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18 November 2015

Phylogenetic Relationships among Species of Phellinus sensu stricto, Cause of White Trunk Rot of Hardwoods, from Northern North America

Center for Agriculture, Food and the Environment, University of Massachusetts, Amherst, MA 01002, USA
Forests2015, 6(11), 4191-4211;https://doi.org/10.3390/f6114191
This article belongs to the Special Issue Joint IUFRO 7.02.02 “Foliage, shoot and stem diseases of forest trees” and 7.03.04 “Diseases and insects in forest nurseries” Working Parties Meeting
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Abstract

Species in Phellinus s.s. are some of the most important wood-decaying fungal pathogens in northern temperate forests, yet data on species incidence in North America remains limited. Therefore, phylogenetic analyses were performed using four loci (ITS, nLSU, tef1 and rpb2) with isolates representing 13 species. Results of phylogenetic analyses using maximum likelihood and Bayesian inference revealed that eight species of Phellinus s.s. occur in North America, and include: P. alni, P. arctostaphyli, P. betulinus, P. lundellii, P. nigricans, P. tremulae and two undescribed species, P. NA1 and P. NA2. Meanwhile, P. tuberculosus, P. igniarius s.s., P. populicola, P. laevigatus s.s. and P. orienticus were not detected and appear restricted to Europe and/or Asia. The tef1 dataset outperformed all other loci used and was able to discriminate among all 13 of the currently known Phellinus s.s. species with significant statistical support. The internal transcribed spacer (ITS) region performed well but a high level of intraspecific variation could lead to inflated taxa recognition. Phellinus alni exhibited the broadest host range, as demonstrated previously, and appears to be the most common species in northern hardwood (Acer-Betula-Fagus), northern floodplain (Fraxinus-Populus-Ulmus) and coastal alder (Alnus) forests of North America.

1. Introduction

Based on several independent studies that utilized the morphological, biological and phylogenetic species concepts, it is clear that Phellinus sensu stricto (s.s.) is composed of a limited number of species that cause a delignifying trunk rot of deciduous hardwoods, with minor exceptions [1,2,3,4,5,6,7,8,9,10,11,12]. Numerous other species previously classified as Phellinus [13] are now members of various segregate genera (e.g., Fomitiporia, Fuscoporia, Inonotus, Onnia, Phellinidium and Porodaedalea, among others) [7,8,9].
White trunk rot of hardwoods, caused by Phellinus s.s., has been described as the single most important rot of hardwoods in North America [14]. Boyce ([15] p. 413) noted: “The false tinder fungus causes more loss than any other wood destroyer of hardwoods”. In North America, Phellinus s.s. is known to be most destructive in northern hardwood (Acer-Betula-Fagus) and aspen (Populus) forest types [14,15,16]. The presence of a single basidiocarp on the trunk of an infected tree can indicate a column of decay approximately 2 m in length for American beech (Fagus grandifolia) and sugar maple (Acer saccharum), and nearly 3 m in length for yellow birch (Betula alleghaniensis) [17]. Phellinus tremulae, one of the first species to be distinguished from the P. igniarius sensu lato (s.l.) complex [18], acts as an aggressive pathogen of aspen (Populus; especially P. tremuloides) and has been shown to cause significant losses of merchantable timber in the U.S. Lake States and Intermountain West [19,20].
For decades, white trunk rot of hardwoods was attributed to the actions of a single species, P. igniarius, even though several varieties or types were described [21,22]. While still referred to as the “P. igniarius group”, there are up to nine species of Phellinus s.s. known or suspected to occur in North America, and include: P. alni, P. acrtostaphyili, P. igniarius s.s., P. laevigatus, P. lundellii, P. nigricans, P. populicola, P. tremulae, P. tuberculosus [8,9,16,23,24]. There are additional species still described as “Phellinus” in North America (P. bicuspidatus and P. spiculosus, for example) that prior studies have shown are not members of Phellinus s.s. [8,10]. Ironically, it has been hypothesized that P. igniarius s.s. is a European species restricted to Salix [6,9]. However, a broader survey of species incidence by Tomšovský et al. [12] demonstrated that while P. igniarius s.s. is most common on Salix, it also occurs on Populus and Malus in Europe and Asia, signifying this species could potentially occur on all members of the Salicaceae and Rosaceae growing in northern temperate regions. An additional Phellinus species found only on Populus, P. populicola, is genetically similar to P. igniarius s.s. and is present throughout northern Europe and Asia [6,12]. To date, it remains unclear if either P. igniarius s.s. or P. populicola occur in North America.
Phylogenetic analyses have vastly improved the ability to discriminate genera and species in Phellinus s.l., yet most studies that include Phellinus s.s. have primarily utilized isolates collected from Europe [6,7,8,9,12], leaving North American researchers to speculate about species distribution and host specificity. The goals of this study were to determine the incidence and host distribution of Phellinus s.s. species using isolates that originated from northern temperate forests in North America. It was hypothesized that eight species of Phellinus s.s. occur in North America: P. alni, P. arctostaphyli, P. nigricans, P. laevigatus, P. lundellii, P. populicola, P. tremulae and P. tuberculosus.

