Phylloporus

VOLUME 2 DECEMBER 2018 PAGES 341–359 Fungal Systemaꢀcs and Evoluꢀon doi.org/10.3114/fuse.2018.02.10 Phylloporus and Phylloboletellus are no longer alone: Phylloporopsis gen. nov. (Boletaceae), a new smooth-spored lamellate genus to accommodate the American species Phylloporus boleꢀnoides A. Farid1*§, M. Gelardi2*, C. Angelini3,4, A.R. Franck5, F. Costanzo2, L. Kaminsky6, E. Ercole7, T.J. Baroni8, A.L. White1, J.R. Garey1, M.E. Smith6, A. Vizzini7§ 1Herbarium, Department of Cell Biology, Micriobiology and Molecular Biology, University of South Florida, Tampa, Florida 33620, USA 2Via Angelo Custode 4A, I-00061 Anguillara Sabazia, RM, Italy 3Via Cappuccini 78/8, I-33170 Pordenone, Italy 4Naꢀonal Botanical Garden of Santo Domingo, Santo Domingo, Dominican Republic 5Wertheim Conservatory, Department of Biological Sciences, Florida Internaꢀonal University, Miami, Florida, 33199, USA 6Department of Plant pathology, University of Florida, Gainesville, Florida 32611, USA 7Department of Life Sciences and Systems Biology, University of Turin, Viale P.A. Maꢂoli 25, I-10125 Torino, Italy 8Department of Biological Sciences, State University of New York – College at Cortland, Cortland, NY 1304, USA *Authors contributed equally to this manuscript §Corresponding authors: [email protected], [email protected] Key words: Boletales lamellate boletes molecular phylogeny taxonomy Abstract: The monotypic genus Phylloporopsis is described as new to science based on Phylloporus boleꢀnoides. This species occurs widely in eastern North America and Central America. It is reported for the first ꢀme from a neotropical montane pine woodland in the Dominican Republic. The confirmaꢀon of this newly recognised monophyleꢀc genus is supported and molecularly confirmed by phylogeneꢀc inference based on mulꢀple loci (ITS, 28S, TEF1-α, and RPB1). A detailed morphological descripꢀon of P . b oleꢀnoides from the Dominican Republic and Florida (USA) is provided along with colour images of fresh basidiomata in habitat, line drawings of the main anatomical features, transmiꢁed light microscopic images of anatomical features and scanning electron microscope images of basidiospores. The taxonomic placement, ecological requirements and distribuꢀon paꢁerns of P . b oleꢀnoides are reviewed and the relaꢀonships with phylogeneꢀcally related or morphologically similar lamellate and boletoid taxa such as Phylloporus, Phylloboletellus, Phyllobolites and Bothia are discussed. Xerocomoideae Published online: 23 October 2018. INTRODUCTION 2015, 2017). Species of Phylloporus form ectomycorrhizal (ECM) associaꢀons with plants in the Casuarinaceae, Dipterocarpaceae, Fabaceae, Fagaceae, Myrtaceae, and Pinaceae (Heinemann 1951, Corner 1970, Heinemann & Rammeloo 1987, Singer 1986, Watling 2008, Neves & Halling 2010, Neves et al. 2012, GarcíaJiménez 2013, Zeng et al. 2013, Ye et al. 2014, Hosen & Li 2015, 2017). Five species of Phylloporus have been described from North America (Neves 2007, Neves et al. 2010). Phylloporus boleꢀnoides is a lamellate to subporoid bolete that was formerly described based on material collected by Harry D. Thiers from Alachua Co., northern Florida (Smith & Thiers 1964) and subsequently reported from all along the Atlanꢀc Coast of eastern and south-eastern USA south to the Gulf Coast (Singer et al. 1990, Singer & Williams 1992, Both 1993, Besseꢁe et al. 2000, 2016, Jusꢀce 2008), down into Central America (Orꢀz-Santana et al. 2007, Neves & Halling 2010), although not reported by Orꢀz- Santana et al. (2007) as occurring in the Dominican Republic. It is a putaꢀve ECM partner of several American pine trees (Pinus spp., Pinaceae), but is also found in mixed pine and oak (Quercus spp., Fagaceae) forests (Smith & Thiers 1964, Singer et al. 1990, Both 1993, Besseꢁe et al. 2000, 2016, Neves & Halling 2010). Phylloporus is a genus of lamellate fungi in the family Boletaceae that is primarily distributed throughout the tropics (Kirk et al. 2008, Neves & Halling 2010, Neves et al. 2012, Zeng et al. 2013). Singer (1945b) moved Phylloporus to the subfamily Xerocomoideae due to its Phylloporus-type hymenophoral trama and olive-brown spore print. Bresinsky & Besl (2003) synonymised Phylloporus with Xerocomus based on molecular data, though only a few taxa were sampled in that study. Recent molecular phylogeneꢀc analyses with more extensive sampling support the monophyly of Phylloporus, showing that Xerocomus is highly polyphyleꢀc (Binder & Hibbeꢁ 2006, Šutara 2008, Nuhn et al. 2013, Wu et al. 2014, 2016) and inferring the placement of Phylloporus in an expanded Xerocomoideae that now also includes taxa with Boletus-type hymenophoral trama and pale yellow to brown spore prints (Šutara 2008, Wu et al. 2014, 2016). Neves & Halling (2010) esꢀmated the genus Phylloporus to include about 70 species, but several subsequent studies have described a number of addiꢀonal taxa (Neves et al. 2010, 2012, García-Jiménez 2013, Zeng et al. 2013, Ye et al. 2014, Hosen & Li Fungal Systemaꢀcs and Evoluꢀon is licensed under a Creaꢀve Commons Aꢁribuꢀon-NonCommercial-ShareAlike 4.0 Internaꢀonal License © 2018 Westerdijk Fungal Biodiversity Insꢀtute 341 Farid et al. <ul style="display: flex;"><li style="flex:1">Singer et al. (1990) re-described the type collecꢀon and placed </li><li style="flex:1">as a rotaꢀon ellipsoid [V = (π.L.W2)/6 ± standard deviaꢀon]. The </li></ul>notaꢀon [n/m/p] indicates that measurements were made on “n” randomly selected basidiospores from “m” basidiomata of “p” collecꢀons. The width of each basidium was measured at the widest part, and the length was measured from the apex (sterigmata excluded) to the basal septum. Metachromaꢀc, cyanophilic and iodine reacꢀons were tested by staining the basidiospores in Brilliant Cresyl blue, Coꢁon blue and Melzer’s reagent, respecꢀvely. Line drawings of microstructures were traced free hand based on digital photomicrographs of rehydrated material. Scanning electron micrographs have been obtained using a JEOL JSM IT300LV (High Vacuum – Low Vacuum 10/650 Pa - 0.3-30kV) Scanning Electron Microscope (SEM) operaꢀng at 10 kV. Selected air dried lamellae from the hymenophore were fixed on pin stubs using a Carbon Conducꢀve Cement glue and then spuꢁer-coated with carbon to a thickness of 400 Å. the species in Phylloporus sect. Fibulaꢀ. The species was originally assumed to represent an intermediate taxon between Suillus and Boleꢀnus (Smith & Thiers 1964). Singer et al. (1990) tentaꢀvely placed P . b oleꢀnoides in Phylloporus sect. Manausenses but later argued that it belonged to a new secꢀon that was never formally established (Singer & Williams 1992). As a maꢁer of fact, this species has always been regarded as retaining an isolated posiꢀon within the genus because of its peculiar morphological features with special reference to the pallid-coloured hymenophore and unusual macro-chemical reacꢀons. Several collecꢀons of P . b oleꢀnoides from Belize, the Dominican Republic and Florida were carefully examined using morphological and molecular approaches. Outcomes revealed strong morphological aꢃniꢀes with other members of Phylloporus, however, phylogeneꢀc analysis of fungal DNA sequences from four gene regions (ITS, 28S, TEF1-α, and RPB1) indicate they are distantly related and support the recogniꢀon of Phylloporopsis as a unique and as yet monotypic generic lineage in the Boletaceae, in phylogeneꢀc sister inference to the poroid genus Bothia and the sequestrate genus Solioccasus. DNA extracꢀon, PCR ampliꢁcaꢀon and DNA sequencing DNA extracꢀon and PCR amplificaꢀon were performed from dried basidiomata (Table 1) or from