Kalapuya brunnea gen. & sp. nov. and its relationship to the other sequestrate genera in Morchellaceae Trappe, M. J., Trappe, J. M., & Bonito, G. M. (2010). Kalapuya brunnea gen. & sp. nov. and its relationship to the other sequestrate genera in Morchellaceae. Mycologia, 102(5), 1058-1065. doi:10.3852/09-232 10.3852/09-232 Mycological Society of America Accepted Manuscript http://cdss.library.oregonstate.edu/sa-termsofuse 1 1 Kalapuya brunnea gen. & sp. nov. and its relationship to the other sequestrate genera in the 2 Morchellaceae 1 3 Matthew J. Trappe 4 James M. Trappe 5 Department of Forest Ecosystems and Society, Oregon State University, Corvallis, Oregon, 6 97331-5752, U.S.A. 7 Gregory M. Bonito 8 Department of Biology, Duke University, Durham, North Carolina, 27708, U.S.A. 9 Running Title: Kalapuya brunnea 10 Abstract: Kalapuya is described as a new, monotypic truffle genus in the Morchellaceae 11 known only from the Pacific Northwestern United States. Its relationship to other 12 hypogeous genera within the Morchellaceae is explored by phylogenetic analysis of the 13 LSU rDNA and EF1α protein coding genes. The type species, K. brunnea, occurs in 14 Douglas-fir forests up to about 50 yrs old on the west slope of the Cascade Mountains in 15 Oregon and in the Coast Ranges of Oregon and northern California. It has a roughened, 16 warty, reddish brown to brown peridium, a solid whitish gleba that develops grayish brown 17 mottling as the spores mature, and produces a cheesy-garlicky odor by maturity. Its 18 smooth, ellipsoid spores resemble those of Morchella spp. but are much larger. Kalapuya 19 together with Fischerula, Imaia, and Leucangium, the other hypogeous genera in the 20 Morchellaceae, appear to form a lineage within the family that is distinct from the epigeous 21 genera, Morchella and Verpa. Locally known as the Oregon brown truffle, Kalapuya has 22 been commercially harvested for culinary use. TRAPPE ET AL.: KALAPUYA GEN. & SP. NOV. 2 23 Key words: Ascomycota, Pezizales, truffle, hypogeous fungus, Leucangium, 24 Morchellaceae, Fischerula, taxonomy, LSU rDNA, EF1α, Douglas-fir, truffle 25 INTRODUCTION 26 A brown to reddish brown ascotruffle collected in northwestern Oregon for several years has 27 been locally known as the “Oregon Brown Truffle.” Because it resembles the Oregon Black 28 Truffle [Leucangium carthusianum (Tul. & C. Tul.) Paol.] in size, texture, glebal appearance and 29 habitat, it had been assumed to be an undescribed Leucangium sp. However, its spore shape 30 differs from that of L. carthusianum, and molecular analyses clearly indicate that it represents an 31 undescribed genus. Here we describe this genus and its only known species, Kalapuya brunnea, 32 and discuss its relationship to other genera within the Morchellaceae. 33 MATERIALS AND METHODS 34 Sections were prepared for light microscopy by hand and mounted in dH2O, Melzer’s reagent 35 and cotton blue as well as by microtoming of paraffin-embedded specimens and staining the thin 36 sections in safranin-fast green. All microscopic measurements were made in dH2O mounts at 37 400x or 1000x with a Zeiss GSL research microscope. Melzer’s reagent was used to test for 38 amyloid reactions and cotton blue for cyanescent reactions. 39 Glebal tissue samples were sequenced at the Institute for Genome Sciences and Policy at 40 Duke University. Clean fungal tissue was removed from within sporocarps, placed in 41 microcentrifuge tubes, and ground with micropestles. DNA was extracted with 24:1 42 chloroform:isoamyl alcohol and PCR amplified by use of the primer sets 897R-NS24 (SSU), 43 ITS5 – ITS4 (ITS) and LROR - LR5 (LSU), RPB2_5F-RPB2_7R (RPB2), and 1577F-2218R 44 (EF1α). Information on primers can be found at: http://www.aftol.org/primers.php. PCR 45 products were visualized on 1% agarose gels stained with SYBR safe (Invitrogen, Carlsbad, TRAPPE ET AL.: KALAPUYA GEN. & SP. NOV. 3 46 CA). Successful amplicons were cleaned with the enzymes exonuclease I and alkaline 47 phosphotase (New England Biolabs, Ipswich, MA). Bidirectional sequencing was performed 48 with the above primers and the Big Dye Sequencing Kit v.3.1 (Applied Biosystems, Foster City, 49 CA) on an ABI3730 capillary sequencer (Applied Biosystems). Sequences were edited with 50 Sequencher v.4.1 (Gene Codes Inc., Ann Arbor, MI) and aligned to reference sequences with the 51 software MacClade 4.0 (Maddison & Maddison, 2002). Phylogenetic analyses were conducted 52 with parsimony and maximum likelihood optimization criteria in PAUP* 4.0b10 (Swofford, 53 2002) and by Bayesian inference with MrBayes (Ronquist & Huelsenbeck, 2003). Sequences 54 produced in this study have been accessioned by Genbank (GQ119349 - GQ119360; GU596456 55 – GU596477) (Table 1). Although not included in our phylogenetic analyses, generated ITS, 56 SSU, and RPB2 sequences for the taxon we describe here have also been accessioned to facilitate 57 future phylogenetic and fungal community studies. In developing the LSU phylogram, along 58 with six collections of the new taxon we included Fischerula subcaulis Trappe 1975, Imaia 59 gigantea (Imai 1933) Trappe & Kovács, two species of Leucangium, two species of Verpa, three 60 species of Morchella, and taxa in the Discinaceae and Tuberaceae as outgroups. For the EF1α 61 phylogeny we included taxa within the Helvellaceae and Tuberaceae as outgroups. 