2. Experimental Section

2.1. Isolates Used, DNA Extraction, PCR, and DNA Sequencing

A total of 57 isolates, representing eight presumed species from northern North America, Europe and Asia were chosen for this study and include: P. alni, P. arctostaphyli, P. nigricans, P. laevigatus, P. lundellii, P. populicola, P. tremulae and P. tuberculosus. In addition, isolates representing segregate genera in Phellinus s.l. and known to infect deciduous hardwoods were also used and include: Fuscoporia spp., Inocutis dryophila, Inonotus vaninii and Phellinopsis conchata. Fomes fomentarius was chosen to represent the outgroup. Most isolates were either field-collected by the author or obtained from the USDA Forest Service, Center for Forest Mycology Research (CFMR) herbarium. Isolates from the CFMR herbarium where chosen based on presumed species, geographic disparity and host diversity from northern North American forests that encompass the northern hardwood, aspen and alder forest types, among others (Table 1).
DNA extraction and PCR protocols have been described previously [25]. To generate PCR amplicons, the following primer pairs were used: ITS1-F and ITS4 [26] for the internal transcribed spacer (ITS); LR0R [27] and LR5 [28] the nuclear large subunit (nLSU) domains one to three; EF595F and EF1160R for partial elongation factor 1-alpha (tef1) [29]; and bRPB2-6F and bRPB2-7.1R [30] for partial RNA polymerase II, second largest subunit (rpb2) sequences. PCR products were visualized on 1% agarose gels stained with SYBR Safe (Life Technologies, Carlsbad, CA, USA) to ensure the presence of single amplicons. After visualizing the tef1 amplicons, six of the ten P. alni isolates chosen for study failed to amplify using the EF595F/EF1160R primer pair. Additional tef1 primers frequently used in phylogenetic studies of Basidiomycetes (983F/2218R) [31] were then used in the following combination to generate amplicons: (i) 983F/1160R; and (ii) EF595F/2218R. The first primer pair again failed, while the second primer pair produced single amplicons, approximately 1120 bp in size. The sequence analysis (described below) showed that isolates of P. alni can have two C/T mismatches in the EF1160R priming region, preventing successful annealing.
Prior to sequencing, PCR products were purified using ExoSAP-IT (USB, Cleveland, OH, USA). Isolates were sequenced using the Big Dye Sequencing Kit v. 3.1 on an ABI 3130xl capillary sequencer (Applied Biosystems, Foster City, CA, USA) at the Genomics Resource Laboratory, University of Massachusetts, Amherst. GenBank accession numbers are listed in Table 1.