ꢀssues preserved in CTAB buffer as described by Alvarado et al. (2015). Primers ITS1F and ITS4 (White et al. 1990, Gardes & Bruns 1993) were used for the ITS region; primers LR0R and LR5 (Vilgalys & Hester 1990, Rehner & Samuels 1994) were used for the 28S rDNA, EF1-983F and EF1-1567R (Rehner & Buckley 2005) for the translaꢀon elongaꢀon factor 1-α (TEF1-α) gene. Amplificaꢀons of DNA- directed RNA polymerase II subunit 1 region (RPB1) were aꢁempted using the fungal primer pair RPB1-Af/RPB1-Cr and the bolete-specific primer pair RPB1-B-F/RPB1-B-R (Wu et al. 2014) but were unsuccessful. To increase amplificaꢀon success, RPB1-primers specific to the Bothia clade were developed (Table 2). A touchdown PCR was used to amplify the RPB1 region with the newly developed primer pairs RPB1-32-F/RPB1- 835-R, RPB1-147-F/RPB1-1091-R. The cycle parameters were as follows: (1) 94 oC for 2 min, (2) 94 °C for 40 s, (3) 66 °C for 40 s, minus 1 °C every cycle, (4) 72 °C 90 s, (5) repeat steps 2–4 for nine addiꢀonal cycles, (6) 94 °C for 45 s, (7) 56 °C for 90 s, (8) 72 °C for 90 s, (9) repeat steps 6–8 for 35 cycles, (10) 72 °C for 300 s. The PCR products were purified with the Wizard SV Gel and PCR Clean-Up System (Promega) following manufacturer’s instrucꢀons and sequenced forward and reverse by MACROGEN Inc. (Seoul, Republic of Korea). This study also widens the geographical extension of P . boleꢀnoides to the Dominican Republic, where it is reported for the first ꢀme and is found in associaꢀon with Pinus occidentalis, although several specimens have been spoꢁed directly growing on decayed wood or even on trunks of living trees, determining some uncertainty relaꢀve to its trophic status. MATERIALS AND METHODS Collecꢀon site and sampling Specimens from Belize, Dominican Republic, and the USA were examined from several public herbaria (CFMR, F, FLAS, JBSD, MICH, USF), as well as private herbaria [personal herbaria of Claudio Angelini (ANGE) and Maꢁeo Gelardi (MG)]. Acronyms of the public herbaria follow Thiers (2018), Herbarium numbers are cited for all collecꢀons from which morphological features were examined. Author citaꢀons follow the Index Fungorum, Authors of Fungal Names <a href="/goto?url=http://www.indexfungorum.org/authorsoffungalnames.htm" target="_blank">(www.indexfungorum.org/ </a><a href="/goto?url=http://www.indexfungorum.org/authorsoffungalnames.htm" target="_blank">authorsoffungalnames.htm). </a>Morphological studies Sequence alignment, data set assembly and phylogeneꢀc Macroscopic descripꢀons, macro-chemical reacꢀons (30 % NH4OH, 30 % KOH, FeSO4·7H2O) and ecological informaꢀon, such as habitat, ꢀme of fruiꢀng and associated plant communiꢀes accompanied the detailed field notes of the fresh basidiomata. Colour terms in capital leꢁers (e.g. Light Buff, Plate XV) are from Ridgway (1912). Microscopic anatomical features were observed and recorded from revived dried material. Secꢀons were rehydrated in water, 5 % potassium hydroxide (KOH), or in anionic soluꢀon saturated with Congo Red. All anatomical structures were measured from preparaꢀons in anionic Congo Red. Colours and pigments were described aﬅer examinaꢀon in water and 5 % KOH. Measurements were made at 1000 × using an ocular micrometer. Basidiospores weremeasureddirectlyfromthehymeniumofmaturebasidiomata, dimensions are given as (minimum) average ± standard deviaꢀon (maximum), Q = length/width raꢀo with the extreme values in parentheses, Qm = average quoꢀent (length/width raꢀo) ± standard deviaꢀon and average spore volume was approximated analyses The sequences obtained in this study were checked and assembled using Geneious v. 11.1.4 (Kearse et al. 2012) and compared to those available in GenBank database by using the Blastn algorithm (Altschul et al. 1990). Chromatograms were examined and manually edited for