embedded in silt (FIG. 1). Basidiomata often grew out of, or close to, aquatic mosses (Scleropodium obtusifolium [Jaeg.] Kindb. in Mac. & Kindb.) and cyanobacteria (Anabaena) (FIG. 3). The stipe bases of several specimens origi- 100 MYCOLOGIA FIGS. 1–6. Psathyrella aquatica. 1. Underwater mushroom fruiting in silt near waterlogged wood. 2. Underwater mushroom with dark spores on gills and gas bubbles on cap and stipe. 3. Mushroom growing with aquatic moss (Scleropodium obtusifolium) emerging from water. 4. Mushroom with undulating gas bubble under pileus. 5. Stipe initiating growth on underside of submerged twig. Arrows point to base of developed stipe and to two primordia. 6. Cap lifted above water showing rafts of spores from burst gas pocket; gills are white after spore discharge. nated from the underside of a piece of gravel or stick (FIG. 5, arrow) before curling around to elongate upward. Gas bubbles were observed on stipes and pilei (FIG. 2), and gas pockets were trapped beneath the pilei (FIG. 4). Some underwater mushrooms with white gills apparently already had shed spores (FIG. 6), but most retained spores on the gills (FIG. 2). Basidiospores collected at the water-gas interface beneath the pileus in some specimens. When specimens were lifted gently from the water FRANK ET AL: AQUATIC PSATHYRELLA 101 FIGS. 7–10. Psathyrella aquatica. 7. Basidiospores. 8. Basidium with spores attached, also pleurocystidia. 9. Pleurocystidium. 10. Cheilocystidia. Bars 5 10 mm. the gas pocket remained intact for a few seconds and undulated from the movement (FIG. 4). Then the gas pocket burst open, releasing wedge-shape rafts of spores that adhered to each other, the stipe and our fingers (FIG. 6). Habitat. Aquatic vegetation near the attachment of the underwater mushrooms included dense beds of Scleropodium obtusifolium and abundant gelatinous masses of cyanobacteria, Anabaena sp., with heterocysts. Terrestrial vegetation on surrounding stream banks was dominated by Pseudotsuga menziesii with Pinus monticola in the canopy and understory trees, Alnus rubra, Acer circinatum and Cornus nuttallii. Upland vegetation and riparian vegetation reached the water’s edge at all times of the year, including the late summer during the period of lowest flow. Organic carbon in the water sample totaled 0.52 mg/L, nitrate measured less than the testing equipment limit of 0.2 mg/L and total phosphorus less than 0.05 mg/L. Water temperature during field observations was 7–13.1 C; ambient air was 23–32.8 C. Known distribution. Oregon. Etymology. In reference to the aquatic habitat. HOLOTYPE: USA. OREGON: Jackson County. North of Prospect (42u519420N, 122u309280W), underwater in the Rogue River on wood, 8 Aug 2007, R.A. Coffan (D. Southworth 1097 MICH). Other specimens examined. See TABLE I. 102 MYCOLOGIA TABLE II. Maximum identity match to the ITS region of Psathyrella aquatica and classification, according to Smith (1972), of Psathyrella collections examined Psathyrella species P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. fontinalis FJ899614 aff. brooksii EU664994 superiorensis FJ899623 atomata FJ899610 superiorensis FJ899622 prona FJ899618 brooksii EU664993 subincarnata FJ899621 alluviana FJ899609 ramicola FJ899620 hydrophila FJ899615 marcescibilis FJ899617 rainierensis FJ899619 uskensis FJ899625 nitens FJ968757 intermedia FJ899616 calvinii FJ899611 tenera FJ899624 cf. gracilis FJ235146 candolleana FJ899612 conopilea FJ899613 velutina FJ899626 Subgenus/section/subsection/series Length (b.p.) Query coverage (%) Max ident (%) Psathyrella/Psathyrella/Psathyrellae/Psathyrellae Pannucia/Pannucia/Mixtae Psathyrella/Psathyrella/Psathyrellae/Psathyrellae Psathyrella/Psathyrella/Psathyrellae/Psathyrellae Psathyrella/Psathyrella/Psathyrellae/Psathyrellae Atomatae/ Pannucia/Pannucia/Mixtae Psathyrella/Psathyrella/Psathyrellae/Tenerae Psathyrella/Psathyrella/Psathyrellae/Tenerae