2.2. Sequence Alignment and Phylogenetic Analysis

Raw sequences were edited using CodonCode v. 4.0.4 (CodonCode Corporation, Dedham, MA) and aligned with MAFFT v. 7 using the L-INS-i option for ITS sequences and the FFT-INS-i option for the remaining datasets [32]. Phylogenetic reconstructions were performed using maximum likelihood (ML) and Bayesian inference (BI). For ML and BI, the best-fit nucleotide substitution model was chosen using log-likelihood (-lnL) scores generated using jModelTest v. 2 [33,34]. For all data sets, the model that produced the highest -lnL score was the general time reversible (GTR) substitution model [35]. Sequences generated in this study were combined with data from previous studies [7,9,12,36,57]. Information for these isolates is provided in Table 1.
Table
Table 1. Information and GenBank accession numbers for isolates of Phellinus s.s..
Table 1. Information and GenBank accession numbers for isolates of Phellinus s.s..
Species & CodeHostState/ProvinceCountryReferenceGenBank Accession Nos.
ITSnLSUrpb2tef1
Phellinus alni
DLL2009-140Acer rubrumMNUSAThis studyKU139159KU139211KU139282KU139331
NJB2011-SM1Acer rubrumMAUSAThis studyKU139161KU139217KU139279KU139324
NJB2011-SM4Acer rubrumMAUSAThis studyKU139163KU139219KU139284KU139326
NJB2011-SM3Acer saccharumMAUSAThis studyKU139162KU139218KU139286KU139325
FP-134638-SpAlnus sp.IDUSAThis studyKU139167KU139213KU139280KU139330
HHB-15085-SpAlnus sp.AKUSAThis studyKU139160KU139216KU139285KU139328
NJB2011-WEN2Betula lentaMAUSAThis studyKU139164KU139220KU139283KU139327
FP-125027-TBetula papyriferaNHUSAThis studyKU139166KU139212KU139281KU139329
FP-70831-TFagus grandifoliaMIUSAThis studyKU139165KU139214KU139278KU139322
NJB2011-GR3Fagus grandifoliaMAUSAThis studyKU139168KU139215KU139287KU139323
BRNM 714864Alnus incanaMikkeli, KakrialaFinland[12]GQ383775 GQ383821
BRNM 714865Alnus incanaČervený KláštorSlovakia[12]GQ383730 GQ383840
BRNM 714891Betula sp.VyborgRussia[12]GQ383770 GQ383853
MJ 51/96Fagus sylvaticaHavlíčkův BrodCzech Republic[12]GQ383756 GQ383831
BRNM 714881Sorbus cf. intermediaUppsalaSweden[12]GQ383732 GQ383848
TN3301Betula sp.n/aFinland[9]AY340040
TW-162Laburnum anagyroidesn/aGermany[7]AF311025
Phellinus arctostaphyli
FP-94140-RArctostaphylos manzanitaORUSAThis studyKU139143KU139250KU139264KU139348
FP-94186-RArctostaphylos patulaCAUSAThis studyKU139145KU139252KU139266KU139350
M-92-2Arctostaphylos patulaORUSAThis studyKU139144KU139251KU139265KU139349
Phellinus laevigatus s.l.
P. betulinus
FP-105325-SpBetula alleghaniensisWVUSAThis studyKU139154KU139239KU139312KU139369
DLL2009-143Betula alleghaniensisMNUSAThis studyKU139146KU139236KU139308KU139371
DVB-BetulaBetula nigraVAUSAThis studyKU139151KU139246KU139314KU139365
NJB2009-FpGBetula papyriferaMAUSAThis studyKU139153KU139248KU139311KU139368
NJB2009-FpEBetula papyriferaMAUSAThis studyKU139152KU139247KU139307KU139370
DLL2009-175Betula papyriferaMNUSAThis studyKU139155KU139237KU139309KU139367
RLG-5835-TBetula papyriferaMTUSAThis studyKU139150KU139240KU139306KU139364
RLG-645-TBetula papyriferaMTUSAThis studyKU139147KU139238KU139313KU139366
P. laevigatus s.s.
NJB2011-PLa1-FBetula pubescensHameFinlandThis studyKU139148KU139241KU139305KU139372
NJB2011-PLa2-FBetula pubescensHameFinlandThis studyKU139149KU139242KU139310KU139373
BRNM 714875Betula sp.Bohemian ForestCzech Republic[12]GQ383779GQ383857
BRNM 714867Betula sp.Bohemian ForestCzech Republic[12]GQ383778GQ383856
BRNM 714877Betula sp.UppsalaSweden[12]GQ383777GQ383855
TN-3260Betula pubescensn/aFinland[7]AF311034
P. orienticus
TN-6392Betula sp.JilinChinaThis studyKU139156KU139243KU139374
TN-6432Betula costaJilinChinaThis studyKU139158KU139245KU139376