accuracy. Sequences were submiꢁed to GenBank <a href="/goto?url=http://www.ncbi.nlm.nih.gov/genbank/" target="_blank">(hꢁp://www.ncbi.nlm.nih.gov/genbank/) </a>and accession numbers are reported in Table 1. Homologous sequences from vouchered specimens and from environmental samples were selected and retrieved from Halling et al. (2007), Trappe et al. (2013), Zeng et al. (2013), Zhu et al. (2015) and Orihara & Smith (2017). A general combined Maximum likelihood tree including all the Boletaceae sequences deposited in GenBank and UNITE <a href="/goto?url=http://unite.ut.ee/" target="_blank">(hꢁp://unite.ut.ee/) </a>databases was generated to detect the phylogeneꢀc posiꢀon of our collecꢀons in the major clades of Boletaceae as circumscribed by Wu et al. © 2018 Westerdijk Fungal Biodiversity Insꢀtute 342 Phylloporopsis gen. nov. Table 1. Specimens and sequences used for the molecular phylogeneꢀc analyses (i.e., the ITS dataset and the 28S + TEF1-α and 28S + RPB1 combined datasets). Sequences newly generated for this study are highlighted in bold. <ul style="display: flex;"><li style="flex:1">Taxon </li><li style="flex:1">Voucher No. </li></ul>00-436 <ul style="display: flex;"><li style="flex:1">Locality </li><li style="flex:1">ITS </li><li style="flex:1">28S </li></ul>TEF1-α RPB1 Afroboletus luteolus Afrocastellanoa ivoryana Australopilus palumanus Austroboletus fusisporus Austroboletus gracilis Austroboletus mutabilis Austroboletus subvirens Baorangia bicolor <ul style="display: flex;"><li style="flex:1">Africa </li><li style="flex:1">-</li></ul>---------- KF030238 KX685721 JX889650 JX889720 DQ534624 KP242263 KP242227 KF030246 KY418895 KJ184558 KF112356 <ul style="display: flex;"><li style="flex:1">KF030397 </li><li style="flex:1">-</li></ul>Arora 126 REH 6791 HKAS75207 112/96 Mukuvisi, Zimbabwe Queensland, Australia China KX685715 -JX889691 -JX889718 -<ul style="display: flex;"><li style="flex:1">MA, USA </li><li style="flex:1">KF030425 </li><li style="flex:1">KF030358 </li></ul>KP242078 KP242045 KF030370 KJ184564 KF030369 - BRI AQ0795793 Queensland, Australia BRI AQ0794171 Queensland, Australia --<ul style="display: flex;"><li style="flex:1">MB07-001 </li><li style="flex:1">NY, USA </li></ul>China -Baorangia pseudocalopus HKAS 63607 HKAS 75739 HKAS 75081 -<ul style="display: flex;"><li style="flex:1">China </li><li style="flex:1">-</li></ul>Nanhua County, Yunnan Prov., China KF112168 Boletus aereus Boletus edulis <ul style="display: flex;"><li style="flex:1">REH 8721 </li><li style="flex:1">Humboldt County, California, </li></ul>USA <ul style="display: flex;"><li style="flex:1">-</li><li style="flex:1">KF030339 </li><li style="flex:1">-</li><li style="flex:1">KF030377 </li></ul><ul style="display: flex;"><li style="flex:1">Be3 </li><li style="flex:1">Bavaria, Germany </li><li style="flex:1">-</li><li style="flex:1">KF030282 </li></ul>HQ161848 KF030261 KF030248 KF030352 JQ327014 JQ928617 DQ867117 -GU187682 - BD380 DS4514 4414 <ul style="display: flex;"><li style="flex:1">Colorado, USA </li><li style="flex:1">-</li><li style="flex:1">-</li><li style="flex:1">-</li></ul>Boletus pulchriceps Chiricahua Mnts, AZ, USA Chestnut Ridge Park, NY, USA Auckland, New Zealand <ul style="display: flex;"><li style="flex:1">-</li><li style="flex:1">KF030409 </li><li style="flex:1">-</li></ul>Boletus rufomaculatus Boletus semigastroideus Boletus variipes var. fagicola Borofutus dhakanus Bothia castanella -<ul style="display: flex;"><li style="flex:1">KF030406 </li><li style="flex:1">KF030369 </li></ul>PBM3076 <ul style="display: flex;"><li style="flex:1">-</li><li style="flex:1">KF030430 </li><li style="flex:1">-</li></ul><ul style="display: flex;"><li style="flex:1">A.H. Smith 4249 Cheboygan Co, Michigan, USA </li><li style="flex:1">-</li><li style="flex:1">JQ327017 </li><li