Psathyrella/Umbonatae Pannucia/Appendiculatae/Hydrophilae Candolleana/ Psathyrella/Psathyrella/Mesosporae Psathyrella/Psathyrella/Psathyrellae/Tenerae Psathyrella/Psathyrella/Mesosporae Psathyrella/Psathyrella/Psathyrellae/Tenerae Psathyrella/Psathyrella/Psathyrellae/Tenerae Psathyrella/Psathyrella/Psathyrellae/Tenerae Psathyrella/Psathyrella/Psathyrellae/Psathyrellae Candolleana/ Psathyrella/Subatratae Lacrymaria/ 556 564 402 666 531 455 453 628 507 593 268 445 213 514 540 330 437 491 528 551 414 467 94 90 98 76 94 100 99 81 89 83 100 99 98 98 91 100 100 99 93 84 78 94 99 99 98 98 96 95 95 95 94 94 93 93 93 93 93 92 92 92 92 91 87 85 Molecular analyses.— DNA sequences from eight specimens of P. aquatica confirmed that only one species was present; six ITS sequences and three 28S sequences of P. aquatica specimens were identical. Sequences of the ITS1 and ITS2 regions, the 5.8S ribosomal gene and the 28S ribosomal gene from six specimens of P. aquatica have been deposited in GenBank (TABLE I). ITS sequences were obtained from 18 other species of Psathyrella from 19 herbarium collections (two collections of P. superiorensis yielded distinct ITS sequences) and from three lyophilized extracts (T ABLE II). Sequences of the 28S region were generated from 12 species. Of 528 total characters in the phylogenetic alignment of 28S sequences, 490 were constant, 24 were considered parsimony informative and 14 variable characters were considered parsimony uninformative. Both the parsimony and the maximum likelihood consensus trees generated from our 28S alignment placed P. aquatica near P. superiorensis (Ammirati 2251, holotype), P. aff. brooksii (Padamsee 098, but not Brooks 1594, holotype) and P. fontinalis with P. superiorensis and P. aff. brooksii (Padamsee 098) closer than P. fontinalis (FIG. 11, TABLE II). Pairwise analysis of ITS sequences show P. aquatica to be closer to P. fontinalis and P. aff. brooksii than to P. superiorensis (holotype) (TABLE II). That two collec- tions identified by A.H. Smith as P. superiorensis both from Michigan differed by more than 2% in the ITS sequence highlights the problems of species identification in Psathyrella. Molecular data show that P. aquatica is close to several species in series Psathyrellae and aligns into Clade A v Psathyrella sensu stricto (Padamsee et al 2008). Commentary.—Psathyrella aquatica belongs to subgenus Psathyrella based on these characteristics: smooth unornamented spores; absence of fasciculate hymenial cystidia; not parasitic on Coprinus; glabrous pileus; absence of granulose veil; pleurocystidia with wall in neck up to 0.5 mm, apex smooth or with only finely granular incrustations; cheilocystidia not lecythiform; and veil thin to rudimentary or absent, not well developed (Smith 1972). Among the 177 species in subgenus Psathyrella, P. aquatica belongs to section Psathyrella based on these characters: not coprophilous, subacute to obtuse pleurocystidia and with spores at least 9–12.5 mm long. Distinguishing between subsections Mesosporae and Psathyrellae depends on basidiospore dimensions in which there is a slight overlap, with spore length in subsection Mesosporae 9–12.5 mm and in Psathyrellae 11–17 mm. A mean basidiospore length of 12.3 mm and extreme lengths of 10–14 mm place P. aquatica among the 27 species in subsection Psathyrellae. Within subsection FRANK ET AL: AQUATIC PSATHYRELLA FIG. 11. Phylogenetic tree using parsimony for 28S data (with GenBank numbers) showing the position of P. aquatica in Psathyrella sensu stricto, with 1000 bootstrap replicates; bootstrap numbers greater than 50% are included above branches. Psathyrellae two series, Psathyrellae and Tenerae, are distinguished by the color of the pileus margin and gill edges. Species in series Psathyrellae have pink gill edges, lacking in series Tenerae. Pink tints were not observed on any specimens of P. aquatica at any stage from immature with veil still attached to post spore discharge. Morphology places P. aquatica in series Tenerae; DNA sequences place P. aquatica in series Psathyrellae (TABLE II, FIG. 11). Morphological comparison with closely related species.