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. Farid^1*§, M. Gelardi^2*, C. Angelini^3,4, A.R. Franck⁵, F. Costanzo², L. Kaminsky⁶, E. Ercole⁷, T.J. Baroni⁸, A.L. White¹, J.R. Garey¹, M.E. Smith⁶, A. Vizzini^7§

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.

• Singer et al. (1990) re-described the type collecꢀon and placed
• as a rotaꢀon ellipsoid [V = (π.L.W²)/6 ± standard deviaꢀon]. The

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 (www.indexfungorum.org/

authorsoffungalnames.htm).

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 (hꢁp://www.ncbi.nlm.nih.gov/genbank/)

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 (hꢁp://unite.ut.ee/) 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.

• Taxon
• Voucher No.

00-436

• Locality
• ITS
• 28S

TEF1-α

RPB1

Afroboletus luteolus Afrocastellanoa ivoryana Australopilus palumanus Austroboletus fusisporus Austroboletus gracilis Austroboletus mutabilis Austroboletus subvirens Baorangia bicolor

• Africa
• -

----------
KF030238

KX685721 JX889650 JX889720 DQ534624 KP242263

KP242227

KF030246

KY418895 KJ184558

KF112356

• KF030397
• -

Arora 126 REH 6791

HKAS75207

112/96

Mukuvisi, Zimbabwe Queensland, Australia China

KX685715

-

JX889691

-

JX889718

-

• MA, USA
• KF030425
• KF030358

KP242078 KP242045 KF030370

KJ184564 KF030369

-
BRI AQ0795793 Queensland, Australia BRI AQ0794171 Queensland, Australia
--

• MB07-001
• NY, USA

China
-

Baorangia pseudocalopus

HKAS 63607

HKAS 75739 HKAS 75081
-

• China
• -

Nanhua County, Yunnan Prov., China

KF112168

Boletus aereus Boletus edulis

• REH 8721
• Humboldt County, California,

USA

• -
• KF030339
• -
• KF030377

• Be3
• Bavaria, Germany
• -
• KF030282

HQ161848 KF030261

KF030248 KF030352 JQ327014

JQ928617 DQ867117

-

GU187682

-
BD380 DS4514 4414

• Colorado, USA
• -
• -
• -

Boletus pulchriceps

Chiricahua Mnts, AZ, USA Chestnut Ridge Park, NY, USA Auckland, New Zealand

• -
• KF030409
• -

Boletus rufomaculatus Boletus semigastroideus Boletus variipes var. fagicola Borofutus dhakanus Bothia castanella

-

• KF030406
• KF030369

PBM3076

• -
• KF030430
• -

• A.H. Smith 4249 Cheboygan Co, Michigan, USA
• -
• JQ327017
• -

HKAS 73792 MB 03-053 NY28003
Bangladesh MA, USA

• -
• JQ928575
• -

DQ867110 DQ867111 DQ867112 DQ867113 DQ867114 KM269196 KM269195

-

• KF030421
• KF030382

• NY, USA
• -
• -

NY8669

• NY, USA
• -
• -
• -

• NY28002
• NY, USA
• -
• -
• -

MB 03-067 HKAS 82693 HKAS 82694 HKAS 76674

• MA, USA
• -
• -
• -

Bothia fujianensis

Fujian Prov., China Fujian Prov., China

• -
• -
• -

KM269193

KF112350

KM272860

KF112277
-

Buchwaldoboletus lignicola

Yichun, Heilongjiang Prov., China

KF112642

Butyriboletus appendiculatus

Butyriboletus roseoflavus Caloboletus firmus

• Bap1
• Bavaria, Germany
• -

-----

AF456837 JX290184

KF030278 JQ327013

DQ534648 JX889648

JQ327025 -
-
HKAS 54099

MB 06-060 MB 06-044

MB 04-001 REH 9502

• China
• KF739741

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

• REH 9455
• Cooloola, Queensland,

Australia
-

• JX889673
• JX889683

-

Gyrodon lividus

• Gl1
• Bavaria, Germany
• -

--
AF098378 KF112437

KX685724

GU187701 KF112270

KX685718

---

Harrya chromapes Heliogaster columellifer

• HKAS 50527
• Dêqên, Yunnan Prov., China

KPM-NC 23012 Odawara, Kanagawa Pref.,
Japan

Hourangia cheoi

HKAS 52269

China China
--
KF112385

KF112286 KP136923

--

Hourangia microcarpa

• HKAS 83763
• KP136945

(Wu1324)

Hourangia nigropunctata Hourangia sp.

HKAS 76657 HKAS 68178

xb2

• China
• -

--
KF112388 KF112453 KF030357
KF112287 KF112301 KF030422
---
China

Imleria badia

Bavaria, Germany

© 2018 Westerdijk Fungal Biodiversity Insꢀtute

343

Farid et al.

Table 1. (Conꢀnued).

• Taxon
• Voucher No.

S-F119691

• Locality
• ITS

-

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

• -
• KC215225

KM605167

-
Heqing, Yunnan Prov., China Erie Co., NY, USA
-

KM605152 KM605163

Lanmaoa carminipes Leccinellum aff. griseum Leccinellum cremeum Leccinellum crocipodium