TN-6425Betula sp.JilinChinaThis studyKU139157KU139244KU139375
Phellinus lundellii
NJB2011-SM2Betula alleghaniensisMAUSAThis studyKU139184KU139235KU139299KU139335
NJB2011-WEN1Betula alleghaniensisMAUSAThis studyKC551835KC551859KU139302KC551884
DLL2011-321Betula alleghaniensisMIUSAThis studyKU139181
NJB2011-GR1Betula lentaMAUSAThis studyKU139182KU139232KU139301KU139336
JJW-694Betula sp.NYUSAThis studyKU139183KU139233KU139298KU139334
NJB2011-PLu-FBetula pubescensHameFinlandThis studyKU139185KU139234KU139300KU139337
BRNU 604719Betula carpaticaJeseníky MountainsCzech Republic[12]GQ383704
Phellinus nigricans
HHB-15513-TBetula nanaAKUSAThis studyKU139171KU139226KU139295KU139347
FP-62186-TBetula occidentalisWAUSAThis studyKU139176KU139227KU139290KU139343
FP-140068-TBetula papyriferaMNUSAThis studyKU139175KU139225KU139292KU139345
DMR-94-13Betula papyriferaMNUSAThis studyKU139173KU139221KU139291KU139344
FP-135209-RBetula papyriferaMIUSAThis studyKU139174KU139224KU139293KU139346
OKM-3356Betula papyriferaIDUSAThis studyKU139178KU139228KU139288KU139342
HHB-12617-TBetula papyriferaAKUSAThis studyKU139172KU139230KU139294KU139338
RLG-5844-SpBetula sp.MTUSAThis studyKU139177KU139229KU139289KU139341
NJB2011-PA1-FBetula pubescensHameFinlandThis studyKU139169KU139222KU139297KU139340
NJB2011-PA2-FBetula pubescensHameFinlandThis studyKU139170KU139223KU139296KU139339
MJ 557/94Betula sp.RondaneNorway[12]GQ383724GQ383801
BRNM 714879Betula sp.UppsalaSweden[12]GQ383721GQ383803
BRNM 714883Betula sp.VyborgRussia[12]GQ383719GQ383851
85-917Betula pubescensn/aGermany[7]AF311027
Phellinus populicola
TN-526 (1) (ATCC 36122; CBS 638.75)Populus tremulaUusimaaFinlandThis studyKU139179KU139231KU139303KU139333
BRNM 714885Populus tremulaUppsalaSweden[12]GQ383706GQ383785
BRNM 714890Populus tremulaUppsalaSweden[12]GQ383707GQ383786
MJ 92/96Populus albaLanžhotCzech Republic[12]GQ383705GQ383787
84-61Populus alban/aGermany[7]AF311038
Phellinus igniarius s.s.
MJ 19/94Populus nigraJihlavaCzech Republic[12]GQ383718GQ383793
BRNM 714866Populus nigraZábřehCzech Republic[12]GQ383710GQ383792
BRNM 714889Salix albaBrnoCzech Republic[12]GQ383709GQ383791
MJ 40/07Salix capreaJihlavaCzech Republic[12]GQ383712GQ383790
BRNM 714884Salix capreaUppsalaSweden[12]GQ383715GQ383795
83-1110aSalix fragilisn/aGermany[7]AF311033
Phellinus tremulae
FP-135820-TPopulus grandidentataWIUSAThis studyKU139136KU139206KU139274KU139363
FP-59023-TPopulus tremuloidesNHUSAThis studyKU139135KU139205KU139270KU139358
FP-135202-TPopulus tremuloidesMIUSAThis studyKU139134KU139207KU139271KU139359
FP-140050-TPopulus tremuloidesMNUSAThis studyKU139131KU139204KU139272KU139362
A-17Populus tremuloidesCOUSAThis studyKU139137KU139200KU139273KU139361
FP-105919-RPopulus tremuloidesSDUSAThis studyKU139130KU139203KU139275KU139360
NJB2011-PT1-FPopulus tremulaHameFinlandThis studyKU139132KU139201KU139276KU139357
NJB2011-PT2-FPopulus tremulaHameFinlandThis studyKU139133KU139202KU139277KU139356
MJ 45/07Populus tremulaHavlíčkův BrodCzech Republic[12]GQ383782GQ383860
BRNM 714886Populus tremulaTřeboňCzech Republic[12]GQ383780GQ383862
MJ 32/07Populus tremulaJihlavaCzech Republic[12]GQ383781GQ383861
Dai2352Populus sp.n/aChina[9]AY340063
89-826cPopulus tremulan/aEstonia[7]AF311042
Dai-Ptn/an/aChinaUnpublishedHQ328536
Phellinus tuberculosus
TN-449 (ATCC 38666)Prunus domesticaNauvoFinlandThis studyKU139142KU139254KU139263KU139352
TN-236 (ATCC 38665)Prunus insititiaTammisaariFinlandThis studyKU139141KU139253KU139262KU139351
MJ 47/07Prunus domesticaHavlíčkův BrodCzech Republic[12]GQ383784GQ383859
MJ 44/07Prunus spinosaHavlíčkův BrodCzech Republic[12]GQ383783GQ383858
Phellinus NA1
OKM-4173Populus tremuloidesIDUSAThis studyKU139180KU139249KU139304KU139332
Phellinus NA2
