Mycologia, 108(6), 2016, pp. 1216–1228. DOI: 10.3852/16-106 # 2016 by The Mycological Society of America, Lawrence, KS 66044-8897 Phylogenetic assessment of global Suillus ITS sequences supports morphologically defined species and reveals synonymous and undescribed taxa Nhu H. Nguyen1 Collectively, this comprehensive genus-level phyloge- Department of Tropical Plant and Soil Sciences, University netic integration of currently available Suillus ITS ‘ ‘ of Hawai iatMa¯noa, Honolulu, Hawai i 96822 molecular data and metadata will aid future taxonomic Else C. Vellinga and ecological work on an important group of ectomy- Thomas D. Bruns corrhizal fungi. Department of Plant and Microbial Biology, University of Key words: Boletales, bolete, geography, molecular California, Berkeley, California 94720 phylogenetics, Suillaceae, suilloid, systematics, taxonomy Peter G. Kennedy INTRODUCTION Departments of Plant Biology and Ecology, Evolution, and Behavior, University of Minnesota, Saint Paul, The genus Suillus Gray comprises approximately 100 Minnesota 55108 mushroom-forming species that are widely distributed throughout the Northern Hemisphere. Suillus species associate almost exclusively with members of the family Abstract: The genus Suillus represents one of the most Pinaceae and exhibit strong patterns of host specificity recognizable groups of mushrooms in conifer forests among different genera (Smith and Thiers 1964a, throughout the Northern Hemisphere. Although for Klofac 2013). They can also be found where hosts decades the genus has been relatively well defined mor- have been planted and have subsequently invaded in phologically, previous molecular phylogenetic assess- the Southern Hemisphere (Chapela et al. 2001, Dickie ments have provided important yet preliminary insights et al. 2010, Walbert et al. 2010, Hayward et al. 2015), as into its evolutionary history. We present the first large- well as Pacific islands (Hynson et al. 2013). Given its scale phylogenetic study of the boundaries of each broad distribution and notable host specificity pat- species in the genus Suillus based on the most current terns, the genus Suillus has emerged as a model system internal transcribed spacer (ITS) barcode sequences for studying linkages among ectomycorrhizal fungal available in public databases, as well as sequencing of diversity, ecosystem function, population genetics, 224 vouchered specimens and cultures, 15 of which and host-symbiont coevolution (Dahlberg and Finlay were type specimens from North America. We found 1999, Wu et al. 2000, Branco et al. 2015). that species boundaries delimited by morphological Although many species need taxonomic revisions data are broadly congruent with those based on ITS or descriptions, the genus Suillus is well circumscribed, sequences. However, some species appear to have and its species boundaries are seldom disputed (Smith been described several times under different names, and Thiers 1964a, 1971; Kretzer et al. 1996; Nguyen several species groups cannot be resolved by ITS et al. 2012; Klofac 2013). The most comprehensive sequences alone, and undescribed taxa are apparent, morphological work on the genus that focused on especially in Asia. Therefore, we elevated S. tomentosus North American taxa was published over 50 y ago var. discolor to S. discolor; proposed synonymies of S. (Smith and Thiers 1964a). This work is still considered neoalbidipes with S. glandulosipes, S. borealis with S. brun- authoritative and has long served as the backbone for nescens, Boletus serotinus and B. solidipes with Suillus elben- Suillus systematics. Recently, Klofac (2013) published sis, S. lactifluus with S. granulatus, S. himalayensis with S. an overview of the nomenclatural history of the genus, americanus; and proposed usage of the names S. clinto- in which he extensively referenced the existing litera- nianus in the place of the North American S. grevillei, ture on Suillus, presented a list of taxa that he accept- S. weaverae for North American S. granulatus, S. ampli- ed, and provided keys to those taxa based mostly porus in the place of the North American S. cavipes, on morphological data. He accepted 94 species in and S. elbensis in place of the North American S. viscidus. Suillus. Since then, S. indicus B. Verma & M.S. Reddy, We showed that the majority of Suillus species have S. triacicularis B. Verma & M.S. Reddy, S. himalayensis strong affinities for particular host genera. Although B. Verma & M.S. Reddy, S. marginielevatus S. Sarwar, deep node support was low, geographic differentiation Khalid & Dentinger, S. lariciphilus K. Das, D. Chakr., was apparent, with species from North America, K.P.D. Latha & Cotter, and S. adhikarii K. Das, Eurasia, and Asia often forming their own