— Morphological traits differentiate P. aquatica from described species in series Psathyrellae (TABLE III). Among species most closely related based on molecular phylogenetic analysis, P. fontinalis has a glabrescent stipe, longer spore maximum length (16 mm), longer pleurocystidia, cylindric to clavate caulocystidia and lighter spore color in KOH. Psathyrella superiorensis has longer pleurocystidia, smaller spores 103 and shorter stipe. Neither P. fontinalis nor P. superiorensis have been reported outside Michigan. Psathyrella aff. brooksii differs in having a thick white fibrillose veil when young and shorter yellow-brown stipe (M. Padamsee pers comm). Psathyrella atomata lacks pleurocystidia; P. preatomata has hyaline cells among the cheilocystidia; and P. gracilis and P. opacipes have longer, more acute pleurocystidia. Among species for which no DNA sequences were obtained, P. filamentosa has clavate to vesiculose cells along the gill margin with cheilocystidia. Species in series Tenerae, which are less close to P. aquatica based on ITS and 28S sequences, similarly lack pink tints (Smith 1972). In addition there are other morphological differences. Psathyrella calvinii has incrustations on stipe hyphae, P. subincarnata has vinaceous lamellae when young, P. tenera has brachybasidioles and P. alluviana has a rugulose pileus. Pleurocystidia in P. intermedia are small and infrequent. Both P. uskensis and P. coloradensis are notably small and fragile, even for this genus. In subsection Mesosporae, P. nitens and P. rainierensis not only differ by DNA but also have smaller spores; P. subhepatica has longer pleurocystidia (44– 70 mm). In subgenus Atomatae pleurocystidia are rare in P. prona. In subgenus Pannucia P. hydrophila has much smaller spores. Spores of P. brooksii are slightly larger (12–15 3 7–9 mm) than those of P. aquatica (10–14 3 6–8 mm); cystidia are subclavate to broadly ventricose, and the veil of P. brooksii is more or less well developed and remains attached to the cap. Pleurocystidia in P. ramicola, subgenus Umbonatae, are utriform. Psathyrella aquatica is distinct from the two Psathyrella species that occur in terrestrial sites along the Rogue River in southern Oregon (TABLE III). Psathyrella quercicola in section Fatuae, collected 34 km downriver, has smaller basidiospores; P. rogueiana in subgenus Candolleana, collected 84 km downriver, has smaller basidiospores, rare pleurocystidia and clavate to utriform cheilocystidia. DISCUSSION In a genus as large as Psathyrella, determining a new species is complex because species are similar in both morphology and DNA sequences. Macromorphological characters can be insufficient to identify specimens to species, and micromorphological characters, especially cystidial shape and the color of gill edges, are variable. Kits van Waveren (1985) considered the character of red underlining of gill edges to be unreliable, although Smith (1972) and Breitenbach and Kränzlin (1995) used red underlining as a key character. Vašutová et al (2008) considered cystidial Spore size Cystidia Clamps Stipe Suprapellis cuticle aquatica 10–14 3 6–8 av. 12.3 3 6.9 2 fibrillose, wefty at base 1 layer, pedicellate white 4.5–10/0.8–1.5 atomata 11–13(–15) 3 6–7 + fragile, pulverulent to glabrous 2–3 layers, vesiculose pink 3–5/1–2.5 filamentosa 11–14 3 5.5–7 + naked, pruinose near apex 1–2 layers, inflated cells white or pink 2–5/0.5–1.2 fontinalis 11–14(–16) 3 6–7.5 (–8.5) + glabrescent palisade, clavate pedicellate pink gracilis (10–)11–14 (–15) 3 6.5–8 + fragile, fibrillose above glabrous below palisade, pyriform and vesiculose to elliptic pink opacipes (12–)13–16.5 3 6–8 + glabrescent pruinose 