pp. l'v1vcu/ogia, 102(5),2010, 1058-1065.001: 10.3852/09,232 by The Mycolog-icai Society of America, Lawrence, ( 2010 KS 66044-8897 Kalapuya brunnea gen. & sp. nov. and its relationship to the other sequestrate genera in Morchellaceae Matthew J. Trappe' it from Leucangium and other known genera. Here James M. Trappe we describe this genus and its only known species, co.',�s,tenH and Society, Oregon Kalapuya 1Yrunnea, and discuss its relationship with Oregon 97331,5752 other genera \\ithin the Morchellaceae. Gregory M. Bonito Department oj Biology, Duke Durham, MATERIALS AND y[ETHODS North Carolina 27708 Sections were prepared for light microscopy by hand and mounted in dH20, Melzer's reagent and cotton blue as well as by microtoming of paraffin-embedded specimens and Kalapuya is described as a new, monotypic Abstract: staining the thin sections in safranin-fast gTeen. All truffle genus in the Morchellaceae knovm only from microscopic measurements were made in dH20 mounts at the Pacific northwestern United States. Its relationship 400X or 1000X with a Zeiss GSL research microscope. to other hypogeous genera within Morchellaceae is Melzer's reagent was used to test for amyloid reactions and explored by phylogenetic analysis of the ribosomal LSU cotton blue for cyanescent reactions. EFlcx Glebal tissue samples were sequenced at the Institute for and protein coding region. The type species, K lxrunnea, occurs in Douglas-fir forests up to about 50 y Genome Sciences and Policy at Duke University. Clean old on the west slope of the Cascade Range in Oregon fungal tissue was removed from within sporocarps, placed in and in the Coastal Ranges of Oregon and northern microcentrifuge tubes and ground with micropestles. DNA California. It has a roughened, warty, reddish bro"\\TI to was extracted with 24: 1 chloroforrn ; isoamyl alcohol and PCR amplified with the primer sets 897R-NS24 (SSU), ITS5- brown peridium, a solid whitish gleba that develops LR5 (ITS and LSD), RPB2_5F,RPB2_7R (RPB2) and 1577F, grayish brown mottling as the spores mature, and 2218R (EFIOl"). Information on primers can be found at produces a cheesy-garlicky odor at maturity. Its smooth, http;!/www. aftol.org/primers.php. PCR products were ellipsoid spores resemble those of 1'v1orchella spp. but viewed on 1 % agarose gels stained with SYBR safe are much larger. The four hypogeous genera kno'wTI in (Invitrogen, Carlsbad, California). Successful amplicons the Morchellaceae, Kalapuya, Fischerula, Imaia and were cleaned with the enzymes exonuclease I and alkaline Leucangium, are distinct from the epigeous genera phosphotase (New England Biolabs, Ipswich, Massachu­ lv10rchella and Verpa, but it is uncertain whether they setts). Bidirectional sequencing was performed with the resulted from a single transition to a hypogeous fruiting above primers and the Big Dye Sequencing Kit 3.1 (Applied habit or from multiple independent transitions. Biosystems, Foster California) on an ABI3730 capillary Kalapuya, locally known as the Oregon brmvn truffle, sequencer (Applied Biosystems). Sequences were edited with Sequencher 4.1 (Gene Codes Inc., Ann Arbor, has been commercially harvested for culinary use. Michigan) and aligned to reference sequences with Key Ascomycota, Douglas-fir, EFlex, words: MacClade 4.0 soft\\'are (Maddison and Maddison 2002). Fischerula, hypogeous fungus, Leucangium, LSU Phylogenetic analyses were conducted with parsimony and rDNA, Morchellaceae, Pezizales, taxonomy, truffle maximum likelihood optimization criteria in PAUP* 4.0blO (Swofford 2002) and by Bayesian inference with MrBayes INTRODUCTION (Ronquist and Huelsenbeck 2003) under a GTR + G + I model of nucleotide substitution. Although not included in A brown to reddish brown ascotruffle collected in our phylogenetic analyses, ITS, SSU and RPB2 sequences northwestern Oregon for several years has been were generated for the taxon described here. All sequences locally known as the "Oregon Brown Truffle". produced in this study have been accessioned by GenBank; Because it resembles the Oregon Black Truffle those used in our phylogenetic analysis (ITS, LSU, SSU, RPB2, EFl:x) are summarized (TABLE I) with GenBank (Leucangium carthusianum [Tul. & C. Tul.] Pao!.) accession numbers. In developing the LSD phylogram, in size, texture, glebal appearance and habitat, it had along with six collections of the new taxon, we included been assumed to be an undescribed Leucangium sp. Fischerula subcaulis Trappe 1975, Imaia gigantea (Imai However its spore shape differs from that of L. 1933) Trappe & Kovacs, two species of Leu cangium, two carthusianum and molecular analyses also distinguish species of Verpa, three species of lWorchella and taxa in Discinaceae and Tuberaceae as outgroups. vVe included Submitted Sep accepted for publication 18 2009; 4 M<!E· 2010.. taxa within the Helvellaceae and Tuberaceae as outgroups 1 Corresponding- author. E.mail: [email protected] for the EFlOl" phvlogeny. 