62 RESULTS 63 Phylogenetic analysis of LSU rDNA and EF1α genes both indicate that the new taxon is 64 nested with the Morchellaceae and related to but distinct from other known hypogeous genera in 65 this family (e.g Fischerula, Imaia, and Leucangium) and epigeous genera (Morchella, Verpa, 66 Disciotis) (FIG. 1). However, the relationships between genera within the Morchellaceae remain 67 unresolved. Morphological characters also clearly distinguish this species from others examined. 68 Accordingly, we designate a new genus, Kalapuya, to accommodate this new species. TRAPPE ET AL.: KALAPUYA GEN. & SP. NOV. 4 69 70 Kalapuya M. Trappe, Trappe, & Bonito, gen. nov. 71 MycoBank 513040, GenBank GQ119354 72 Ascomata hypogaea, stereothecia, subglobosa. Peridium porphyreum vel brunneum, 73 verrucosum, verrucis fissuris tenuibus separatae. Gleba solida, albida, contextu fertili griseo- 74 brunneo maculato. Asci ellipsoidei vel globosi. Sporae ellipsoideae, laeves, juventute hyalinae, 75 maturitate sucineae. 76 TYPE SPECIES: Kalapuya brunnea M. Trappe, Trappe, & Bonito. 77 Etymology. Kalapuya, after the native American tribe that inhabited the range of this genus, 78 from the western foothills of the Cascade Range to the Pacific coast. 79 80 Kalapuya brunnea M. Trappe, Trappe, & Bonito, sp. nov. 81 MycoBank 513040, GenBank GQ119354 82 Ascomata hypogaea, stereothecia, subglobosa vel globosa , 12–80 × 10–45 mm. Peridium 83 porphyreum vel brunneum, verrucosum, verrucis fissuris tenuibus separatae. Gleba solida, 84 albida, contextu fertili griseo-brunneo maculato. Asci globosi, 70–90 × 65–90 µm, pariete 1–3 85 µm crassis, 6-8 sporis. Sporae ellipsoideae, 32–43 × 25–33 µm, laeves, juventute hyalinae, 86 maturitate sucineae. 87 Macrocharacters. ASCOMATA (FIG. 2) hypogeous stereothecia, subglobose to lobed and 88 furrowed, 12–60 (–80) × 10–45 mm, with a subcartilagenous, dendroid, basal attachment that 89 easily breaks off when specimens are removed from soil. PERIDIUM (FIG. 3) light yellowish 90 brown to orange brown, reddish brown or brown, often with darker patches or becoming blackish 91 on the upper surface in age, rough to granular, up to 2 mm thick, unevenly covered with patches TRAPPE ET AL.: KALAPUYA GEN. & SP. NOV. 5 92 of flat to rounded warts 0.5–3 mm broad, the larger warts often in turn beset wilth minute warts, 93 polygonal in face view and separated by narrow fissures, in age often rimose-areolate. GLEBA 94 (FIG. 4) solid, firm, whitish to or yellowish gray, with grayish brown mottling of fertile pockets 95 surrounded by sterile, undifferentiated veins. TASTE and ODOR mildly garlicky-cheesy, the 96 odor reminiscent of mature Camembert cheese. 97 Microcharacters. ASCOSPORES (FIG. 5) ellipsoid, 32–43 × 25–38 µm (Q=1.09–1.68), the 98 surface smooth, containing a large, central guttule with crowded tiny droplets inside the spore at 99 its ends and on the sides of the central guttule; spore walls 1–3 µm thick, hyaline in youth 100 becoming amber to pale olive by maturity, nonreactive in Melzer’s reagent, stongly bluing in 101 cotton blue. ASCI (FIG 5) 6–8 spored, ellipsoid to globose or irregular, nonamyloid, 70–110 × 102 60–100 µm, with a stem 10–40 × 6–10 µm and having a forked base; walls hyaline, in youth up 103 to 3 µm thick, thinning to about 1 µm by maturity. ECTAL EXCIPULUM (FIG. 6) with warts 104 up to 150 µm tall of rounded to polyhedral cells 10–28 (–50)× 10–25 (–50) µm, the walls thin to 105 thickened up to 5 µm near the surface, reddish brown in dH2O. ENTAL EXCIPULUM (FIG. 7) 106 near the ectal excipulum of rounded, thin-walled cells 10–45 × 10–30 µm mixed with thin- 107 walled hyphae 5–13 µm broad at septa, hyaline in dH2O, towards the gleba grading to tightly 108 interwoven, hyaline, thin-walled hyphae 5–13 µm broad at septae. GLEBA of loosely 109 interwoven, ascus-bearing, thin-walled, hyaline hyphae 5–13 µm broad at the septae plus 110 scattered inflated cells. 111 Etymology. Brunnea (Latin): referring to the brown peridium. 112 Habitat, distribution and season. Hypogeous under Douglas-fir (Pseudotsuga menziesii) up 113 to ca 50 years old in the top 2–10 cm of mineral soil, beneath scant litter layers at elevs from 114 near sea level to ca 500 m in the Cascade Mountains in Oregon and Coast Ranges of Oregon and TRAPPE ET AL.: KALAPUYA GEN. & SP. NOV. 6 115 northern California (Humboldt County). Fruiting October through March, but occasionally as 116 early as September and as late as July.