SRM-158-SpPrunus americanaNEUSAThis studyKU139140KU139210KU139267KU139353
FP-103293-RPrunus angustifoliaSCUSAThis studyKU139138KU139208KU139268KU139355
FP-105670-RPrunus sp.GAUSAThis studyKU139139KU139209KU139269KU139354
Inonotus vaninii
DMR-95-1-TPopulus tremuloidesMNUSAThis studyKU139198KU139258KU139318KU139380
DLL2010-102Populus tremuloidesMNUSA[57]KU139197
Inocutis dryophila
DLL2012-001Quercus albaWIUSAThis studyKU139186KU139255KU139317
Phellinopsis conchata
DLL2009-149Acer sp.MNUSA[57]KU139187KU139256KU139316KU139378
L-7601Fraxinus nigraNYUSAThis studyKU139188KU139257KU139315KU139377
89-1014Salix sp.—Germany[7]AF311028
Fuscoporia ferrea
FP-133592-SpAlnus sp.ORUSAThis studyKU139189KU139259KU139319KU139379
DLL2009-035Populus tremuloidesMNUSA[57]KU139190
Fuscoporia gilva
DLL2011-109Acer saccharumWIUSA[36]KU139195
DLL2011-147Acer saccharumWIUSA[36]KU139196
Fuscoporia sp. 1
DLL2009-025Populus tremuloidesMNUSA[57]KU139193
DLL2011-211Prunus serotinaWIUSA[36]KU139194
Fuscoporia sp. 2
DLL2011-140Acer saccharumWIUSA[36]KU139192
DLL2011-256Ostrya virginianaWIUSA[36]KU139191
Fomes fomentarius
NJB2011-KD3Fagus grandifoliaMAUSAThis studyKU139199KU139260KU139320KU139382
NJB2011-GR2Populus grandidentataMAUSAThis studyKU139261KU139321KU139381
(1) Type specimen.
ML analyses were carried out in MEGA v. 6.06 [37] using the GTR+I+G substitution model with the subtree-pruning-regrafting (SPR level 5) heuristic search method and the branch swap filter set to “moderate”. The gamma shape parameter value was estimated directly from the data within MEGA. Significance was assessed over 1000 bootstrap replicates and clades with BS values ≥70% were considered significant. BI of phylogenies was determined using MrBayes 3.2.4 [38]. The analyses were performed using the GTR substitution model with gamma distributed rate variation and a proportion of invariable sites (invgamma). Posterior probability distributions were obtained with two independent runs of Markov Chain Monte Carlo (MCMC), each with four chains (three heated and one cold) for 10,000,000 generations and a sampling frequency every 1000th generation. Posterior probability (PP) values were calculated after excluding 25% (burn-in) of the trees produced during the MCMC analysis. Estimated Sample Size (ESS) values for the parameters were then assessed in Tracer v. 1.6.0 (http://tree.bio.ed.ac.uk/software/tracer/) to assure convergence. Sufficient sampling was accomplished if ESS values >200. Trees sampled after convergence of the two runs (when average standard deviation of split frequencies <0.01 and average potential scale reduction factor for parameter values >1) were used to reconstruct a 50% majority rule consensus tree and obtain PP values. Trees were viewed in FigTree v. 1.4.0 (http://tree.bio.ed.ac.uk/software/figtree/) and clades supported by PP values ≥0.95 were considered significant.
The disparity index (ID) test of pattern heterogeneity [39] was performed to determine if significant differences in substitution rates existed among the chosen loci. Only the isolates for which all datasets were available (ITS, nLSU, tef1 and rpb2) were used. The test was performed using 1000 Monte-Carlo replications with significance assessed at p = 0.05. Pairwise analysis of all sequences in each dataset determined that two sets of loci evolved with similar substitution rates: (i) ITS and tef1; and (ii) nLSU and rpb2. However, phylogenetic reconstructions carried out separately for each dataset highlighted significant differences in tree topology (BS values ≥70% and PP values ≥0.95 at the nodes; see Results). Therefore, none of the individual datasets were concatenated for further phylogenetic analysis.