style="flex:1">-</li></ul>HKAS 73792 MB 03-053 NY28003 Bangladesh MA, USA <ul style="display: flex;"><li style="flex:1">-</li><li style="flex:1">JQ928575 </li><li style="flex:1">-</li></ul>DQ867110 DQ867111 DQ867112 DQ867113 DQ867114 KM269196 KM269195 -<ul style="display: flex;"><li style="flex:1">KF030421 </li><li style="flex:1">KF030382 </li></ul><ul style="display: flex;"><li style="flex:1">NY, USA </li><li style="flex:1">-</li><li style="flex:1">-</li></ul>NY8669 <ul style="display: flex;"><li style="flex:1">NY, USA </li><li style="flex:1">-</li><li style="flex:1">-</li><li style="flex:1">-</li></ul><ul style="display: flex;"><li style="flex:1">NY28002 </li><li style="flex:1">NY, USA </li><li style="flex:1">-</li><li style="flex:1">-</li><li style="flex:1">-</li></ul>MB 03-067 HKAS 82693 HKAS 82694 HKAS 76674 <ul style="display: flex;"><li style="flex:1">MA, USA </li><li style="flex:1">-</li><li style="flex:1">-</li><li style="flex:1">-</li></ul>Bothia fujianensis Fujian Prov., China Fujian Prov., China <ul style="display: flex;"><li style="flex:1">-</li><li style="flex:1">-</li><li style="flex:1">-</li></ul>KM269193 KF112350 KM272860 KF112277 -Buchwaldoboletus lignicola Yichun, Heilongjiang Prov., China KF112642 Butyriboletus appendiculatus Butyriboletus roseoflavus Caloboletus firmus <ul style="display: flex;"><li style="flex:1">Bap1 </li><li style="flex:1">Bavaria, Germany </li><li style="flex:1">-</li></ul>-----AF456837 JX290184 KF030278 JQ327013 DQ534648 JX889648 JQ327025 - - HKAS 54099 MB 06-060 MB 06-044 MB 04-001 REH 9502 <ul style="display: flex;"><li style="flex:1">China </li><li style="flex:1">KF739741 </li></ul>KF030368 KF030362 Chestnut Ridge Park, NY, USA Erie Co., NY, USA KF030408 JQ327020 Caloboletus inedulis Chalciporus piperatus Fistulinella prunicolor Rutland State Park, MA, USA GU187690 GU187453 Fraser Island, Queensland, Australia JX889690 -Gymnogaster boletoides <ul style="display: flex;"><li style="flex:1">REH 9455 </li><li style="flex:1">Cooloola, Queensland, </li></ul>Australia -<ul style="display: flex;"><li style="flex:1">JX889673 </li><li style="flex:1">JX889683 </li></ul>-Gyrodon lividus <ul style="display: flex;"><li style="flex:1">Gl1 </li><li style="flex:1">Bavaria, Germany </li><li style="flex:1">-</li></ul>-- AF098378 KF112437 KX685724 GU187701 KF112270 KX685718 ---Harrya chromapes Heliogaster columellifer <ul style="display: flex;"><li style="flex:1">HKAS 50527 </li><li style="flex:1">Dêqên, Yunnan Prov., China </li></ul>KPM-NC 23012 Odawara, Kanagawa Pref., Japan Hourangia cheoi HKAS 52269 China China -- KF112385 KF112286 KP136923 --Hourangia microcarpa <ul style="display: flex;"><li style="flex:1">HKAS 83763 </li><li style="flex:1">KP136945 </li></ul>(Wu1324) Hourangia nigropunctata Hourangia sp. HKAS 76657 HKAS 68178 xb2 <ul style="display: flex;"><li style="flex:1">China </li><li style="flex:1">-</li></ul>-- KF112388 KF112453 KF030357 KF112287 KF112301 KF030422 --- China Imleria badia Bavaria, Germany © 2018 Westerdijk Fungal Biodiversity Insꢀtute 343 Farid et al. Table 1. (Conꢀnued). <ul style="display: flex;"><li style="flex:1">Taxon </li><li style="flex:1">Voucher No. </li></ul>S-F119691 <ul style="display: flex;"><li style="flex:1">Locality </li><li style="flex:1">ITS </li></ul>-28S TEF1-α KJ806969 KF112190 -RPB1 Sweden KJ806971 KF112374 KM605140 KM605142 JQ327001 JN378508 - -Imleria obscurebrunnea Lanmaoa angusꢀspora Lanmaoa asiaꢀca HKAS 52557 HKAS 74759 HKAS 63592 MB 06-061 Ning’er, Yunnan Prov., China China <ul style="display: flex;"><li style="flex:1">-</li><li style="flex:1">KC215225 </li></ul>KM605167 - Heqing, Yunnan Prov., China Erie Co., NY, USA -KM605152 KM605163 Lanmaoa carminipes Leccinellum aff. griseum Leccinellum cremeum Leccinellum crocipodium

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