1 layer, vesiculose or pedicellate white or vinaceous 3–7/1–3.5 praeatomata 11–14 (–15) 3 5.5–7 2 naked above, scattered fibrils below 2–3 layers, pedicellate and vesiculose pink 2–5/0.8–1.5 superiorensis 11–13 3 5.5–6.5 Pl: ventricose, subacute 25–40 3 10–13 Ch: ventricose, subacute 25–40 3 10–18 Ca: + Pl: absent to rare Ch: fusoid-ventricose 35–48 3 9–14, acute to subacute, long neck Ca: ? Pl: fusoid ventricose, apex obtuse to subacute with adhering granules, 43–58 3 10–17 Ch: fusoid ventricose with vesiculose cells Ca: ? Pl: Ventricose-elongate, short neck, obtuse apex to fusoid-ventricose, subacute apex 38–65 3 10–16 Ch: similar or ventricose to clavate 15–26 3 8–12 Ca: + Pl: subaciculate to fusoid-ventricose, acute to subacute, neck flexuous 54–75 3 10–16 Ch: Shorter, more obtuse Ca: + Pl: fusoid-ventricose, acute to subacute 38– 70 3 9–16 Ch: saccate to clavate 18–26 3 10–15 Ca: + Pl: Ventricose with neck, subacute, 36–48 3 10–15 Ch: Subfusoid to clavate to fusoid-ventricose 8–12 wide Ca: ? Pl: Fusoid-ventricose 50–85 3 10–15 long neck, obtuse Ch: smaller or clavate Ca: ? + fibrillose, pallid above, brown below 1–2 layers, vesiculose pink 2–4/0.6–1.4 Psathyrella species Height/cap diam (cm) (3–)5–10/1–3 6–12/1.5–3.5 MYCOLOGIA Pl, pleurocystidia; Ch, cheilocystidia; Ca, caulocystidia; +, present; 2, absent; ?, not mentioned. Gill edge color 104 TABLE III. Diagnostic characters among described species of Psathyrella in series Psathyrellae including those with close DNA matches to P. aquatica. Data from species other than P. aquatica from Smith (1972). Spore and cystidia dimensions in micrometers; clamp connections in pileus FRANK ET AL: AQUATIC PSATHYRELLA shapes as homoplasic and insufficient to determine phylogeny. In a test of sorting within the P. gracilis group Kemp (1985) sent six split collections of P. gracilis to A.H. Smith and E. Kits van Waveren; taxonomic agreement was reached on only one. Padamsee et al (2008) found disagreement between molecular and morphological information among several pairs of Psathyrella species. Studies show the value of molecular data in determining phylogenetic relationships within genus Psathyrella (Padamsee et al 2008, Vašutová et al 2008, Larsson and Örstadius 2008). Our analysis of 28S DNA sequences placed P. aquatica into Clade A v. Taken together morphological and molecular evidence support the hypothesis that the underwater mushroom is a new species of Psathyrella most closely related to P. fontinalis, P. superiorensis, P. atomata and the P. gracilis group. Psathyrella aquatica is characterized by a relatively long fibrillose stipe that is not fragile, relatively small cap, a thin veil disappearing at maturity, nonpink gill edges and ventricose pleuro- and cheilocystidia with subacute apices. The habitat of P. aquatica appears unique among species of Psathyrella, none of which have been reported in running water. Most species of Psathyrella occur in terrestrial habitats, often on dung. However some, including several in subgenus Psathyrella section Psathyrellae, are associated with damp or wet soil, wet or drying muck or the margins of wetlands where the mycelium might grow underwater (Smith 1972). For example P. filamentosa and P. opacipes were found in damp habitats or at the edges of marshes (Smith 1972). Of the six species closest to P. aquatica, three (P. atomata, P. fontinalis and P. superiorensis) have been collected from damp habitats (e.g. muck, drying drainage ponds and damp soil). The other three (P. aff. brooksii, P. gracilis and P. ramicola) occur in dry terrestrial habitats on soil or wood. Although 28s and ITS sequences failed to separate P. aquatica from P. aff. Brooksii, morphological differences do not justify considering P. aff brooksii a conspecific specimen. Species in other subgenera, for example P. typhae (subgenus Pannucia), also grow on wetland plant debris and silt and on