3. Results

3.1. Phylogenetic Analysis and Species Identification

Successful amplification and sequencing was achieved for nearly all isolates used in this study.For certain isolates representing segregate genera in Phellinus s.l., only ITS sequences were generated (Table 1). Based on the results of the analyses performed here, 13 phylogenetic species in Phellinus s.s. were differentiated from isolates that originated from northern North America, Europe and Asia (Table 1; Figure 1, Figure 2, Figure 3 and Figure 4). Of these 13 species, eight are present in North America, and include: P. alni, P. arctostaphyli, P. betulinus, P. lundellii, P. nigricans, P. tremulae and two undescribed species, hereafter referred to as P. NA1 and P. NA2 (Figure 1, Figure 2, Figure 3 and Figure 4).
Forests 06 04191 g001 1024
Figure 1. Maximum likelihood tree based on rDNA ITS sequences of Phellinus s.s. species. Bootstrap support values ≥70% and PP values ≥0.95 are shown next to the nodes. Thickened branches represent BS/PP values ≥99%/1. Intraspecific variation by geographic origin is defined by the following codes: NA, North America; EU, Europe; AS, Asia; NA-NC, north-central North America; NA-AK, Alaska; NA-W, western North America.
Forests 06 04191 g002 1024
Figure 2. Maximum likelihood tree based on nLSU sequences of Phellinus s.s. species. Bootstrap support values ≥70% and PP values ≥0.95 are shown next to the nodes. Thickened branches represent BS/PP values ≥99%/1.
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Figure 3. Maximum likelihood tree based on rpb2 sequences of Phellinus s.s. species. Bootstrap support values ≥70% and PP values ≥0.95 are shown next to the nodes. Thickened branches represent BS/PP values ≥99%/1.
Forests 06 04191 g004 1024
Figure 4. Maximum likelihood tree based on tef1 sequences of Phellinus s.s. species. Bootstrap support values ≥70% and PP values ≥0.95 are shown next to the nodes. Thickened branches represent BS/PP values ≥99%/1.
The results demonstrate that three species suspected of having a Holarctic distribution are, based on this dataset, restricted to Europe and Asia with a sister species present in North America. Specifically, P. laevigatus s.s., P. populicola and P. tuberculosus were not detected from North America (Table 1; Figure 1, Figure 2, Figure 3 and Figure 4). In their place, P. betulinus, P. NA1 and P. NA2 occur with the same host preference: Betula, Populus and Prunus (Table 1; Figure 1, Figure 2, Figure 3 and Figure 4). Based on the tef1 dataset, P. laevigatus s.l. is composed of three unique phylogenetic species (P. betulinus, P. orienticus and P. laevigatus s.s.) (Figure 4). The remaining three loci were unable to fully discriminate the P. laevigatus s.l. complex, although the ITS dataset did distinguish P. orienticus as distinct from P. betulinus and P. laevigatus s.s. (Figure 1, Figure 2 and Figure 3). Finally, P. igniarius s.s. was not detected from the North American dataset, although one of its primary hosts in Europe (Salix) was not analyzed here. However, a closely-related species, P. NA1, was discovered but is represented by only a single isolate (Figure 1, Figure 2, Figure 3 and Figure 4).
Overall, the four loci used produced similar tree topologies with one significant difference. Trees reconstructed using the ITS, nLSU and rpb2 datasets all grouped isolates representing the P. laevigatus s.l. complex distal to all other Phellinus s.s. species (Figure 1, Figure 2 and Figure 3). Only the tef1 dataset was not in agreement (Figure 4), grouping P. laevigatus s.l. in a superclade with P. tremulae, P. NA2, P. tuberculosus and P. arctostaphyli. Additionally, all four loci demonstrated a close relationship between two sets of species: (i) P. tremulae and P. NA2 and; (ii) P. tuberculosus and P. arctostaphyli. These four species consistently grouped together regardless of locus or analysis method (Figure 1, Figure 2, Figure 3 and Figure 4).

3.2. Phylogenetic Relationships by Locus

The four loci used in this study (ITS, nLSU, rpb2 and tef1) had varying levels of utility in discriminating Phellinus s.s. species. The ITS dataset was the most extensive in the number of sequences available from different geographic regions and hosts (Table 1; Figure 1). While ITS sequences demonstrated an ability to differentiate most Phellinus s.s. species, this dataset also exhibited high intraspecific variation. For example, isolates representing P. alni, P. nigricans and P. igniarius s.s. were divided into subclades with significant statistical support based on their geographic origin (Figure 1). In other cases, ITS provided only limited utility in differentiating several closely-related species groups, such as P. igniarius s.s.-P. populicola-P. NA1 and P. tremulae-P. NA2 (Figure 1). The ITS dataset was able to distinguish P. orienticus as a unique phylogenetic species from the P. laevigatus s.l. group but could not differentiate between P. betulinus and P. laevigatus s.s. (Figure 1).
Much like the ITS dataset, the 5′ region of the nLSU is able to differentiate among most Phellinus species, but support for some clades was weak with both ML and BI (Figure 2). As expected, there was limited intraspecific variation in the nLSU compared to the ITS region, but closely-related species are once again undifferentiated (Figure 2). Sequences from domains six to seven in the rpb2 gene comprised the lowest number of total sequences due to the lack of additional data in public databases (Figure 3). This dataset provided higher BS and PP values compared to the ITS and nLSU datasets but again, lacked resolution among closely-related species (Figure 3).
The tef1 dataset was superior to all other loci used in this study (Figure 4). Nearly all of the species analyzed were supported with significant BS and PP values with most supported with very high confidence (BS ≥ 99% and PP = 1). In conjunction with sequences generated by Tomšovský et al. [12], tef1 grouped Phellinus s.s. species into two main superclades (Figure 4). The tef1 dataset was also able to discriminate members among the closely-related P. laevigatus s.l. complex (P. betulinus, P. laevigatus s.s. and P. orienticus) with strong statistical support (Figure 4). Despite the high overall resolution, this dataset, like all others, failed to differentiate P. igniarius s.s., P. populicola and P. NA1 with consistent, significant support (Figure 4).