clades. D. Chakr. & Cotter have been described from Pakistan Submitted 12 May 2016; accepted for publication 31 Aug 2016. and the Himalayan regions of India and Nepal (Verma 1 Corresponding author. E-mail: [email protected] and Reddy 2014a, 2014b, 2015; Adamcˇík et al. 2015; 1216 NGUYEN ET AL.: GLOBAL SUILLUS ITS PHYLOGENY 1217 Das et al. 2015) along with the very unusual S. foetidus Verma and Reddy 2014a, 2015; Qi et al. 2016). Y. Li & L.L. Qi from northeastern China (Qi 2016). Sequencing of holotype specimens has been shown to The description of many new species in a relatively be essential in assigning correct names to phylogenetic short time suggests the possibility of a number of addi- clades and delimiting species. For example, Nguyen tional undescribed species in currently understudied et al. (2012) showed that by sequencing holotype areas. specimens S. ponderosus A.H. Sm. & Thiers and S. caer- Smith and Thiers (1964a) divided the genus Suillus ulescens A.H. Sm. & Thiers each contained a variety into three sections, Paragyrodon, Boletinus, and Suillus, of the species S. imitatus A.H. Sm. & Thiers, whereas although they stated that these sections were recog- Bruns et al. (2009) produced sequences from holo- nized “partly for historical reasons and for conve- types to indicate that S. quiescens T.D. Bruns & Vellinga nience in dealing with the species in contrasting indeed had not been described before. The widespread groups even if these are based mainly on a single char- application of DNA sequencing has also produced acter” (Smith and Thiers 1964a). Section Paragyrodon numerous sequences from environmental samples was monotypic and included only Paragyrodon sphaeros- (i.e. either ectomycorrhizal root tips or soil). These porus (Peck) Singer, a Quercus-associated species that samples cannot be accurately assigned to species with- Singer (1942) had previously placed in its own genus. out the use of molecular phylogenetics, so placing An early mitochondrial mapping study subsequently these data in the context of a comprehensive global confirmed that it did not belong in Suillus (Bruns specimen-based phylogeny is necessary to assign taxo- and Palmer 1989). Section Boletinus, which was recog- nomic names properly (Bonito et al. 2010; Nguyen nized as a distinct genus (Singer 1975), was included et al. 2012, 2013). in Suillus and described as having either stipes with a Molecular data has been useful in delineating and fibrillose zone or a distinct membranous annulus or a identifying Suillus species when macroscopic charac- distinctly fibrillose-squamulose pileus. Section Suillus ters are plastic or hard to interpret (Nguyen et al. was further broken down into series Hirtellini (mis- 2012). Most species distinctions are based on appear- spelled as Hirtellinii) and Suilli. Species in series Hirtel- ance of fresh collections, host associations, and geogra- lini have a glandular-dotted stipe, although the glands phy. Microscopic differences in cystidial position and often appear as smears rather than dots. The imma- clustering, pileipellis structure, or spore color distin- ture (button) pileus is covered by a tomentum that in guish some major groups within the genus. However, fully expanded mature specimens aggregates into tufts distinctions among closely related species are generally or becomes glabrous in some species. For species of limited to subtle differences in spore sizes that are not series Suilli, the veil forms a well developed annulus based on extensive measurements of multiple collec- or it adheres to the pileus margin when it breaks tions and rarely hold up to scrutiny. Although most open, leaving a “cottony roll” on the pileus margin; species are distinct when young, many older specimens young specimens may have a distinct false veil (i.e. look similar to each other and their appearances one not attached to the stipe but initially almost cover- are affected by environmental factors such as heavy ing the tube cavity). Species with dark brown spores rain, drying winds, or sun exposure that can lead to were excluded by Smith and Thiers (1964a) from misidentifications. Developmental changes in color Suillus and treated as members of a separate genus are also common. For example, some species always Fuscoboletinus Pomerl. & A.H. Sm., but mitochondrial have white basidiocarps, whereas others start out white mapping results disagreed with that segregation but darken to various colors, while a third group is (Bruns and Palmer 1989). made up of white variants of typically colored species. The ability to sequence DNA started the modern era These variations often make identification based solely of molecular phylogenetic studies, with the first com- on morphology difficult to impossible. One frequently prehensive molecular