1058 TRAPPE IT AL: K'iL1PUYA BRUNVE;\ 1059 TABLE 1. Collection information for specimen and sequences generated in this_study GenBank numbers Species name Voucher Location ITS" LSU" sse RPB2 EFla Balsamia GU596458 sp. )'1£S84 Yolo Co., CA, USA Balsamia GU596459 sp. SRC868 Yuba Co., CA, USA sp. .JTT27686 :-JS';\', Australia GU596461 Dingleya sp. JT27860 Victoria, Australia GU596462 Fischerula subcaulis (H) OSC131366 Tillamook Co., OR, USA GU596467 Helvella lacunosa GC596456 MES2I8 Riverside Co., CA, CSA Helvella lacunosa MSNorCall Yuba Co. , CA.. , USA GU596457 lm,aia gigantea JTl7444 Ha)wood Co., :-.JC, USA GC596468 un£nnea GB309 Benton CO.,OR, USA GQl19349 GQ1l9349 GU596476 brunnea GB310 Benton Co., OR, USA GQl19350 GQ1l9350 GU596477 Kalapuya !mmnea JT30484 Benton Co., OR, USA GQ1l9351 GQ1l9351 GC596473 bT'clnnea JTl7311 Linn Co., OR, USA GQ1l9352 GQ1l9352 brunnea JT30506 Linn Co., OR, USA GQ1l9353 GQ1l9353 GU596474 Kalapuya bmnnea (H) JT32730 Benton Co., OR, USA GQ1l9354 GQ1l9354 GU596475 carthusianum GB311 Benton Co. OR, USA GQ1l9355 GQ1l9355 carthusianum JT23195 Polk Co., OR, CSA GQ1l9356 GQl19356 carthusia-num JT27191 Mason Co., WA, USA GQ1l9357 GQ1l9357 GU596471 carthusianum M1248 Italy GQ3797I9 GQ379720 GU596472 sp. JT17223 OR, USA GU596469 sp. JTl7201 Polk Co., OR, USA GQ1l9358 GQ119358 Leucangium sp. JT22831 Polk Co., OR, USA GQl19359 GQll9359 sp. JT22764 Tillamook Co., OR, CSA GQ119360 GQl19360 sp. JT23195 Polk Co., OR, USA GU596470 sp. AWC4985 Victoria, Australia GU596460 Tuber gibbosum JT30580 Clackamas Co., OR, USA GU596463 Tuber melanosporwn GB200 Italy GU596464 (H) = Holotype. a In most cases ITS and LSU were submitted as a single sequence, thus they share a single accession number. RESULTS TYPE SPECIES: Kalapuya brunnea M. Trappe, Trappe, Bonito. Phylogenetic analysis of LSD rDNA and EFh genes & Etymology. Kalapuya, after the native American tribe both indicate the new taxon is nested with Morch­ whose ancestral lands encompassed the range of this ellaceae and related to but distinct from other known genus, from the western foothills of the Cascade Range hypogeous genera in this family (e.g. Fischerula, Imaia to Coastal Ranges. and Leucangium) and epigeous genera UVIorchella, Verpa, Disciotis) (FIG. 1). However the relationships M. Trappe, Trappe, Bonito, sp. among genera within Morchellaceae remain unre­ Kalapuya brunnea & solved (FIG. 2). Morphologicai characters also clearly nov. distinguish this species from others examined. We MycoBank MB 513040, GenBank GQ1l9354 accordingly designate a new genus, Kalapuya, to Ascomata hypogaea, stereothecia, subglobosa vel globosa, 12-80 X 10-45 mm. Peridium porphyreum vel bnmneum, accommodate this new species. verrUCOSUffi, verrucis fissuris tenuibus separatis. Gleba solida, aibida, contextu fertili griseo-bmnneo mandato. M. Trappe, Trappe, Bonito, gen. nov. Asci globosi, 70-90 X 65-90 �m, pariete 1-3 �m crassis, 6-8 Kalapuya & MycoBank MB513040, GenBank GQ1l9354 sporis. Sporae ellipsoideae, 32-43 X 25-33 ,urn, jaeves, juventute hyalinae, matm-itate succineae. Ascomata hypogaea, stereothecia, subglobosa. Peridium (FIG. porphYTeum vel brunneum, verrUCOSUffi, vernlcis fissuris Macrocharacters. ASCOMATA 3) hypogeous tenuibus separatis. Gleba solida, albida, contextu fertili stereothecia, sub globose to lobed and furrowed, 12- griseo-bmnneo mandato. Asci ellipsoidei vel globosi. 60(-80) X 10-45 mm, with a subcartilagenous, Sporae ellipsoideae, iaeves, juventute hyalinae, maturitate dendroid, basal attachment that easily breaks offwhen succineac. specimens are removed from soil. PERIDIUM (FIG. 4) 1060 MYCOLOGIA Kalapuya brunnea GQ1 19353 Ka/apuya GQ119352 brunnea KBlapuya brunnea GQ119349 100 Kalapuya brunnea GQl19350 100 KBlapuya brunnea GQ119351 78 Kalapuya brunnea GQ119354 HOLOTYPE '----- Fischerula subcaulis U42673 Leucangium GQ119358 sp. Leucangium GQl19359 sp. s:: Leucangium GQ119360 sp. o Leucangium carthusianum GQ119356 � Leucangium carthusianum 100 GQ119355 ::I: m Leucangium carthusianum M1248 Italy r­ r- Leucangium carthusianum GQ119357 � lmaia gigantea EU327202 � Imaia gigantea EU327201 m Imaia gigantea EU327200 venosa AJ698472 Disciotis Morchel/a AY533016 99 escu/enta ,.--f- Morche/la spongioJa AJ698463 100 Morchella AJ698462 crassipes FJ176853 96 Verpa bohemica 99 conica Verpa AY544666 ] Gyromitra californica AY544673 Hydnotrya cubispora DQ200845 DISCINACEAE cro ora Discina ma sp U42678 Gyromitra esculenta 176906 FJ verrucosa Dingleya U42686 76 AF43582 Choiromyces alveolatus TU BERACEAE 77 '------ Tuber regimontanum EU375838 HOLOTYPEJ - 5 changes FIG. 1. One of 137