3.3. Phylogenetic Relationships by Host

The results show that most Phellinus s.s. species primarily infect a single host genus (Table 2). Five species (P. betulinus, P. laevigatus s.s., P. orienticus, P. lundellii and P. nigricans) are specific to Betula and three (P. betulinus, P. lundellii and P. nigricans) have overlapping host ranges in North America. Prunus has two associated species (P. tuberculosus and P. NA2) while Populus has three (P. populicola, P. tremulae and P. NA1). Lastly, P. arctostaphyli is known only from the woody shrub genus Arctostaphylos. Two remaining Phellinus species, meanwhile, are notable exceptions to this trend: (i) P. alni, which has a broad host range that includes Acer, Alnus, Betula and Fagus and in this particular study; and (ii) P. igniarius s.s., which is still unconfirmed from North America.
Table
Table 2. Known range, host genera and host families of Phellinus s.s. species.
Table 2. Known range, host genera and host families of Phellinus s.s. species.
Phellinus SpeciesKnown RangeKnown Host GeneraKnown Host Families
P. alniHolarcticAcer, Alnus, Betula, Carpinus, Corylus, Fagus, Fraxinus, Juglans, Laburnum, Malus, Padus, Prunus and SorbusBetulaceae, Fagaceae, Juglandaceae, Oleaceae, Rosaceae, Sapindaceae & Ulmaceae
P. lundelliiHolarcticBetulaBetulaceae
P. nigricansHolarcticBetulaBetulaceae
P. tremulaeHolarcticPopulusSalicaceae
P. arctostaphyliNorth AmericaArctostaphylosEricaceae
P. betulinusNorth AmericaBetulaBetulaceae
P. NA1North AmericaPopulusSalicaceae
P. NA2North AmericaPrunusRosaceae
P. igniarius s.s.EuropePopulus, Salix and MalusSalicaceae and Rosaceae
P. populicolaEurope & AsiaPopulusSalicaceae
P. tuberculosusEurope & AsiaPrunusRosaceae
P. laevigatus s.s.Europe & West AsiaBetulaBetulaceae
P. orienticusEast AsiaBetulaBetulaceae