floating matter (Redhead 1979, 1981; Schulz et al 2005). Other Psathyrellaceae, such as Coprinopsis kubickiae (reported as Coprinus amphibius) and Coprinellus congregatus (reported as Coprinus alkalinus), have been isolated from submerged wood, but fruiting bodies were not formed underwater (Anastasiou 1967, Redhead and Traquair 1981). The spore discharge mechanism remains enigmatic in Psathyrella aquatica. Spore prints were obtained, suggesting that basidiospores are discharged as ballistospores when conditions are appropriate. Fur- 105 thermore basidiospores show the asymmetrically positioned hilar appendix, a feature compatible with forcible discharge (McLaughlin et al 1985). The wedge-shape rafts of spores released by the bursting gas bubble resemble sections of a spore print deposited on the gas-water interface beneath the cap. The ballistospore discharge mechanism involving a water droplet and a water film could not occur if gills were totally in contact with water (Money 1998, Pringle et al 2005). Gas pockets were trapped under many P. aquatica pilei; a similar bubble was observed under the hymenophore of Gloiocephala menierii collected from Carex stems near the mud-air interface (Redhead 1981, Desjardin et al 1995, Redhead pers comm). Trapped gases might provide the atmosphere needed for ballistosporic discharge in underwater environments. A fungus growing in a fast flowing stream would encounter spore dispersal problems because the current tends to wash spores downstream. Psathyrella aquatica fruitbodies retain released basidiospores at the air-water interface of the pileal gas pocket until disturbed. Even then the spores do not disperse individually but appear hydrophobic, attracted to each other or to other hydrophobic surfaces. Desjardin (1995) recognized the problem of underwater agaric spore dispersal and hypothesized that windinduced water currents or aquatic animals might disperse spores to nearby vegetation within a lake. Dispersal by currents in a turbulent stream however are unidirectional downstream, transporting spores to different and possibly unsuitable habitats. Adherence of Psathyrella spores to stipe and gills might counteract currents that would wash the spores downstream. In addition aquatic invertebrates might graze these fungi, keeping spores in the same habitat and dispersing them nearby, even upstream. Retention of spores near fruitbodies would allow for reinoculation in suitable habitats. The growth of this fungus on alluvial gravel as well as on submerged wood suggests that it might obtain carbon from the film of bacteria, algae and sediment that collects on the surface of submerged substrates as well as from decomposing wood. It might obtain nitrogen from cyanobacteria colonies. The relatively constant conditions of the aquatic environment might help to explain the exceptionally long fruiting period. Growth below 20 C classifies the species as psychrophilic (Kendrick 2000). Reproductive isolation could account for speciation because underwater fruiting would limit opportunities for genetic exchange between aquatic and terrestrial individuals. Combining evidence from DNA sequences, morphology and habitat, we conclude that the underwater mushrooms are a new 106 MYCOLOGIA species, P. aquatica, in Clade A v Psathyrella sensu stricto (Padamsee et al 2008). The conditions of this stream are distinctive, although not unique. This particular river habitat combines the characteristics of steady spring-fed flows, clear, cold, aerated water with woody debris in shallow depths on fine volcanic substrate. This underwater environment is a new habitat for gilled mushrooms. ACKNOWLEDGMENTS This study was supported by National Science Foundation Grants DEB-0516229 through Research at Undergraduate Institutions and DBI-0115892 to the Biotechnology Center at Southern Oregon University. 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