4. Discussion

The primary objective of this study was to better understand the assemblage of Phellinus s.s. species present in North America using a multilocus dataset composed of isolates representing a broad geographic area. As hypothesized, at least eight species of Phellinus s.s. occur in North America, yet the specific species assemblage was not accurately predicted. Up to this point, nearly all of the work aimed at elucidating the species in Phellinus s.s. has been performed in Europe, albeit with some isolates originating from North America [4,6,7,8,9,12,40]. While some Phellinus s.s. species have Holarctic distributions, the results of this study demonstrate clear differences in the North American assemblage that were not previously known.
In North America, P. alni has the widest host range of any Phellinus s.s. species, occurring on Acer, Alnus, Betula and Fagus. Including its known host range in Europe, P. alni inhabits at least 14 host genera, including Fraxinus, Juglans, Prunus and Ulmus [6,12]. This broad host range is unique within the genus, as most members of Phellinus s.s. exhibit a very narrow host range (see Table 2). Considering this, P. alni is likely the most common species found in northern hardwood (Acer-Betula-Fagus), northern floodplain (Fraxinus-Populus-Ulmus) and coastal Alnus forests in North America. It is most likely widespread in Quercus-dominated forests as well, since many of the minor tree species in the Quercus forest types are hosts for P. alni [41]. Phylogenetically, all four loci grouped P. alni with P. lundellii, P. igniarius s.s., P. NA1, P. nigricans and P. populicola. Of this group, P. igniarius s.s., P. NA1 and P. populicola are known only from woody plants in the Salicaceae and Rosaceae. Interestingly, both Fischer [6] and Tomšovský et al. [12] found that P. alni does not infect members of the Salicaceae in Europe and this trend was observed in North America as well. Yet, Populus was the only member of Salicaceae examined in this study and more targeted sampling of Salix and Populus species is necessary to conclude if P. alni can attack these hosts. Additionally, this targeted sampling will help to conclude if P. igniarius s.s. and P. populicola are indeed present in North America. At present, these two species are known only from Europe and Asia. Attempts to utilize “Phellinus” specimens from the CFMR herbarium that were collected from Salix proved these isolates belong to segregate genera such as Fuscoporia (results not shown).
Based on variations in morphological characters, Parmasto [40] proposed that P. laevigatus s.l. is composed of two species: P. laevigatus s.s. from Europe and P. betulinus from North America and Asia. Furthermore, Parmasto believed that P. betulinus can be further divided into two subspecies: ssp. betulinus from North America and ssp. orienticus from Asia. The results presented here show that P. laevigatus s.s., P. betulinus and P. orienticus each represent a unique phylogenetic species. While this assertion is supported by only one dataset (tef1), prior studies have shown tef1 to be the only marker capable of discriminating between very closely-related species in basidiomycete genera like Armillaria and Trametes [42,43,44]. Additionally, the ITS dataset was able to discriminate P. orienticus from P. betulinus-P. laevigatus s.s. with a high level of support (BS/PP = 99/1). Thus, the results presented here support the elevation of P. orienticus and P. betulinus to species status. Phellinus orienticus has been previously treated as a distinct species based solely on morphological characters [45]. Additionally, Wagner and Fisher [8] treated P. orienticus as a unique species in their study to deconstruct Phellinus s.l. using nLSU sequences. In this study, nLSU sequences were unable to discriminate among members of P. laevigatus s.l.
Isolates collected from Prunus in North America that were thought to represent P. tuberculosus were instead shown to be a separate phylogenetic species, referred to here as P. NA2. What is perhaps more interesting is that each locus examined in this study showed that P. NA2 is more closely-related to P. tremulae than to P. tuberculosus. Meanwhile, the results also reveal a close genetic relationship between P. tuberculosus and P. arctostaphyli. Regardless of its closest relative, the results suggest that P. tuberculosus is restricted to Europe and Asia and that P. NA2 requires formal description as a new North American species. Overholts [22] (pp. 64–65) did note that specimens of P. tuberculosus from Europe had larger basidiospores compared to specimens from North America and that: “The American plant should perhaps be designated as a distinct variety”. Basidiospore variation between P. NA2 and P. tuberculosus was also confirmed by Niemelä [46] and Gilbertson and Ryvarden [5].
The first recognized description of Phellinus from Prunus was performed by Baumgarten, who described the species as Boletus tuberculosus [47]. This name was soon forgotten though, in favor of the epithet B. pomaceus, which appeared in the literature less than a decade later [48]. Both of these early descriptions utilized European strains of the pathogen. Numerous, subsequent descriptions of this species were made in North America and Europe, all using Phellinus pomaceus [5,22,46,49,50] until it was determined to be a synonym of P. tuberculosus, with the latter having precedence [51]. After this point, Riffle and Conway [52] adopted the use of P. tuberculosus while Ryvarden and Gilbertson [53] preferred use of the well-established P. pomaceus. Phylogenetic studies of Phellinus s.l. have consistently used P. tuberculosus over P. pomaceus [7,8,10,12], but the use of P. pomaceus still continues, albeit infrequently [54,55]. Adding to the confusion is the fact that P. alni can also be found on Prunus [6] along with Fomitiporia prunicola [56].
One of the most fascinating aspects of Phellinus s.s. is that five species are known to occur primarily on Betula (P. betulinus, P. laevigatus s.s., P. lundellii, P. nigricans, and P. orienticus). Three of these species (P. betulinus, P. lundellii and P. nigricans) are present in North American forests and have overlapping host ranges. Not only do these three species occupy the same geographic area but they appear to occupy the same ecological niche, with no apparent preference for a particular species of Betula. Clearly, these species represent an interesting model for studies on sympatric speciation. At the same time, P. nigricans and P. lundellii were somewhat partitioned in their distribution in North America, with P. nigricans present in western and north-central North America while P. lundellii was identified from north-central and eastern North America. Because their ranges do overlap in the Lake States region and Betula species occur contiguously across northern North America, it’s most likely both species are present across the continent as well.
A single isolate collected from Idaho in 1966 by Orson Miller, and presumed to be P. tremulae, represents a sister species to P. igniarius s.s. and P. populicola. This species, P. NA1, remains undescribed here pending further study on its distribution and host range. While white trunk rot of aspen is primarily the result of infection by P. tremulae, this study shows that P. NA1 must be considered as an additional causal agent. Further, Inonotus vaninii (formerly Phellinus vaninii) can also be found on Populus [57]. Even if P. NA1 and I. vaninii are uncommon on Populus compared to P. tremulae, a complex of Phellinus s.l. species should now be considered to cause white trunk rot of aspen.
It is entirely reasonable to believe that Phellinus s.s. species described here as host-specific may, at times, be found on non-preferred hosts. Because of decades of confusion and misinterpretation in the taxonomy of Phellinus s.s. [13], numerous reports exist of exceptions in the host-specificity reported here and in previous studies [6,12]. Yet, these reports of incidence on non-preferred hosts should be carefully scrutinized as future studies shed further light on the incidence and host range of Phellinus s.s. across northern temperate forests.

Acknowledgments

I am grateful to Daniel Lindner and Tuomo Niemelä for their assistance in obtaining isolates used in this study. Funding was provided, in part, by the USDA Forest Service, Center for Forest Mycology Research.

Conflicts of Interest

The author declares no conflict of interest.

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